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NIBGE-FAO Workshop on GMO Detection
CAPACITY BUILDING IN BIOSAFETY OF
GENETICALLY MODIFIED CROPS:
GMOS
(GENETICALLY
MODIFIED
ORGANISMS)
DETECTION
Yusuf Zafar
Muhammad Asif
Ahmad M. Khalid
NATIONAL INSTITUTE FOR BIOTECHNOLOGY AND GENETIC
ENGINEERING
P.O.Box 577, Jhang Road, Faisalabad, Pakistan
Tel: +92-41-651475-9
Fax: +92-41-651472
Email: [email protected];
Website: www.nibge.org
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NIBGE-FAO Workshop on GMO Detection
SPONSOR: -GCP/RAS/185/JPN- Food and Agriculture Organization (FAO)
© 2004 National Institute for
Engineering, Faisalabad, Pakistan
Biotechnology
and
Genetic
All rights reserved. No Part of this publication may be reproduced, stored in a retrieval system or
transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise,
without the prior written permission of the publisher, National Institute for Biotechnology and Genetic
Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan.
Tel: +92-41-651475-9
Fax: +92-41-651472
Email: [email protected]
[email protected]
[email protected]
No responsibility is assumed by the National Institute for Biotechnology and Genetic
Engineering (NIBGE), for any injury and /or damage to the persons or property as a matter
of products liability, negligence or otherwise, or from by use or operation of any methods,
products, instructions or ideas contained in the material herein.
ISNB: 969-8189-10-8
Price:
NIBGE-FAO Workshop on GMO Detection
3
PREFACE
Since the last 30 years recombinant DNA technology is being used for selection of
agronomically important genes and to introduce them in diverse plant species of
agricultural importance. The plants transformed with genes from sources other than their
own genes are genetically modified to express those foreign genes. This technology of
“Genetic Engineering” has enabled scientists to enhance or introduce desired traits such
as resistance to insects, viral/fungal diseases, herbicide tolerance, drought/ salinity
tolerance and improved nutritional traits. There has been quantum jump in development,
adaptation and commercialization of GM crops all over the world.
The introduction of foreign genes into host plants is associated with selectable markers
that may be an antibiotic or herbicide resistant gene. These genes, all together, in a
genetically modified organism impose certain threats to the biosafety of our environment.
Evaluation and release of GM crops is regulated through various international bodies
(WTO, CBD, CPB, UPOV, Codex, Alimentaris) which have been involved in developing
framework for release/trade of GM crops/food. In Pakistan, GM crops have been
developed by NIBGE and few other public sector organizations. However, the
regulations for evaluation and release of GM crops have not yet been promulgated in the
country.
Capacity building in biosafety is an integral activity for the field release of GM crops.
NIBGE being the premier biotech research body in the country and is pleased to
announce the first ever short-term training workshop on GMOs detection in
collaboration with FAO. The workshop is structured in a manner that extensive lectures
by the resource persons would be followed by actual practical to induce an intriguing
environment for co-operative learning. It is anticipated that valuable comments by the
participants from different agricultural businesses will help to improve the future
perspectives in connection with GMOs and knowledge sharing.
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NIBGE-FAO Workshop on GMO Detection
TABLE OF CONTENTS
Sr. No.
Contents
Page
1.
List of Contributors
3
2.
Lectures
5
3.
Advancement in Biotechnology
6
4.
Capacity Building in Biosafety of GM Crops in Asia
11
5.
DNA Technology and GM Crops
16
6.
Global Issues of GM Food and derived products
21
7.
Methods of GMOs Production
30
8.
GMO Detection Methods
37
9.
Polymerase Chain Reaction
57
10.
Real Time PCR for GMO Detection
60
11.
Development of Biosafe Transgenic Plants
64
12.
Sampling for Detection of GMO
70
13.
Development of IR-Cotton: A Case Study
79
14.
Capacity Building in Biosafety of GM Crops in Pakistan
90
15.
Lab Protocols
97
16.
Plant DNA Extraction
98
17.
GMO Detection Methods
109
18.
Cartagena Protocol
131
19.
Glossary
163
20.
Organizing Committees
167
21.
List of Participants
168
22.
Suppliers
171
23.
Authors Index
172
NIBGE-FAO Workshop on GMO Detection
LIST OF CONTRIBUTORS
Dr. Kauser A. Malik
Member Biosciences/ Administrator
Pakistan Atomic Energy commission (PAEC)
P.O. Box, 1114, Islamabad, Pakistan
Ph: + 92-051-9203149 Fax: + 92- 051-9204908
Dr. Anwar Nasim
Chairman
National Commission on Biotechnology
Ministry of Science & Technology
3-Constutution Avenue Sector, G-5/2
Islamabad
Fax: 051-9220265/9205264
Dr. Ahmad Mukhtar Khalid
Director
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Dr. Nobuyuki Kabaki
Plant Biotechnology/Biosafety Specialist
Capacity Building in Biosafety of GM Crops in Asia
FAO Regional Office for Asia and the Pacific
Maliwan Mansion, Phra Atit Road
Bangkok 10200, Thailand
Dr. Yusuf Zafar
Chief Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Dr. Akhlaq Hussain Shah
Director General
Federal Seed Certification & Registration Department (FSC&RD)
Mauve Area, G-9/4, Islamabad
Fax: 051-9260234
Dr. Zabta Khan Shinwari
IABGR
National Agriculture Research Centre (NARC)
P.O. N.I.H, Park Road, Islamabad
Fax: 051-9255201
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NIBGE-FAO Workshop on GMO Detection
Mr. Muhammad Anwar Khan
Deputy Director (WTO)
Ministry of Food Agriculture and Livestock
B-Block, Pak Secretariat
Islamabad
Fax: 051-9221246
Dr. Shahid Mansoor
Principal Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Dr. Aftab Bashir
Principal Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Dr. Muhammad Sarwar Khan
Senior Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Dr. Mehboob-ur-Rahman
Senior Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
Mr. Muhammad Asif
Scientific Officer
National Institute for Biotechnology and Genetic Engineering, (NIBGE)
P.O. Box, 577, Jhang Road, Faisalabad 38000, Pakistan
Phone: + 92-41-651475-9 Fax: + 92-41-651472
6
NIBGE-FAO Workshop on GMO Detection
L
E
C
T
U
R
E
S
7
NIBGE-FAO Workshop on GMO Detection
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Advancement in Biotechnology
Kauser Abdullah Malik, PhD
Member Biosciences
Pakistan Atomic Energy Commission
P.O. Box 1114,
Islamabad- Pakistan
In its broadest sense, biotechnology can be described as the use of living organisms or
biological processes to produce substance or process useful to mankind. In this sense it is
far from new one. One simple example is composting which builds soil fertility by
encouraging microorganisms to break down crop residues. Another example is the
production and use of vaccines to control diseases. Bread, Cheese, Wine and Yogurt
making are among the many practical uses of biotechnology. Thus fermentation, tissue
culture, biopesticides and biofertilizer production constitute as traditional or
conventional biotechnology.
New or modern biotechnology grew out of advances in biological sciences such as
genetics, microbiology, biochemistry and information technology. Biotechnology and
genetic engineering is a fast emerging science. The past 20 years have seen
unprecedented progress in the field of biotechnology, which made revolutionary impacts
on every aspects of human activities which included agriculture, livestock, industry and
medicine. In agriculture it is envisaged that the next green revolution or more
appropriately EVER GREEN REVOLUTION will be due to recent developments in
plant molecular biology, genetic engineering and rapid advancement in genomics.
Biotechnology in Pakistan
Traditional biotech activities particularly related to plant tissue culture have been carried
out in few academic and research institutes since 1970s. An exclusive National Center of
Molecular Biology (CEMB) was established in 1983-4 in Zoology Department,
University of the Punjab, Lahore. In 1986, Government of Pakistan approved building of
National Institute for Biotechnology and Genetic Engineering (NIBGE) in Faisalabad,
which was formally inaugurated in 1994 at a cost of Rs. 340 million. Except these two
major inputs, all other activities remained peripheral. However, during the last 3-4 years,
there is renewed interest in establishing Biotech centers in major cities. Provincial
Governments also initiated some programmes during the same period. Despite all these
developments, there is yet no coherent national policy regarding biotechnology. The
number of biotech centers in the country now exceed to over twenty. Lack of clear
national objectives/priorities, resulted in duplication of work and dilution of efforts.
In contrast to developed world, the biotech research in Pakistan is exclusively carried out
in public sector research centers. Multinational companies (MNCs) are conducting
business but R&D activities of these MNCs in Pakistan are negligible, if any.
For background information and national status of biotechnology in Pakistan, readers are
requested to consult already published reviews on this topic (Broerse, 1990; Desalvia,
NIBGE-FAO Workshop on GMO Detection
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1997; Khan, 1997; Khan & Afzal, 1997; Masood, 1995; Persley & MacIntyre, 2002;
Zafar, 1997 and Zafar, 2002).
Biotechnology consists of a gradient of technologies, ranging from the long-established
and widely used techniques of traditional biotechnology (for example, food fermentation
and biological control), through to novel and continuously evolving techniques of modern
biotechnology.
During the 1970s scientists developed new methods for precise recombination of portions
of deoxyribonucleic acid (DNA), the biochemical material in all living cells that conveys
the instructions that govern inherited characteristics, and for transferring portions of DNA
from one organism to another. This set of enabling techniques is referred to as
recombinant DNA technology or genetic engineering. Modern biotechnology currently
includes the various uses of new techniques of recombinant DNA technology,
“monoclonal antibodies” and new cell and tissue culture methods.
Over the past two decades the number of significant advances made in modern
biotechnology has increased dramatically. It is this increase in the use of new techniques
for understanding and modifying the genetics of living organisms that has led to greatly
increased interest and investment in biotechnology. This has been accompanied by
increasing concerns as to the power of the new technologies and the safety of their use,
both to human health and to the environment.
Evolution of Modern Genetics
Mendel’s laws of genetics were rediscovered in 1900. Mendel had published his original
work on inheritance patterns in pea in 1865, but it took 35 years for others to grasp their
significance. Since 1900, we have witnessed steady progress in our understanding of the
genetic make-up of all living organisms, ranging from microbes to humans. A major step
in human control over genetic traits was taken in the 1920s when Muller and Stadler
discovered that radiation can induce mutations in animals and plants.
In the 1930s and 1940s several new methods of chromosome and gene manipulation were
discovered and used in plant improvement. These included the use of colchicines to
achieve a doubling in chromosome number, commercial exploitation of hybrid vigour in
maize and other crops, use of chemicals such as nitrogen mustard and ethyl methane
sulphonate to induce mutations and techniques such as tissue culture and embryo rescue
to get viable hybrids from distantly related species.
The double-helix structure of DNA, the chemical substance of heredity, was discovered
in 1953 by James Watson and Francis Crick. This combined with the development of
recombinant DNA technology, triggered explosive progress in every field of genetics.
Today, there is a rapid transition from Mendelian to molecular genetic application in
agriculture, medicine and industry.
This brief review of genetic progress from 1900 to 1999 (see Swaminathan, 2000)
stresses that knowledge and discovery represent a continuum, with each generation taking
NIBGE-FAO Workshop on GMO Detection
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our understanding of the complex web of life to a higher level. It would therefore be
unwise to either adopt or discard experimental tools or scientific innovations because
they are either old or new. Just as it took 35 years for biologists to understand fully the
significance of Mendel’s work, it may take a decade or more to understand fully the
benefits and risks associated with living modified organisms (LMOs), including
genetically improved foods (Persley and Siedow, 1999).
The gene revolution
The 1990s have seen dramatic advances in our understanding of how biological
organisms function at the molecular level, as well as in our ability analyses, understand
and manipulate DNA molecules, the biological material from which the genes in all
organisms are made. The entire process has been accelerated by the Human Genome
Project, which has invested substantial public and private resources into the development
of new technologies to work with human genes. The same technologies are directly
applicable to all other organisms, including plants and animals. The first complete
sequence of a plant genome – the flowering plant Arabidpsis thaliana- is now completed
(Arabidopsis Genome Initiative, 2000). The rice genome is also very close to
completions. The new scientific discipline of genomics has arisen, which has contributed
to powerful new approaches to identify the structure and functions of genes and their
applications in human health, agriculture and the environment. These new discoveries
and their commercial applications have helped to promote the biotechnology industry,
mainly in North America and Europe.
Commercial Applications of Biotechnology
The greater specificity in the handling of genes since the 1970s has meant that inventors
could protect their discoveries by means of patents and other forms of intellectual
property rights (IPR). This has led to an explosion of private investment in the
biosciences, leading to what has been called a biotechnology revolution. Most modern
biotechnology applications are in health care, where they offer new hope to patients with
AIDS, genetically inherited diseases, diabetes, influenza and some forms of cancer.
Biotechnology-based processes are now used routinely in the production of most new
medicines, diagnostic tools and medical therapies. The global market of these products is
approximately US$ 13 billion.
New development in agricultural biotechnology are being used to increase the
productivity of corps, primarily by reducing the costs of production by decreasing the
needs for inputs of pesticides and herbicides, mostly in crops grown in temperate zones.
The applications of agricultural biotechnology are developing new strains of plants that
give higher yields with fewer inputs, can be grown in a wider range of environments,
give better rotations to conserve natural resources and provide more nutritious harvested
products that keep much longer in storage and transport, and continued low-cost food
supplies for consumers.
Private industry has dominated research, accounting for approximately 80% of all R & D.
Consolidation of the industry has proceeded rapidly since 1996, with more than 25 major
acquisitions and alliances, worth US$ 15 billion. During the past decade, the commercial
NIBGE-FAO Workshop on GMO Detection
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cultivations of transgenic plant varieties became well established, particularly during the
latter part of the decade. In 2002, it is estimated that approximately 143 million acres of
land were planted with transgenic varieties of over 20 plants species, the most
commercially important of which were cotton, maize, soybean and rape-seed. The value
of the global market in transgenic crops grew from US$ 75 million in 1995 to US$ 1.64
billion in 1998.
The traits these new varieties contain include insect resistance (cotton, maize), herbicide
resistance (maize, soybean) and delayed fruit ripening (tomato). The benefits of these
new crops are better weed and insect control, higher productivity and more flexible crop
management. These benefits accrue primarily to farmers and to farmers and
agribusinesses, although there are also economic benefits accruing to consumers in terms
of maintaining food production at low prices. Health benefits for consumers are also
emerging from new varieties of maize and rap-seed with modified oil content and
reduced levels of potentially carcinogenic mycotoxins. The broader benefits to the
environment and the community through reduced use of pesticides contribute to more
sustainable agriculture and improved food security.
Other corps/input trait combinations currently being field-tested include virus-resistant
melon, papaya, potato, squash, tomato and sweet pepper; “insect-resistant rice,” soybean
and tomato; disease-resistant potato; and “delayed-ripening chilli pepper”. There is also
work in progress to use plants such as maize, potato and banana as mini-factories for the
production of vaccines and biodegradable plastics.
Several large corporations in Europe and the USA have made major investments to adapt
the new discoveries in the biological sciences to commercial purposes, especially to
produce new plant varieties of agriculture importance for large-scale commercial
agriculture. The same technologies also have important potential applications to address
problems of poverty reductions, food security, environmental conservation and trade
competitiveness in developing countries (Tzotzos and Skryabin, 2000; US National
Academy of Sciences, 2000).
Scientific advances
Further scientific advances will probably result in corps with a wider range of traits, some
of which are likely to be of more direct interest to consumers- for example, by having
traits that confer improved nutritional quality in food. Corps with improved output traits
could have nutritional benefits for millions of people who suffer from malnutrition and
deficiency disorders. Genes have been identified that can modify and enhance the
composition of oils, proteins, carbohydrates and starch in food /feedgrains and root crops.
A gene encoding β -carotene/ vitamin A formation, for example, has been incorporated
experimentally in rice. This is being evaluated for the feasibility of using vitamin-A
enriched rice to enhance the diets of the 180 million children who suffer form vitamin A
deficiency. Similarly, introducing genes that increase available iron levels in rice
threefold is a potential remedy for iron deficiency, which affects more than 2 billion
people and causes anaemia in about half that number.
NIBGE-FAO Workshop on GMO Detection
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Applications of biotechnology in agriculture are in their infancy. Most current genetically
engineered plant varieties are modified only for a single trait, such as herbicide tolerance
or pest resistance. The rapid progress being made in genomics may enhance plant
breeding as the functions of more genes and how they control particular traits are
identified. This may enable more successful breeding for complex traits, such as drought
and salt tolerance. This would be of great benefit to those farming in marginal and rainfed lands worldwide, because breeding for such traits, has had limited success with
conventional breeding of the major staple food crops.
Suggested Readings
Broerse, J.E.W. (1990) Country case study Pakistan. Supplement to Bunders et al: Nature
376 (24th August) : 631-638
Persley, G.J. and L.R. MacIntyre (2002) Agricultural Biotechnology: Country Case
Studies: A Decade of Development CABI Publishing, UK.
Zafar, Y. (1997) Status of Biotechnology in Agriculture. In Expert Group Meeting on
Strategies for the Development and Application of Biotechnology for
Economic Growth. Nov.12-13, 1997, Pakistan Council for Science & Technology
(PCST), Islamabad. Pp: 26-50
Zafar, Y. (2002): Pakistan: In: Agricultural Biotechnology: Country Case Studies-A
Decade of Development. Eds: G.J. Persley and L.R. MacIntyre. CABI Publishing, UK.
pp:95-103.
NIBGE-FAO Workshop on GMO Detection
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Capacity Building in Biosafety of GM Crops in Asia
Nobuyuki Kabaki, PhD
FAO Regional Office for Asia and the Pacific
1. Role of genetically modified (GM) crops in food security
World population is reaching 6.4 billion in which 1.2 billion people (one fifth) live in a
state of absolute poverty and more than 842 million people are undernourished.
Although the growth rate of world population is gradually decreasing, it still keeps the
annual number of increase to be over 80 million, which leads to the estimate of the
population of over 10 billions in 21th century. Increase of farmland for food production,
on the contrary, has come to the limiting point worldwide to cause the degradation of
environment with the prolonged development. Increase of food production should
logically depend on the increase of production per unit area of the existent farmland.
“Green Revolution” in 20th increased the overall production of crops with the
development of high yielding varieties and adoption of agricultural commodities such as
fertilizer and pesticides, contributing greatly to increase of food security in the world.
However, it was mainly successful where the conditions of cultivation were sufficient to
raise the yield, leaving the unfavorable large regions with low productivity remained
unimproved. Besides, excessive use of fertilizer and pesticides caused pollution in some
areas, which obliged farmers to resume traditional method of cultivation.
In view of these situation described above, mission of agricultural production in
21th century is grave to achieve the food security through the increase of productivity
with special reference to reduce the burden on environment, to enhance adaptability to
unfavorable conditions and to raise the quality and value of the products.
Biotechnology, especially, gene manipulation, opens a new horizon to increase food
security in 21th century with the introduction of valuable traits with the creation of
genetically modified (GM) crops with four groups categorized from the strategy of
production. The first group raises the tolerance of crops to pests such as insects, diseases
and weeds, which alleviates the burden on the environment reducing the amounts of
chemicals. The second group endows crops with the tolerance to environmental (abiotic)
stress such as drought, salinity, high and low temperature, which enables increase of
production in unfavorable regions. The third enhances the yield and quality with higher
photosynthesis, control of maturity and nutritional value, which increase food security
and reduce the malnutrition. The fourth adds value and diversifies the use of crops other
than food and feed such as medical and industrial purposes, which raises the value of
crops increasing the income of farmers with the development of new agro-industries.
2. Situation of GM crop development
Development of GM crops was initiated from 1980s and large scale commercial
production has started in late 1990s. Recently, ISAAA (International Service for the
NIBGE-FAO Workshop on GMO Detection
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Acquisition of Agri-biotec Application) summed up the situation in 2003. The GM crops
are cultivated in eighteen countries in the world with total area of 67.7 million ha which
showed 15% of increase rate from the last year. Top six countries dominate 99% of the
area with the order of United States (42.8 million ha, 63% of the total area), Argentine
(13.9, 21%), Canada (4.4, 6%), Brazil (3.0, 4%), China (2.8, 4%) and South Africa (0.4,
1%). The other countries consist of Australia, Uruguay, Mexico, Colombia, Honduras,
India, Philippines, Indonesia, Romania, Spain, Germany and Bulgaria. The area in
United States is outstanding and north and south America dominates more than 90% of
the total area.
Crop species used for GM were limited to four with soybean (41.4 million ha, 61% of
the total area), corn (15.5, 23%), cotton (7.2, 11%) and canola (3.6, 5%). They are
classified with the traits of tolerance to herbicides (49.7 million ha, 73% of total area),
resistance to insects (12.2, 18%) and combination of tolerance-resistance to herbicides
and insects (5.8, 8%). The share of GM crops in whole crop species has increased to be
55% in soybean, 16% in canola and 11% in corn, which poses significant effects on the
world trade of crops.
GM crops have been developed in other crops and approved for cultivation in
somewhere in the world. Those are tomato (delayed maturity), potato (resistance to
insect or virus), squash (resistance to virus), papaya (resistance to virus), sugar beet
(tolerance to herbicide), sunflower (tolerance to herbicide), tobacco (quality), wheat
(tolerance to herbicide), melon (delayed maturity), rice (tolerance to herbicide), chicory
(tolerance to herbicide) and carnation (flower color, long-life). Variation of the traits in
the research stage enlarges widely exhibiting the unlimited potentials such as tolerance to
salinity, frost, drought, nutritional composition and growth characteristics.
3. Problems associated with GM crops
While GM crops bears huge possibilities to increase food security and sustainable
agriculture, they also pose problems to be thoroughly considered. One of those are
potential risks to human and animal health, and the environmental consequences. The
risks to human and animal health will be presented by producing toxins or allergic
compounds. Potential risks to the environment include the possibility of endangering the
biodiversity of existing floras and faunas, for example , with the emergence of more
aggressive weeds or upsetting the ecosystem balance. Therefore, establishment of
science-based risk assessment and management is indispensable to secure biosafety
measure on GM crops.
The other problem associated with GM crops is the gap between user and supplier.
Concern about the safety of GM crops is presented by consumers and has become a big
issue on the cultivation and use of GM crops. Development of the system for public
participation and public awareness are important to deepen the mutual understanding
along with the establishment of reliable risk assessment and management. The another
aspect of the gap is related to the equity of access to modern biotechnology including the
development and use of GM crops. Since the technologies and products of biotechnology
NIBGE-FAO Workshop on GMO Detection
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are patented by the developer in rich countries with intellectual property right, poor
countries and people are disadvantaged in the application. Equitable benefit sharing is
important to narrow the gap between users and suppliers.
4. International instruments and the stance of FAO
To solve the problems associated GM crops, several international instruments have been
raised one after another. Those are divided into two categories, one of which is the issue
in the trade of crops. Treatment of GM crops in trades have been repeatedly taken up in
the subcommittee of WTO (TBT, SPS and TRIPS) with Codex Arimentarius and
International Plant Protection Convention as the standard to evaluate the quality of GM
crops. Another one is the issue related to biodiversity and genetic resources regulated by
Convention of Biological Diversity, Cartagena Protocol and International Treaty on Plant
Genetic Resources.
Cartagena Protocol is the first instrument to take up GM crops as a main object. It
has been ratified by 92 countries becoming the mainstream to deal with GM crops from
both trade and biodiversity issues such as transboundary movement of living modified
organisms (LMO), advance informed agreement for trade and national biosafety
framework for evaluation. Each ratified countries is requested to establish its own
biosafety framework based on biosafety laws / regulations, risk assessment and
management, and public participation and awareness.
In these variant and critical situation, FAO maintains a firm stance on biotechnology
which was issued as a statement at FAO conference 2000. It is summed up into five
items as follows,
to recognize the importance of biotechnology to help increase of
productivity in Agriculture, Forestry and Fisheries, to reduce potential risks on human
and animal health and environments,
to support science-based evaluation system that
determine the benefits and risks,
to ensure that developing countries benefit with
public funding and dialogues, and
to assist member countries while acknowledging
the responsibility rests with themselves.
5. Implementation and progress of FAO project
With these background, the FAO project “Capacity Building in Biosafety of GM Crops in
Asia” was initiated in 2002 with the duration of three years with the budget of US$1.23
million donated from Japanese government. Participating countries consist of ten,
Bangladesh, China, India, Indonesia, Malaysia, Philippines, Sri Lanka, Thailand and
Vietnam.
The general objective is to enhance food and livelihood security in Asia through
sustainable and environmental-friendly, in the yield and quality of agricultural produce
including, where appropriate, the safe and judicious harnessing of modern biotechnology.
To achieve the mandate, three specific objectives were proposed which are composed of
strengthening of national capacities for the biosafety of GM crops, establishment of
an Asian Network on Biotechnology for harmonizing biosafety and
supporting and
NIBGE-FAO Workshop on GMO Detection
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promoting research and technology development for safe and environmentally
sustainable use of GM crops.
For the 1st objective, “Strengthening of national capacities for the biosafety of GM
crops”, two activities, “Creation of Benchmark Document” and “National Stakeholder
Workshop” are carried on. Benchmark document compares the strength, weaknesses and
gaps in the cooperating countries, with which direction for the improvement is to be
elucidated. Draft of Benchmark Document is already completed and currently under
circulation for final confirmation before printing. The National Stakeholder Workshop is
held in every country with all relevant institutions for national legislation, regulation and
capacity coordinated with the enabling activities under the Cartagena protocol.
Philippine and Sri Lanka already finished, with the workshop in Pakistan as the third
ones. The other countries follow from now on.
The 2nd specific objective “Establishment of an Asian Network for Harmonizing
Biosafety” consist of three activities of Annual Steering Committee and Technical Expert
Group Meeting, Regional Consultation and Internet Site for Biosafety. “Annual Steering
Committee and Technical Expert Group Meeting” are held in every year to play a driving
role in the project in which the 2nd meeting was held in March 2004 and approved the
work plan until the end of this phase. “Regional Consultation” is useful to develop
harmonization of biosafety measures, standards and regulations in Asian countries. The
1st Regional Consultation was held in July 2003 with the participation of representatives
from participating countries, donor country, International Research Organizations, NGOs
and private countries, in which the framework of the activities was discussed and decided
for further application. “Internet site for biosafety aspects of GM crops” will become the
information center of the project.
For this purpose, Asia-BioNet website
(http://asabionet.org) was opened in November 2003. Improvement of the contents is
forwarding for GM crop information and development of information exchange
mechanisms.
The 3rd specific obejective “Supporting and promoting research and technology
development” is implementing regional training for the development of human resources
in analyzing, managing and researching biosafety risks of GM crops. Four training
workshop, DNA detection, Public participation and Awareness, Risk Assessment and
Management, and Intellectual Property Right are conducted in which
GMO detection was held at DNA Technology Laboratory of BIOTEC Thailand located at
Kasetsat University KamphaengSaen Campus. Other three training workshops are under
preparation with the cooperation between host countries (Philippines, Japan, etc) and
project secretariat.
6. Perspectives
While Cartagena Protocol regulates transboundary movement of GM crops from the
standpoint of the maintenance of biological diversity, it also requests ratified countries to
set up domestic biosafety framework including enforcement of law/regulation, risk
assessment/management and public participation/awareness. Since it will lead to the
NIBGE-FAO Workshop on GMO Detection
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mainstream of international instruments with the increase of ratified countries,
development and use of GM crops will not be possible without its application in near
future. Implementation of the project is quite well-timed in view of the present situation.
Benchmark Document, a product of collaboration in the project, elucidated the large
unequality in the present status of biosafety capacity development between participating
countries. Harmonization is indispensable to harness the huge possibilities of
biotechnology with the function of regional security system and could be only attained
through the sustained collaboration between participating countries. The utmost
objective of the project is the achievement of regional harmonization of biosafety
framework with legitimate benefit sharing system to formulate Asian group on modern
biotechnology.
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DNA Technology and GM Crops
Yusuf Zafar, PhD
Head Plant Biotechnology Division
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Cells are the structural and functional unit of all living organisms. Some organisms, such
as bacteria, are unicellular, consisting of a single cell. Other organisms, such as humans
and crop palnts are multicellular. Each cell is an amazing world unto itself: it can take in
nutrients, convert these nutrients into energy, carry out specialized functions, and
reproduce as necessary. Even more amazing is that each cell stores its own set of
instructions for carrying out each of these activities. There are two general categories of
cells: prokaryotes and eukaryotes. It appears that life arose on earth about 4 billion
years ago. The simplest of cells, and the first types of cells to evolve, were prokaryotic
cells—organisms that lack a nuclear membrane. Prokaryotes (like bacteria) are
distinguished from eukaryotes on the basis of nuclear organization, specifically their lack
of a nuclear membrane. Eukaryotes include fungi, animals, and plants as well as some
unicellular organisms. The major and extremely significant difference between
prokaryotes and eukaryotes is that eukaryotic cells contain different membrane-bounded
compartments in which specific metabolic activities take place. Inside the cell there is a
large fluid-filled space called the cytoplasm which contain different organelles. Most
important part of the cell is the nucleus, a membrane-delineated compartment that houses
the eukaryotic cell’s DNA. The origin of the eukaryotic cell was a milestone in the
evolution of life. Although eukaryotes use the same genetic code and metabolic processes
as prokaryotes, their higher level of organizational complexity has permitted the
development of truly multicellular organisms. Without eukaryotes, the world would lack
mammals, birds, fish, invertebrates, mushrooms, plants, and complex single-celled
organisms.
The nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the
cell's chromosomes and is the place where almost all DNA replication and RNA
synthesis occurs. During processing, DNA is transcribed, or synthesized, into a special
RNA, called mRNA. This mRNA is then transported out of the nucleus, where it is
translated into a specific protein molecule. In prokaryotes, DNA processing takes place in
the cytoplasm. Ribosomes are found in both prokaryotes and eukaryotes. The ribosome
is a large complex composed of many molecules, including RNAs and proteins, and is
responsible for processing the genetic instructions carried by a mRNA. The process of
converting a mRNA's genetic code into the exact sequence of amino acids that make up a
protein is called translation. For most unicellular organisms, reproduction is a simple
matter of cell duplication, also known as replication. But for multicellular organisms,
cell replication and reproduction are two separate processes. Multicellular organisms
replace damaged or worn out cells through a replication process called mitosis, the
division of a eukaryotic cell nucleus to produce two identical daughter nuclei. To
reproduce, eukaryotes must first create special cells called gametes—eggs and sperm—
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that then fuse to form the beginning of a new organism. Meiosis is a specialized type of
cell division that occurs during the formation of gametes.
Perhaps the most fundamental property of all living things is their ability to reproduce.
All cells arise from pre-existing cells, that is, their genetic material must be replicated and
passed from parent cell to progeny. Likewise, all multicellular organisms inherit their
genetic information specifying structure and function from their parents. Two different
kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). Most organisms are made of DNA, but a few viruses have RNA as their genetic
material. The biological information contained in an organism is encoded in its DNA or
RNA sequence. Every organism, has a genome that contains all of the biological
information needed to build and maintain a living example of that organism. The
biological information contained in a genome is encoded in its DNA and divided into
discrete units called genes and code for proteins. The "Central Dogma" a fundamental
principle of molecular biology states that genetic information flows from DNA to RNA
to protein. Ultimately, however, the genetic code resides in DNA
Mitochondria are self-replicating organelles with their own genome that occur in various
numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. Chloroplasts are
similar to mitochondria but are found only in plants. Both organelles have their own
DNA and are involved in energy metabolism. Chloroplasts convert light energy from the
sun into ATP through a process called photosynthesis.
All organisms suffer a certain number of small mutations, or random changes in a DNA
sequence, during the process of DNA replication. These are called spontaneous
mutations and occur at a rate characteristic for that organism. Genetic recombination
refers more to a large-scale rearrangement of a DNA molecule. This process involves
pairing between complementary strands of two parental duplex, or double-stranded
DNAs, and results from a physical exchange of chromosome material. The position at
which a gene is located on a chromosome is called a locus. In a given individual, one
might find two different versions of this gene at a particular locus. These alternate gene
forms are called alleles. During Meiosis, when the chromosomes line up along the
metaphase plate, the two strands of a chromosome pair may physically cross over one
another. This may cause the strands to break apart at the crossover point and reconnect to
the other chromosome, resulting in the exchange of part of the chromosome.
Recombination is the main source of variation in organisms and plant breeders exploit
this variation for crop improvement.
Molecular genetics is the study of the agents that pass information from generation to
generation. These molecules, our genes, are long polymers of deoxyribonucleic acid, or
DNA. Just four chemical building blocks—guanine (G), adenine (A), thymine (T), and
cytosine (C)—are placed in a unique order to code for all of the genes in all living
organisms. The word "cloning" refers to making multiple, exact copies of a particular
sequence of DNA. To make a clone, a target DNA sequence is inserted into what is
called a cloning vector. A cloning vector is a DNA molecule originating from a virus,
plasmid, or the cell of a higher organism into which another DNA fragment of
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appropriate size can be integrated without interfering with the vector's capacity for selfreplication. The target and vector DNA fragments are then ligated, or joined together, to
create what is called a recombinant DNA molecule. These Recombinant DNA
molecules are usually introduced into Escherichia coli, or E. coli—a common laboratory
strain of a bacterium— by transformation, the natural DNA uptake mechanism
possessed by bacteria. Within the bacterium, the vector directs the multiplication of the
recombinant DNA molecule, producing a number of identical copies. These new tools of
DNA technology for creating variation and introducing desired traits has a great potential
for crop improvement in agriculture by combating various diseases and different other
stresses.
Status of GM Crops
Modern biotechnology can contribute to sustainable gains in agricultural productivity and
to food security, especially in the developing world. Breeding and selecting for crops
with increased resistance to pests and disease, vigorous growth and higher yields has
been a primary objective throughout the history of agriculture. Genes identified in wild
germplasm or recovered as spontaneous or induced mutations have been incorporated
through cross-breeding into cultivated varieties of many major crop species. In the past
20 years, the application of molecular biological tools has allowed the production of
genetically modified (GM) plants with traits that could not have been introduced through
traditional breeding techniques. The deliberate release of transgenic crops is often taken
to be an indicator of agricultural biotechnology development. On a global scale, more
than 50 different plant species have been genetically engineered and deliberately released
in about 35 different countries. The projected value of transgenic plants was estimated to
reach about 2-3 billion US$ in the year 2000 and will double by 2005. Field releases in
developing countries account for less than 10% of the total and have been mainly carried
out by transnational companies, usually for the purpose of accelerating seed production in
the counter season. Several reasons account for the limited number of field releases of
transgenic crops in developing countries, like lack of infrastructure and initiative; lack of
adapted biosafety guidelines (a prerequisite for technology transfer); uncertainties in
analysing or predicting potential impacts on ecology, health, environment and
biodiversity.
In 2003, the global area of transgenic crops continued to grow for the seventh
consecutive year at a sustained double-digit growth rate of 15% compared with 12% in
2002. The estimated global area of GM crops for 2003 was 67.7 million hectares; this
includes a provisional conservative estimate of 3 million hectares of GM soybean in
Brazil (the final hectarage could be significantly higher), officially approved for planting
for the first time in 2003. It is noteworthy that a double-digit rate of 10% growth in GM
crops was sustained in 2003, even excluding the Brazilian hectarage. The 67.7 million
hectares of GM crops in 2003, equivalent to 167 million acres was grown by 7 million
farmers in 18 countries, an increase from 6 million farmers in 16 countries in 2002. The
increase in area between 2002 and 2003 of 15% is equivalent to 9 million hectares or 22
million acres.
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The Potential Contribution of GM Crops
The World Food Program recently reported that the number of people suffering from
malnutrition increased by 25 million from 815 to 840 million. The most compelling case
for biotechnology, and more specifically GM crops, is their capability to contribute to:
• increasing crop productivity, and thus contribute to global food, feed and fiber security;
• conserving biodiversity, as a land-saving technology capable of higher productivity;
• more efficient use of external inputs, for a more sustainable agriculture and
environment;
• increasing stability of production to lessen suffering during famines due to abiotic and
biotic stresses;
• and, to the improvement of economic and social benefits and the alleviation of abject
poverty in developing countries.
The Global Value of GM Crops
• In 2003, the global market value of GM crops is estimated to be $4.50 to $4.75 billion,
having increased from $4.0 billion in 2002 when it represented 15% of the $31 billion
global crop protection market and 13% of the $30 billion global commercial seed market.
The market value of the global transgenic crop market is based on the sale price of
transgenic seed plus any technology fees that apply. The global value of the GM crop
market is projected at $5 billion or more, for 2005.
Developing Countries and GM Crops
Between 1970-90 the Green Revolution brought about greatly improved crop yields in
many, but by no means all, parts of the developing world. Poverty and hunger fell
dramatically. However, Africa and parts of Asia saw little gain, and the initial rate of
improvement of the Green Revolution was not sustained between 1985-90. The best areas
had already been saturated with semi-dwarf wheat and rice. Further yield increases were
held back by water shortages, soil problems, and the emergence of new types of pest and
disease. Population growth had slowed down sharply (in Asia since the mid 1970s, and in
Africa since the mid-1980s). In contrast, the rapid and widespread growth in the numbers
of people of working age was sustained. These trends look set to continue. Food
insecurity prevails, even in developing countries with food surpluses. One proposed
solution, the redistribution of surpluses among and within countries poses serious
practical and political challenges. Food aid programmes and efforts towards land reform
have achieved much and should continue. However, improving the productivity of small
farms is by far the best means of achieving a substantial reduction of food insecurity and
poverty. Many people are poor, and therefore hungry, because they can neither produce
enough food on their small farms, nor obtain enough employment by working on those of
others. Enhancement of yields on small farms tends to increase the demand and hence
rewards for poor labourers.
Concluding Comments and Future Prospectives
Despite the on-going debate in the European Union, there is cause for cautious optimism
that the global area and the number of farmers planting GM crops will continue to grow
in 2004 and beyond. Taking all factors into account, the outlook for the next five years
points to continued growth in the global hectarage of GM crops to approximately 100
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million hectares, with up to 10 million farmers growing GM crops in 25, or more,
countries. The global number and proportion of small farmers from developing countries
growing GM crops is expected to increase significantly. Established GM country markets
are continuing to grow in GM area, with a more diversified portfolio of GM crop
products available. New GM countries from the South, like India and Brazil, have
increased their hectarage of Bt cotton and herbicide tolerant soybean respectively, and
some like Uruguay have also approved new products such as GM maize, already
deployed in other countries. New input trait products from industry that will contribute to
sustained growth include the dual Bt gene (cry1Ac and cry1Ab) in cotton and two new
traits introduced into maize in North America. The cry3Bb1 for corn rootworm control,
and the cry1Fa2 gene in Bt maize, with broader control of lepidopteran pests were both
introduced in the US in 2003. Furthermore, five new Bt and novel gene products for
maize insect resistance are expected to be launched in the next three years. Thus, the
global GM maize area with insect resistance and herbicide tolerance traits, as well as the
stacked traits, is likely to increase significantly in the near to mid-term. With the approval
of GM soybean in Brazil for 2003/04, global GM soybean area is likely to experience
renewed high growth rates in the near to mid-term.
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Global Issues of GM Food and derived products
Zabta K. Shinwari*, Ahmad M. Khalid** and Anwar Nasim***
*IABGR-NARC, ISLAMABAD** NIBGE, Faisalabad
*** COMSTECH, Islamabad
Summary
The use of GM (Genetically Modified) crops is increasing in developing countries. In
USA, the rate of adoption of GM crop plants has overshadowed any other introduction of
an agricultural biotechnology. Agricultural and food biotechnology have generated considerable
interest and controversy in the United States and around the world. Some tout the technology's
benefits while others raise questions about environmental and food safety issues. The proponent
of Biotechnology advocate that this is the technology which help in Feeding the world’s
millions and making the deserts bloom’ with ‘miraculous’, even ‘golden’, new
technology; but the opponents of the technology are of the opinion that due to this
technology concentration of ownership over life and biodiversity by bio-elite enforced
with patents; appropriation of indigenous and local knowledges: ‘biopiracy’ and
‘bioprospecting’; profit-making focus on ‘quick fixes’ that fail to come to terms with
underlying causes of social and environmental problems and high risks associated with
release of novel organisms into environment (‘superweeds’, diseases etc). But the bottom
line is that, science will never tell us everything, what will happen no science will tell us
anything.
The commonly asked questions are:
Could GM-derived food be more toxic, more carcinogenic, or nutritionally less
adequate when compared to other foods?
What is the potential for GM technology to produce foods with enhanced
nutritional content or reduced toxicity compared with their non-GM counterparts?
To summarize the answers; FAO and the WHO have concluded that the food safety
considerations for current GM crops, derived food and feed are fundamentally of the
same nature as those that arise from conventional plant breeding (FAO/WHO, 1996). But
when reliable information is available making it possible to identify potentially
dangerous effects to human health, or when there is scientific uncertainty making it
impossible to correctly assess the potential risks for consumers, it is appropriate to adopt
a precautionary approach in risk assessment and management (EC, 2000). However, USA
believes in equivalency approach.
Our obligation (CBD, Convention on Biological diversity) negotiated under auspices
UNEP which entered into force on 29 December 1993 is approved by more than 130
countries. The preamble recognizes risks and benefits associated with biotechnology and
the need to protect biological diversity. According to Article 19.3 of the CBD: Handling
of Biotechnology and distribution of its benefit.
The parties shall consider the need for and modalities of a protocol setting out appropriate
procedures, including, in particular, advanced informed agreement, in the field of safe
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transfer, handling and use of any living modified organisms (LMOs) resulting from
Biotechnology that may have adverse effect on the conservation and sustainable use of
Biological diversity. The protocol establishes a Biosafety Clearing House (BCH) for
countries to share information about genetically modified organisms (GMOs). Countries
must inform the BCH with in 15 days of the approval of any crop varieties which could
be used in food, animal feed and processing. Developing countries in Asia, Latin
America and Africa have serious doubts about whether GM technology will help with
food security. They also have grave concerns about patenting of life forms, which they
see as having the potential to rob them of the rights to the genetic diversity, which exists
in their countries. GM crops could also directly address environmental or human health
objectives, such as the production of renewable materials, or of foods with reduced
allergenicity. However, the availability of novel techniques to develop biosafe
agricultural and food products using natural genetic material has tremendous potential to
feed the world.
Introduction
This paper addresses many of the issues that arise from the use of Genetic modification
techniques that allow new living modified organisms to be produced that have
characteristics that have not necessarily been found before. Genes are copied from
organisms that are unrelated to those into which they are inserted. This technology has
provided a concern from the very outset of its use, and most countries recognize that it
needs some form of regulation to ensure safety both to human health and the
environment. In many cases no need has been seen for changes to law, in others new laws
have been implemented. The potential uses of genetic modification1 were obvious from
the moment that the techniques that enabled the transfer of genes from one organism to
another unrelated organism were first identified.
There was a recognition in CBD that the technology has many advantages and it was
necessary to foster internationally agreed principles to be applied so as to ensure the
“environmentally sound management of biotechnology, to engender public trust and
confidence, to promote the development of sustainable applications of biotechnology and
to establish appropriate enabling mechanisms, especially within developing countries
through:
a. Increasing the availability of food, feed and renewable raw materials
b. Improving human health
c. Enhancing protection of the environment
d. Enhancing safety and developing international mechanisms for cooperation
e. Establishing enabling mechanisms for the development and the environmentally sound
application of biotechnology.
The Convention on Biological Diversity, agreed in 1992, required parties to establish
national frameworks for ensuring that biotechnology is used safely. Article 8(g) states
that Parties shall as far as possible and as appropriate “Establish or maintain means to
regulate, manage or control the risks associated with the use and release of living
modified organisms resulting from biotechnology which are likely to have adverse
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environmental impacts that could affect the conservation and sustainable use of
biological diversity, taking also into account the risks to human health”.
Following are potential environment benefits of Biotechnology:
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Fever, less toxic or less persistent pesticides
Increased yield
Reduced input (Water, fertilizer, labour)
Reduced soil tillage-improved soil conservation
Stress tolerance
Environmental remediation- metals, radioactive substances
Phytoremediation
Cheep therapeutics and vaccines
Reduced mold toxins in corn
Less phosphorus excretion from farm animals fed transgenic feed
No transgene in animal food
More pulp friendly trees, improved lumber
Renewable resources substitute for petroleum based products
Improved farm worker safety
Rescue of endangered wildlife (American chestnut, elm virus, fungal diseases)
Genetic modification offers the industrial forest sector, with its long-standing
limitations on tree improvement, potential for development that would not have
been thought possible 15 years before.
Biosafety (one term that is used to describe the policies and procedures adopted to ensure
the environmentally safe application of modern technology)
•
•
•
•
Our obligation (CBD= Convention on Biological diversity) negotiated under
auspices UNEP
Tabled at the earth summit at Rio De Janeiro, Brazil on 13-6-92
Entered into force on 29 December 1993
Article 19.3 Handling of Biotechnology and distribution of its benefit
The parties shall consider the need for and modalities of a protocol setting out
appropriate procedures, including, in particular, advanced informed agreement, in
the field of safe transfer, handling and use of any living modified organisms
(LMOs) resulting from Biotechnology that may have adverse effect on the
conservation and sustainable use of Biological diversity
Decision 11/5 COP2
• To develop a protocol on Biosafety specially focus on Transboundry movement
of LMOs from Modern biotechnology thtaq may have adverse effects on the
conservation and sustainable use of Biological diversity, setting out for
consideration, in particular, appropriate procedure for advance informed
agreement (AIA)
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UN Food Biosafety Pact (Washington 2000)
•
Protocol approved by <130 countries
•
Preamble recognizes risks and benefits associated with biotechnology and the
need to protect biological diversity
•
Preamble emphasizes protocol “ shall not be interpreted” as changing the rights
and obligations of countries under other international pacts such as WTO.
•
Preamble also recognizes trade and environmental agreements should be mutually
supportive and the protocol is not subordinate to other international pacts.
•
The protocol establishes a Biosafety Clearing House (BCH) for countries to share
information about genetically modified organisms (GMOs). Countries must
inform the BCH with in 15 days of the approval of any crop varieties which could
be used in food, animal feed and processing.
•
Exporters are required to obtain an importing country’s approval, through a
procedure known as AIA, for initial shipments of GMOs intended for release into
the environment (Seeds, Trees).
•
GMOs intended for food, feed and processing-in other words commodities are
exempted , however must be labeled “may contain”
•
Countries also may consider “socioeconomic factors” such as impact on local
farmer, consistent with their other international obligations when making import
decisions.
•
Exception of AIA (Contained use) Research and Transit
•
Members of the pact will cooperate to help developing countries build human
resources and institutions to make informed decisions on GMOs
•
Issue on liability for any damage has to be negotiated
•
Countries have an obligation to inform affected parties and take other appropriate
action if they discover an unintentional movement of GMO across the borders.
•
Illegal shipments, the affected party can request the shipper to retrieve or destroy
the GMO at its own expense
•
Concern about natural wildlife and vegetation
•
How to protect non-GM organisms
•
RR Canola-Weed-spreading over to neighboring crops
•
Mixing genes with native sp., refusing to die with conventional chemicals
Bioethics
•
Government responsibility to ensure that new products do not threaten
environment or human life
•
Any GMO act
•
If there is one, enough human resource that can implement it?
•
How to ensure adequate safety and risk assessment criteria
•
IPR
Environment Consideration
• Agriculture is intrinsically destructive
• Can destroy biodiversity if ill applied
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•
•
•
•
•
•
•
27
Poorly regulated and controlled commercialization of biotechnology in the forest
sector poses additional risks compared with agriculture
Real risk of genetic pollution and invasiveness should not be underestimated,
more immediate problems (inappropriate plantations)
Since 1988 120 confirmed GM tree trials
To what are GE crops compared
Are risk real or hypothetical (Pine pollen can be dispersed upto 600km)
Are there challenges that biotech seeks to address that cannot be met through
conventional tech
Are appropriate assessment and monitoring tools available
Are the benefits outweigh the risks
Risk
Risk may be defined as the likelihood that an organism introduced into the environment
may cause harm to that environment and can be seen as comprised of two factors,
a. The consequence of a particular event
b. The likelihood of the event occurring
Risk arises from exposure and hazard. There is no risk, regardless of how hazardous a
particular organism may be, if there is no exposure. Hazard may be regarded as the
potential to cause harmful effects.
Risk Management
Risk management, which includes the system by which decisions are made, is considered
to be separate from risk assessments. “Risk management” is the process of identifying,
evaluating, selecting and implementing actions to reduce risk to human health and to
ecosystems. The goal of risk management is scientifically sound, cost effective,
integrated actions that reduce or prevent risk while taking into account, social cultural,
ethical, political and legal considerations.
The processes that need to be instituted to ensure that any living modified organisms
(LMOs) are used safely are complex. A country needs:
1. A system for receiving notifications about the intended introduction of a LMO. That
system must provide for informing the applicant of the receipt of the dossier of
information. This system may be the same for applications received from applicants
working on the modification of organisms within the country or for those from applicants
wishing to import products into the country.
2. The systems set up may be different for different uses – if a product is to be used only
for food and feed, and for which there is no intention to grow the organism, the procedure
may be very different from those instituted with organisms that are intended to be
released into the environment.
3. The administrative system must then examine the dossier submitted by an applicant to
ensure completeness – is all the necessary information provided?
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4. Either the Government performs a risk assessment based on information in the dossier,
or the Government audits the risk assessment provided by the applicant. In either event, it
must set up a system of scientific oversight of the dossiers received. If information is
absent a system for requesting further information from applicants must be put into place.
5. There is an obligation to set up systems to allow public and stakeholder comment to be
considered.
6. A system for evaluating the risk assessment and/or the audit report and any public
comments must be instituted. Changes to the design of the ‘release’ or marketing
programme may have to be requested.
7. The decision making process, including the manner in which the decision is
communicated to the applicant and to the general public must be implemented All of this
must be in place before the first decision is madd.
Minimizing Risks
• Elimination of antibiotic resistance and other marker genes
• Engineering plant chloroplast DNA that is not spread through pollens
• Molecular gene pyramiding to develop cost-effective plants and reduce gene
pollution
• Use of plant-derived genes as selectable markers
• Specific genes that block entry of foreign genes via pollination
• Plant sterility to prevent out crossing
• Improved crop management system
• Protection of geographic center of origin of plants to conserve genes
Views and Different Interests on GMO
i)
USDA
“Biotechnology has the potential to make agriculture more productive. … Scientists
believe that biotech has the potential to increase crop yields by 20% or more with no
greater use of natural resources, even on small farms.”
The overwhelming majority of scientific experts worldwide – both private and public
– based on years of research, believe that biotech foods are safe for people to eat. In fact,
all evidence that we have indicates that biotech foods are as safe as conventional foods,
even those foods that have been around for hundreds of years.
This promising beginning has been marred already by Europe’s refusal to permit
imports of biotech corn varieties approved by competent European authorities. As a
result, American corn producers are now losing some $200 million in legitimate exports
annually.
Alan P. Larson (Undersecretary, US Department of State), “Biotechnology:Food Security
and Safety”, Economic Perspectives UDSA Electronic Journal Vol.4 No. 4 October 1999.
ii)
Consumers International (www.consumersinternational.org)
“Genetically Engineered Foods are Different. It is not same as conventional
foods “.
“We have repeatedly argued in the Codex Labeling Committee that even were the
immediate issues of the safety of GM foods.- both generally and in particular - agreed
within an appropriately comprehensive scientific framework and risk assessment
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programme, mandatory labelling should be introduced to allow consumers to decide for
themselves whether they wish to buy and eat them”.
iii)
Organic Consumers Association (www.purefood.org)
The mysterious DNA was found in the Monsanto Company's Roundup Ready
soybeans by Belgian government and university scientists, who described their findings
in a paper published yesterday in the journal European Food Research and Technology.
Greenpeace called yesterday for countries to re-evaluate the regulatory approvals of
the soybeans, saying that Monsanto did not know as much as it should about its product
(The New York Times, August 16, 2001).
iv) WHO - Director-General (Dr Gro Harlem Brundtland)
“We need to improve the systems that we use to ensure food safety and re-establish
consumer confidence. We must reassess them all the way from the farm to the table.
The Codex Commission needs to ensure that there are clear and useful international
guidelines for genetically modified food (at the opening speech at the Codex
Alimentarius Commission meeting in Geneva (2-7 July 2001).
v)
FAO - Director-General (Jacques Diouf) May 2001) "Genetically modified
organisms (GMOs), like all the new technologies, are instruments that can be
used for good and for bad in the same way that they can be either managed to
the benefit of the most needy or skewed to the advantage of specific
groups,“(www. fao.org, FAO Press, May 2001)
vi)
IFAP (International Federation of Agricultural Producers)
• “The potential benefits of the technology are considerable. However, strict
precautions must be taken to avoid any adverse effects on human health,
the environment and on farmers’ ability to run their operations."
• To build up a consumer confidence in the products placed on the market,
we request labelling of any product containing GMO material.
• IFAP recommends that farmers and the food manufacturers bring nonGMO food to the market. It is essential that publicly-funded research
increases focus on the potentials of biotechnology. Monopoly is a major
threat to agriculture.”(Third draft report on "FOOD SAFETY AND
QUALITY”, July 2001).
vii) Codex (FAO-WHO), Codex takes some steps to tighten GM food safety tests
Geneva, 6 July 2001-The Codex Alimentarius Commission has taken some initial steps
to tightening up the ease with which genetically modified foods are put on the market
globally (www. twnside.org, sg) – The Third Word Network.
Discussions
Biotechnology is not new. It has been practiced by human in activities such as baking
bread, brewing alcoholic beverages, breeding food crops and domestic animals.
Biotechnology has been defined as “the application of scientific and engineering
principles to the processing of materials by biological agents to provide goods and
services.” It has been used for many centuries in agriculture and manufacturing to
produce food, chemicals, beverages and many other products that have been of benefit in
many areas including nutrition and health care. Biotechnology is characterised by a
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number of unique conditions. Firstly, it is a cross-cutting technology. It is subject to
wide application, across sectors and biological boundaries.
The total amount of land under cultivation with GM crops grew 12 per cent in 2002,
continuing the double-digit growth recorded since 1996, said the International Service for
the Acquisition of Agri-Biotech Applications (ISAAA), an organisation promoting the
sharing of GM technology.
Genetically modified food and agricultural biotechnology have generated considerable
interest and controversy in the United States and around the world. Some tout the
technology's benefits while others raise questions about environmental and food safety
issues. The Pew Initiative on Food and Biotechnology is developing a series of fact
sheets on some of the common questions that are frequently asked about genetically
modified food and agricultural biotechnology. Transgenic golden rice does not yet fill the
bowls of hungry Asian children. But the possibility that it will is the bright hope of
scientists and biotech companies beaten down by the consumer backlash against the rapid
and largely covert introduction of genetically modified organisms into global food
supplies. The advertisement for golden rice, widely broadcast, is that it avoids all the
pitfalls associated with the ill-fated "Frankenfoods" that so unsettled the buying public.
Unlike with many of the current genetically modified organisms, golden rice poses no
risk of increased pest resistance to herbicides or insecticides. (Holdrege and Talbott,
2003). But people still have doubts on it.
Labelling of GM food-current situation in Europe“Novel foods” Regulation no
258/97-”food containing a GMO has to be labelled as such”equivalence”
• Council Regulation no 1139/98-”labelling based on presence of DNA and
protein “
• Commission Regulation no 50/2000 (additives and flavourings)”-labelling
based on presence of DNA and protein”
• Principle: labelling if DNA or protein resulting from the genetic modification
is detectable
Commission Regulation 49/2000 (amending regulations 1139/98)
• Adventitious presence of GMOs or GM material in food during cultivation,
harvest, transport or processing.
• Threshold of 1%(on ingredient level) for approved GMOs
• Tries to solve the problem of operators who do not use GMOs
Objectives of the revised labelling schemeFreedom to choose
•
•
•
Avoid misleading the consumer
Build confidence
94,6%want to have a free choice on GM food*
GM TraceabilityTraceability enatils the ability to trace products through the
production and distribution chains. The general objectives are to facilitate:
• a harmonised and coherent traceability framework in the EU;
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•
•
31
targeted monitoring of potential effects on the environment
withdrawl of products should unforeseen risk be established;
control and verification of labelling claims.
Biosafety Guidelines Of Pakistan
Biosafety guidelines document, yet to be approved by the Ministry of environment,
Government of Pakistan, has 13 chapters. Chapter one mainly deals with the concerns
that showed that the development and use of transgenic organisms in the open
environment has to be looked at with caution to ensure safety of user and the
environment. This has raised the need to develop and adopt safety of user and the
environment. This has raised the need to develop and adopt safety protocols during
laboratory experimentation as well as during eventual use of Genetically Modified
Organisms (GMOs) and the products thereof. As a rule, research on GMOs is carried out
by competent researchers who are fully conscious of good laboratory practices and the
acceptable safety of releasing the GMOs into the environment. Therefore, the scope and
necessity of the guidelines has been explained.
Chapter two deals with the laboratory guidelines while chapter 3 for fieldwork
guidelines. Chapter 4 Regulations and containment during field tests and chapter 5 deals
with composition and functions of various biosafety committees. Rest of the chapters
explains the details about various requirements.
References
For CBD please visit http://www.biodiv.org/convention/articles.asp
Holdrege, C and Talbott, S. 2003 GOLDEN GENES AND WORLD HUNGER: LET
THEM EAT TRANSGENIC RICE? http://www.oreilly.com/~stevet/netfuture/
Delgado, C., M. Rosegrant, H. Steinfeld, S.Ehui, and C. Courbois. 1999. Livestock to
2020: The Next Food Revolution.
FAO. 1996. In Investment in Agriculture: Evolution and Prospects. World Food Summit
Technical Background, Document .No. 10
Hollingsworth, W 1998. Release of Genetically modified materials in the Caribbean.
Mc Calla, A.F. 1998. The challenge of Food security in the 21st Century Montreal,
Quebec: Convocation Address, Faculty of Environment Sciences, McGill University
Pinstrup-Andersen, P., R. Pandya-Lorch and M.W Rosegrant. 1999. World food
Prospect: Critical Issues for the early Twenty-First Century. Washington, D.C.
International Food policy Research Institute.
NIBGE-FAO Workshop on GMO Detection
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Methods of GMOs Production
Yusuf Zafar, PhD
Head Plant Biotechnology Division
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Genetically modified (GM) plants are playing an important role in crop germplasm
improvement. Transgenic technologies which are only 20 years old are still in infancy.
Gene transfer through recombinant technology is a rapid mode for introducing desirable
traits into plants.
Transformation refers to procedures of delivering foreign DNA into a living cell. The
event may be transient or integrative. This technology, which has developed rapidly in
recent years, has applications for studies in basic biology allowing the analysis of gene
structure and regulation and most importantly in Genetic Engineering experiments in
bacteria, yeast, animals and plants. In the present article, the emphasis will be on plant
transformation however; the principles are the same and applicable to all other living
systems with slight modifications.
Genetic engineering of crop plants though apparently simple in principle but is a
complex, sophisticated and costly technology, which requires a certain degree of
expertise. Genetic engineering of crops plants comprises of following steps:1)
2)
3)
4)
5)
6)
7)
8)
Availability of gene
Vector construction
Transformation
Efficient regeneration system
Gene expression analysis
Inheritance
Field evaluation
Commercialization
There is little doubt that gene transfer, in combination with techniques for gene isolation,
DNA modification, in-situ hybridization of mRNA etc., will play an essential role in the
future using gene transfer and manipulation techniques for the predicable alternation of
plant genomes, especially those plants of high economic or nutritional importance. Gene
transfer for the genetic engineering of plants impose very specific requirements on the
technology, requiring methods that work efficiently with virtually every variety of every
given plant species.
There are several methods for introducing foreign DNA into plants. Following methods
are commonly being used to improve plants through biotechnology:
A.
B.
Agrobacterium mediated transformation
Direct Delivery of DNA.
NIBGE-FAO Workshop on GMO Detection
B.
Direct delivery of DNA
B.1.
Protoplast based transformation
Chemical based …..PEG
Electroporation
Microinjection
Liposome mediated
-
B.2.
Non-protoplast based transformation
Gene gun
33
Agrobacterium Mediated Transformation:
Engineering crops with desired traits depends upon the reliable and efficient means of
transferring cloned genes into plants. The gram negative soil bacteria, Agrobacterium
tumefaciens and Agrobacterium rhizogenes are natural genetic engineers capable of
transforming a range of dicotyledonous plants by transferring plasmid encoded genes into
recipient plants genomes. Both bacteria contain a large plasmid (more than 200Kb),
which is required for initiation of the neoplasia (crown gall and hairy root). In the case of
A. tumefaciens, the plasmid is called the tumor inducing Ti plasmid whereas it is called
the root inducing or Ri plasmid in the case of A. rhizogenes. During the establishment of
infection, a region of either the Ti and Ri plasmid termed the T-DNA is transferred into
the plant cell and becomes stably integrated into one of the chromosomes in the nucleus.
Another set of transferred genes are responsible for the production of special amino acid
region and sugar derivatives called opines. Another region of the Ti plasmid the
virculence (Vir) encodes most of the functions necessary for T-DNA transfer. The
polarity and position of 25bp long repeats bordering the T-DNA are essential for its
transfer and integration into plant genome. These processes are not dependent on the
expression of the T-DNA encoded genes, but are coordinated by the expression of genes
in the virulence region. This has allowed the construction of a wide variety of plant
transformation vectors and generally these can be classified as being one of two types cointegrative or binary vectors.
Co-integrative vectors are deletion derivatives of the Ti plasmid from which the
majority of the T-DNA between the border repeats has been replaced by a defined
sequence of DNA. Foreign DNA to be inserted into the plants genome is cloned into an
intermediate vector which is able to replicate in E.coli but not in Agrobacterium and
contains appropriate selectable marker genes as well as a sequence homologous to that
located between the border repeats of the cointegrative vector. The intermediate vector is
introduced into Agrobacterium containing the co-integrative vector by conjugation.
Binary vectors are plasmids that contain origins of replication that are active in both
types of bacteria. Flanking a region that allows the insertion of foreign DNA are the TDNA border repeat sequences. Binary vector containing foreign DNA to be inserted into
the plant genome can be introduced into Agrobacterium containing a Ti plasmid from
which the T-DNA has been deleted but retains a functional vir region by either
transformation with electroporation or conjugation.
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The protocols used at NIBGE for the transformation of tobacco and cotton will be
demonstrated. The most critical factors that affect transformation efficiency are plant
genotype, growth conditions, growth stage and bacterial concentration.
Protoplast-mediated Transformation:
Plant regeneration via protoplasts is the epitome of plant cell totipotency because they
can be cultured as single cells that produce multicellular colonies from which plants
devlop. The most commonly used protoplast isolation procedures involving digestion of
plant material (leaves, tissue culture, etc.) with mixtures of purified fungal cellulases.
Since that time, applications of protoplast isolation and culture have been extended to a
diverse number of plant species. In crop plants, rapid initial progress was reported in the
Solanaceous and Brassica species, and some forage legumes. Cereal crops proved to be
most recalcitrant, but through development of embryogenic suspension cultures as
sources of totipotent cells, first rice and then the other cereals have been regenerated from
protoplasts with varying success.
Protoplasts, in principle, are ideal cells for DNA delivery and selection of transgenic
events. Removal of the cell wall eliminates a major barrier to DNA delivery.
Furthermore, protoplast cultures are analogous to bacterial culture systems with the
advantages inherent in culturing large numbers of single cells (often in excess of 106) in
defined medium. Protoplast-derived tissue cultures generally are clonal in origin because
protoplasts exist initially as individual cells. Selection efficiency for recovery of
transgenic events is maximized because cross feeding, and chimerism between transgenic
and wild type cells is minimized in comparison to transformation systems based on
multicellular tissues.
PEG Mediated Transformation:
Development of chemical treatments to introduce DNA into microbial and mammalian
cells preceded adoption of these protocols to plant protoplast culture systems. The most
commonly used procedure for direct DNA delivery into plant protoplasts involves
treatment with polyethylene glycol (PEG) to alter plasma membrane properties by
causing reversible permeabilization that enables exogenous macromolecules to enter the
cytoplasm. The precise mechanism of PEG-mediated membrane permeabilization, and
there by DNA delivery, is not completely understood.
The first reports of direct DNA delivery and stable transformation involved transfer and
expression of Agrobacterium tumefaciens T-DNA genes into tobacco protoplasts via PEG
treatments. The first example of transgenic plants was reported in 1984. Key factors in
the success of this system were:
-
Optimization of the tobacco leaf protoplast-to-regenerated plant protocol;
-
Development of the aminoglycoside phosphotransferase gene II from E.
coli transposon Tn5 as a selectable marker conferring plant cell resistance
to kanamycin and
Optimization of DNA delivery frequency
-
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PEG-mediated DNA delivery has been applied to transformation of other dicots and a
number of monocots such as cell cultures of annual ryegrass (Lolium multiflorum L.) and
wheat (Triticum monococcum L.). More recently, chemical treatments of protoplasts
using Polybrene increased transformation frequencies up to 8%. It is likely that further
modifications in protoplast culture systems and chemical agent likely will increase
frequency of DNA delivery mediated by chemical treatments.
Electroporation Mediated Transformation involves subjecting protoplasts to electrical
pulses of high field strength to cause reversible permeabilization of the plasma membrane
enabling macromolecule delivery. Electroporation is routinely used for transient gene
expression studies in both mammalian cell and plant protoplast systems. Application of
electroporation for stable transformation has been extended to a number of plant species.
Stable transformation of tobacco plants regenerated from electroporated protoplasts
involved combinations of PEG and electroporation treatments. Frequencies of
transformed tissue cultures were 1 to 2% of all colonies recovered without selection.
Stable transformation of corn cell cultures were reported also. In this study,
transformation frequencies were upto 1% of dividing protoplasts were obtained.
Regenerated but sterile transgenic corn and rice plants were produced from
electroporated protoplasts. Rice was the first fertile transgenic cereal species produced
using electroporation.
The primary advantages of electroporation over PEG or other chemical-mediated
treatments are reproducibility of high frequency DNA delivery and simplicity of the
technique. Electroporator devices are easily constructed and also commercially available.
Electroporation of protoplasts suffers from the drawbacks and limitations inherent with
protoplast cultures. Because of the problems with protoplast cultures, electroporationmediated DNA delivery has been extended to intact plant tissues.
Microinjection Mediated Transformation:
Microinjection is an old technology dating to the mid 1880’s. This technique gained
widespread use in the 1940’s when several new micromanipulator designs were
introduced. The first important use of microinjection was amphibian nuclear
transplantation where nuclei from differentiated cells were injected into enucleated egg
cells. Results from these experiments provided evidence for totipotency during
development.
The first genetically transformed animals produced by microinjection. Mice embryos at
the blastocyst stage were injected with SV40 DNA. About 40% of the cells in the
resulting animals were mosaics. Non-mosaic, fully transformed animals were created
when fertilized egg cells were microinjected. When injected 125 molecules of the
thrymidine kinase gene into mouse egg nuclei, approximately 1 cell in 500 was
transformed. The production of transgenic mice via microinjection is now a routine
laboratory practice.
Microinjection of plant cells is far from routine. Plant cells are more difficult to inject
than animal cells for two reasons.
NIBGE-FAO Workshop on GMO Detection
36
*
Plant cells have a cell was composed of thick layers of lignin and cellulose
that are difficult for a glass microneedle to penetrate.
*
The vacuole contains many hydrolase and toxic compounds. If the
vacuolar contents are emptied into the cytoplasm, the cell will most likely
die. In some instances, the cell’s nucleous can be found embedded within
the vacuole, making it impossible to inject.
Several systems are used to inject plant cells or protoplasts. The first system uses two
micromanipulators. One micromanipulator holds the protoplast while the other holds the
injection needle. One can use the holding manipulator to select a protoplast with a
nucleus peripherally exposed to the plasma membrane and in line with the injection
needle.
A disadvantage of this system is that it is very time consuming. If protoplasts are not held
by a micromanipulator during injection, then some other method is required for their
immobilization. Protoplasts can be attached to glass that has been coated with poly-Llysine and subsequently
injected, however, the concentration of poly-L-lysine required for attachent is toxic to
some species. Toxicity can be avoided by embedding protoplasts in agarose. However,
the disadvantages of agarose are reduced visibility and it is difficult to move the injection
needle within this gel.
Removal of the vacuole has been tested to overcome problems with solute toxicity.
Evacuolated protoplasts that retain their nucleus and cytoplasm remain viable. There are
many methods for evacuolating protoplasts. Although microinjection of evacuolated
protoplasts is more efficient, the frequency of regeneration is less than with vacuolated
protoplasts.
The equipment used in microinjection research ranges from simple to complex. A simple
micrometer attached to the plunger of a syringe has been used. A simple micrometer
attached to the plunger of a syringe has been used. A complicated system consisting of
pressurized gas tanks attached to gas-burst regulators allows a more controlled and
uniform injection rate.
Several commercial pressurized microinjection systems are now available. Design of the
micromanipulator also is important to successful microinjection. Several different designs
are commercially available (pneumatic, lever, rack-and-pinion and electronic).
Equally important is the microscope design. A microscope with a long working-distance
lens (Distance between subject and lens) is essential since it gives more room for
manipulation. Another important development is the inverted microscope (objective lens
is under the subject and the condenser lens is above the subject) because of the increased
working distance between the subject and condenser lens. A large number of inverted
microscopes are currently available. There are many reports demonstrating that
protoplasts can survive microinjection. In addition, there are papers that describe the
injection of pollen grains and their successful use in vitro fertilization. The injection of
NIBGE-FAO Workshop on GMO Detection
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L2 germline cells from wheat and their successful regeneration into whole plants also
was reported.
B.2.
Non-Protoplast based Direct Delivery
Biolistic Transformation:
One way to circumvent the limitations of regeneration of protoplasts into plants and
Agrobacterium host specificity is to use micro-projectile bombardment also called
particle gun or biolistic method of transformation. In this method, tissues or cells are
bombarded with DNA typically bound to gold or tungsten particles and the plants are
then regenerated. The merit of this procedure is that almost any kind of tissue having the
potential to regenerate plants can be used as the target/recipient for foreign DNA. Thus
particle bombardment provides best method for achieving genotype independent
transformation in plants. After the development of first apparatus based on gun-powder
charge, different types of guns have been developed/used by varying the mode of the
acceleration of particles like compressed air, nitrogen gas, helium gas and electric
discharge. Recently, a hand held particle gun ‘Heliose’ has been introduced in the market.
The first report of transgenic plant production through particle bombardment appeared in
1988 when transgenic soybean plants were produced. By 1990, transgenic cotton, maize
and tobacco were reported. In the following two years, almost all major crops like rice,
wheat, and sugarcane were transformed using particle bombardment.
Different explants can be used as target cells including embryogenic cell suspensions,
meristems, embryos, immature embryos and pollen.
The Biolistic® PDS-1000 device is the only commercially available particle delivery
system for gene transfer. The first PDS-1000 device marketed by the DuPont Company,
was based on the design of Sanford (1987). The device employed a gunpowder charge to
propel microscopic tungsten particles, called microcarriers, on the face of a plastic
cylinder, called a macrocarrier. The gunpowder design proved successful for genetic
transformation of diverse plant species in numerous laboratories, however, lack of control
over the power of the bombardment as well as substantial damage to target cells limited
the number of stable transformants that could be obtained. The current model, PDS1000/He, which is now marketed by Bio-Rad Laboratories, represents a significant
technical improvement over the gunpowder device. The basic design was developed by
Sanford et al. (1991). The PDS-1000/He device is powered by a burst of helium gas that
accelerates a macrocarrier, upon which millions of DNA coated microcarriers have been
dried. Compared to the gunpowder device, the PDS-1000/He is cleaner and safer, allows
better control over bombardment power, distributes microcarriers more uniformly over
target cells, is more gentle to target cells, is more consistent from bombardment to
bombardment, and yields 4-300 fold more transformants in the species tested (Sanford et
al. 1991).
NIBGE-FAO Workshop on GMO Detection
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Conclusion
DNA delivery methods are described in our review. The ideal transformation system
should be based on a DNA delivery technology that is simple, efficient and inexpensive.
This process should enable efficient, high fidelity integration of transgene sequence(s)
(nuclear, plastid or mitochondrial). Target cells for DNA delivery should be easy to
isolate and exhibit totipotency from a wide array of genotypes within a species. The
tissue culture process required for selection of transgenic cultures and regeneration of
plants therefrom should be minimized to reduce the frequency of deleterious mutations.
These guidelines will differ from species to species. Improvements to these DNA
delivery systems will assist in increasing the frequency of stable transformation and
promote utility of such systems in industry.
Suggested Readings
Hughes, M.A (1996) Plant Molecular Genetic Longman Publishers, U.K.
Hanson, G. and M.S. Wright (1999). Recent advances in the transformation of plants.
Trends in Plant Sciences 4(6): 226-231.
Kikkert, J.T. (1993) The Biolistic® PDS-1000/He device Plant Cell, Tissue and Orgem
Culture 33: 221-226.
Potrykus, I. (1990a) Gene transfer to cereals: An Assessment Bio/technology 535-542.
Potrykus, I. (1990b). Gene transfer to plants: A critical assessment Physiologia Plantarum
(Special Issue) Vol. 79.
Songstad, D.D., D.A. Somers and R..J. Griesbach (1995). Advances in alternative DNA
delivery techniques. Plant Cell, Tissue and Organ Culture 40:1 15.
NIBGE-FAO Workshop on GMO Detection
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GMO Detection Methods
Muhammad Asif
Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Background Information
The detection of genetically modified crops (GMCs) has become necessary to allow
consumers to make an informed choice and to comply with labeling regulations.
Although legislation governs the release of new foods, and a strict approval process
includes the safety assessment of every new GM crop, there is still reluctance on the part
of consumers to knowingly buy plant-derived GM products. Public concern and
awareness of the issue have been significantly increased. Food safety is concerned with
consumers’ right to know what is in the products they buy. Without transparent
information flow of GM products, informed choice can not be sustained. Lack of
information also negatively impacts the development of biotechnology. Although
biotechnology presents considerable potential for food production and crop improvement
efficiency, but food derived from biotechnology will face constant challenges from
consumers around the world until information and evidence of the issues raised can be
provided. The major regulatory and scientific agencies in the world believe that GM
crops pose no greater threat to human health than those posed by traditional crop
breeding approaches. Nevertheless, some countries have introduced mandatory-labeling
legislation of GM foods to give their consumers a choice in selecting the foods they feel
comfortable with. An agreement, the ‘Cartagena Biosafety Protocol’, governs the trade
and transfers of GMOs across international borders and allows governments to prohibit
importation of GM food when there is concern over its safety. Universal legislation
makes it imperative for governments, the food industry, crop producers and the testing
laboratories to develop ways to accurately quantitate GMOs in crops, foods and food
ingredients to assure compliance with threshold levels of GM products required for
labelling.
Basics of GMO Detection
A genetically modified organism (GMO) is a living organism, e.g. a plant, whose genetic
composition has been altered through recombinant DNA technology. The genetic
modification usually involves insertion of a foreign DNA into the genome of the
organism to be transformed. The most common genetic modifications in crops confer
herbicide or insect resistance to the plant. This resistance is achieved through production
of a novel protein encoded by the inserted DNA sequence. In plants that are genetically
modified for commercial agricultural purposes, the recombinant DNAs that are
artificially inserted into the natural plant genome have some common genetic elements.
Each inserted DNA sequence consists of at least a promoter, a protein-coding site (the
structural gene) and a terminator. The promoter is a sequence of DNA that acts like an
"on switch" for the transcription of DNA into mRNA. The terminator marks the end point
for this transcription procedure. The structural gene determines the particular protein that
is to be made. The 35S promoter in the genetic modification is derived from the
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cauliflower mosaic virus. The promoter used in Roundup Ready soy is 35S, while the
novel protein that confers resistance to Roundup is EPSPS taken from the soil bacterium
Agrobacterium tumefaciens. The Nos-terminator used in the Roundup Ready soybean's
GM construct originates from A. tumefaciens as well.
GMCs are increasingly being introduced into the world's food supply. Concerns raised by
consumers and regulatory agencies in various countries have highlighted the need for
reliable and accurate testing for the presence and the amount of genetically modified
components in food. Since, GM products contain an additional trait encoded by
introduced gene(s), which generally produce additional protein(s) that confers the trait of
interest. Therefore, raw materials (e.g. grains) and processed products (e.g. foods)
derived from GM crops might thus be identified by testing for the presence of introduced
DNA, or by detecting expressed novel proteins encoded by the genetic material.
Methods of GMO Detection
There are several commonly used GMO testing protocols, which include mainly
biological tests based on phenotype, as well as protein and DNA based detection. Of
these methods, DNA-based polymerase chain reaction (PCR) with different formats
achieves the greatest sensitivity, selectivity, and ability to screen a wide range of GM
products. While among protein-based, Enzyme-linked immunosorbent assay (ELISA) is a
quicker, less expensive and simpler to perform. An immunological assay is based on the
specific binding between a protein and an antibody and therefore, any conformational
changes in the tertiary structure of the protein render the test ineffective. Such
conformational changes are induced frequently during food processing and hence,
processed foods are generally analysed with PCR method. Although much progress has
been achieved in the development of genetic analysis methods, several other analytical
technologies are emerging that can provide solutions to current technical issues in GMO
analysis. Those include, mass spectrometry, chromatography, near infrared spectroscopy,
micro fabricated devices, DNA chip technology and nanoscale GMO analysis.
A) Phenotype-Based GMO Detection
These tests mainly include germination test, towel test and bioassay mostly used for
herbicide tolerance and insect resistance traits. In germination and towel tests seeds are
placed in a germinating media or a towel, moistened with a diluted solution containing
the herbicide, or spraying the herbicide on seedlings. Seeds or seedlings that test positive
for the presence of the herbicide tolerance trait will germinate and develop normally,
whereas those that die will be GMO-free. For insect resistance trait mostly bioassay is
performed. For this purpose insect larvae are reared on leaves of the crop plants to be
tested. If the larvae die, that plants have the genetically modified trait for insect
resistance, while if the larvae feed on normally then that plants are GMO-free. These
procedures are relatively cheaper, easy to perform and user friendly but time consuming
and limited to the plants producing germinating seeds only because it could not be
applied to crushed grains and processed foods, moreover, identification of specific event
is not possible.
NIBGE-FAO Workshop on GMO Detection
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B) Protein-Based GMO Detection
The specific detection of a novel protein synthesised by a gene introduced during
transformation constitutes an alternative approach for the identification of genetically
modified plants. Protein detection methods are based mainly on immunoassays, which
can be used for the detection of many types of proteins in different matrices when the
target analyte is known. Both monoclonal (highly specific) and polyclonal (often more
sensitive) antibodies can be used. On the basis of typical concentrations of transgenic
material in plant tissue (more than 10 µg per tissue), the detection limits of protein
immunoassays can predict the presence of modified proteins in the range of 1% GMOs.
There are also some limitations of protein based GMO testing because genetic
modification is not always specifically directed at the production of a new protein and
does not always result in protein expression levels sufficient for detection purposes. In
addition, certain proteins may be expressed only in specific parts of the plant or
expressed at different levels in distinct parts or during different phases of the
physiological development. Since immunoassays require proteins with an intact tertiary
or quaternary structure, so these methods are limited to fresh and unprocessed foods.
I) Immunoassays
Immunoassays are analytical measurement systems that use antibodies as test reagents.
Antibodies are proteins produced in the serum of animals in response to foreign
substances (antigens) and specifically bind the substance that elicited their production. In
the case of detection of GMOs, the antigen can be the newly synthesized protein. One of
the major drawbacks of immunochemical assays is that their accuracy and precision can
be adversely affected in complex matrixes, such as processed vegetal and food products.
Indeed, many substances present in food matrixes such as surfactants (saponins),
phenolic compounds, fatty acids, endogenous phosphatases, or enzymes may inhibit the
specific antigen-antibody interaction. Moreover, detection capability may be hampered
when the transgenic protein is expressed at a very low level, or degraded and denatured
by thermal treatment. The newly expressed protein may not be evenly present in all tissue
of the plant.
a) ELISA
The most common type of immunoassay is the Enzyme-Linked ImmunoSorbent Assay
(ELISA), which utilises an enzyme-labelled immunoreactant (antigen or antibody) and an
immunosorbent (antigen or antibody bound to a solid support). The ELISA technique has
been widely applied for evaluating the expression level of the protein(s) synthesized by
the newly introduced gene. With ELISA we can detect the presence of GMOs in raw
material at concentrations ranging between 0.3% and 5%. However, differences may be
observed in the expression level of the protein between crop varieties. There are two
formats in ELISA: the microwell plate (or strip) format and coated tube format. In an
ELISA test for genetically modified agricultural product, the novel protein that is made
by the bioengineered gene, is isolated and antibodies are raised against specific surface
structures (epitopes) of this protein. If proteins are present, they are bound to the walls of
the test kit and react with tagged antibodies resulting in a change of color. In some
commercial kits it is possible to get a quantified reading of the amount of targeted protein
NIBGE-FAO Workshop on GMO Detection
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by preparing a standard curve and using a photometric reader to compare the degree of
color change of the sample to the standard curve.
b) Lateral Flow Strip
Lateral flow strip technology is a variation on ELISA, using strips rather than microtiter
wells. Immobilized double antibodies, specific for the expressed protein, are coupled to a
color reactant and incorporated into a nitrocellulose strip. When the strip is placed in a
plastic eppendrof vial containing an extract from plant tissue or seed harboring a
transgenic protein, it leads to an antibody sandwich with some of the antibody that is
coupled to the color reagent. This colored sandwich flows to the other end of the strip
through a porous membrane that contains two captured zones, one specific for the
transgenic protein sandwich and another specific for untreated antibodies coupled to the
color reagent. Presence of only one (control) line on the membrane indicates a negative
sample, and presence of two lines indicates a positive result.
The lateral flow format gives results within a few minutes, but these results are
qualitative because there is no quantitative internal standard within the assay and no extra
information can be obtained concerning the presence of GMOs at the ingredient level in
food. It is economical, more amenable to point-of-sale application, and suitable as an
initial screening method early in the food chain. These test strips are fast, cheap and
require minimal training and equipment. These strips have been developed commercially
to detect endotoxins expressed by Bacillus thuringiensis that protect against insects and
CP4 EPSPS protein. Commercially available lateral flow strips are currently limited to
few GM products, but strips that can simultaneously detect multiple proteins are being
developed.
c) Other Immunoassay Formats
In addition to microplate ELISA and lateral flow devices, other immunoassay formats use
magnetic particles as the solid support surface. The magnetic particles can be coated with
the capture antibody and the reaction carried out in a test tube. The particles with bound
reactants are separated from unbound reactants in solution by a magnet. Advantages of
this format are superior kinetics and increased precision because the uniform particles are
free to move in reaction solutions. Advances are also being made in combining antibody
methods with instrumental techniques. For example, in addition to the hyphenated
method, such as immunoassay-mass spectrometry, considerable advances have been
made in relative observation of antibody binding to target molecules using biosensors.
II) Western Blot
The western blot is a method that provides qualitative determination whether the target
protein level is below or above a predetermined threshold. It is particularly useful for the
analysis of insoluble protein. This method is considered more suitable for research
applications than for routine GMO testing. The samples to be assayed are solubilized
with detergents and reducing agents, and separated by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The components are transferred to nitrocellulose
membrane, and binding immunoglobulin sites on the membrane are blocked by dried
nonfat milk. The specific sites are then probed with antibodies. Then the bounded
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antibody is stained with Ponceau, silver nitrate or Coomassie, or a secondary
immunological reagent, such as protein A coupled to horseradish- peroxidase (HRP) or
alkaline phosphatase.
Although protein-based tests are practical and effective, some GM products do not
express a detectable level of protein. ELISA is the method of choice to screen for a
particular GMO in raw material, semi-processed foods and processed ingredients,
provided the expressed protein is not degraded and can be detected. However, because
ELISA has lower detection power than PCR methods, it is less sensitive for testing
finished food products with many ingredients, especially if the threshold for detection is
low.
C) DNA-Based GMO Detection
As DNA is a rather stable molecule it is the preferred analyte for almost any kind of
sample (raw materials, ingredients, processed foods). Irrespective of the variety of
methods used for DNA analysis, only PCR in its different formats has been widely
applied in GMO detection/analysis and generally accepted for regulatory compliance
purposes. PCR is very sensitive and therefore, very small amount of material is required
for the analysis. Provided that the laboratory sample is representative for the field sample
and that it has been adequately homogenised aliquots between 100 mg and 350 mg are
adequate for DNA extraction procedures in the laboratory.
I) Southern Blot
This method involves fixation of isolated sample DNA onto nitrocellulose or nylon
membranes, the probing with labeled probes specific to the GMO, and the detection of
hybridization radiographically, fluoremetrically or by chemiluminescence. Earlier probes
were labeled with 32P, however, non- radioactive fluoresceinlabeled DNA, digoxigenin-,
or biotin-labeled DNA probes, with sensitivity equal to 32P probes, are recently used,
obviating the need for radioactivity in the testing laboratory. These non-radioactive
probes reduced detection to less than 1 hr, as opposed to 24 hrs labeling required by 32P.
However, because only one probe is used and no amplification is carried out, this method
is considered less sensitive than PCR. An alternative Southern blot technology with near
infrared (NIR) fluorescent dyes (emitting at 700 and 800 nm) coupled to a carbodiimidereactive group and attached directly to DNA in a 5 min reaction has been attempted
recently. The signals for both dyes are detected simultaneously by two detectors of an
infrared imager, something not yet possible with conventional radioactive or
chemiluminescent detection techniques.
II) PCR-Based Detection
PCR allows the million-fold amplification of a specific target DNA fragment framed by
two primers. In principle, the PCR is a multiple-process with consecutive cycles of three
different temperatures, where the number of target sequences grows exponentially
according to the number of cycles. In the first step, the template, i.e. the DNA serving as
master-copy for the DNA polymerase is separated into single strands by heat denaturation
at ~94°C. In the second step, the reaction mix is cooled down to a temperature of 5065°C (depending on the GC-content) to allow the annealing of the primers to the target
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sequence. In the third step, the annealed primers are extended usually by a Thermus
aquaticus (Taq) polymerase at the optimum temperature of 72°C. With the elongation of
the primers, a copy of the target sequence is generated. The cycle is then repeated about
40 times.
Any PCR-based GMO detection strategy will thus depend on the selection of the
oligonucleotide primers and the detailed knowledge of the molecular structure and
transgenic DNA sequences used in the development of all GMOs. Genetic control
elements such as the cauliflower mosaic virus 35S promoter (P-35S) and the
Agrobacterium tumefaciens nos terminator (nos3’) are present in many GMOs currently
on the market. The first GMO screening method has been originally introduced by Swiss
and German scientists and is based on the detection of the P-35S and nos3’ genetic
elements. However, a few approved GMOs are not screenable/detectable with the P-35S
or the nos3’ primers and additional target sequences are needed to guarantee a complete
screening procedure.
a) Qualitative PCR
PCR exploits the specificity of DNA polymerase to allow the selective amplification of
specific DNA segments occurring at low frequency in a complex mixture of other DNA
sequences. Detection limits are in the range of 20 pg to 10 ng of target DNA and
0.0001% to 1% of the mass fraction of GMOs. In a standard PCR test, two pairs of
primers are used which are designed to hybridize on opposite strands of the sequence of
interest, and amplify the sequence between the primers millions of times through a series
of repetitive cycles. Amplified pieces can be subjected to agarose gel electrophoresis to
separate amplified products according to size, other separation methods, such as high
performance liquid chromatography (HPLC) and capillary electrophoresis (CE), have
also been used. Several food ingredients (e.g. wheat, cotton, soybean, canola, potatoes,
rice, maize and tomatoes) can be analyzed using PCR.
Most currently available GMOs contain any one of three genetic elements: the
cauliflower mosaic virus (CaMV) 35S promoter, the nopalin synthase (NOS) terminator,
and the kanamycin-resistance marker gene (nptII) and others. Product-specific PCR
methods that have been developed for a range of different GM foods can also be carried
out. These methods exploit a set of primer pairs that span the boundary of two adjacent
genetic elements (e.g. promoters, target genes and terminators), or that are specific for
detection of the altered target gene sequence. If PCR assay gives a positive result
verification of the identity of the amplicon is also performed.
Qualitative PCR and Confirmatory Assays
Confirmation is necessary to assure that the amplified DNA is really corresponding to the
chosen target sequence and is not a by-product of un-specific binding of the primers.
Different methods can be used to confirm the qualitative PCR results. If the PCR
products have the exact expected size revealed after gel electrophoresis, even then, there
is a risk that an artifact having the same size of the target sequence may have been
amplified. Therefore, the PCR products should additionally be verified for their
restriction endonuclease profile. Even more reliable is a Southern blot assay, whereby
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the amplicon is separated by gel electrophoresis, transferred onto a membrane and
hybridized to a specific DNA probe. Another possible control is to subject the PCR
product to a second round of PCR cycle in a technique that is called nested PCR. Here,
two different sets of primers, an outer and an inner are being used within the target region
in two consecutive rounds of PCR amplifications. This strategy reduces substantially the
problem of un-specific amplification, as the probability for the inner pair of primers of
finding complementary sequences within the nonspecific amplification products of the
outer pair is extremely low. Temperature and denaturing gradient gel electrophoresis
can also be performed for additional verification. The most reliable way to confirm the
authenticity of a PCR product is its sequencing.
b) Quantitative End-point PCR or QC-PCR
A crucial aspect of analysis of GMOs in food is quantitation, because maximum limits of
GMOs in foods are the basis for labeling in many countries, such as EU, Japan, Korea
and Taiwan. Therefore, quantitative PCR approaches have been developed. PCR is
quantitative if an internal DNA standard is co-amplified with target DNA. Quantitative
competitive (QC)-PCR involves the co-amplification of unknown amounts of a specific
gene target and of known amounts of an internal control template in the same reaction
tube by the identical primer pair. QC- PCR consists of four steps: (1) co-amplification of
standard- and target- DNA in the same reaction tube; (2) separation of the products by an
appropriate method, such as agarose gel electrophoresis and staining the gel by ethidium
bromide; (3) analysis of the gel densitometrically and (4) estimation of the relative
amounts of target and standard DNA by regression analysis. The control template is
typically a deletion product of the target sequence so that identical primers and reaction
conditions can be maintained to generate amplification products that should not differ.
Multiple PCR reactions are needed as each sample is amplified with increasing amounts
of competitor, while maintaining constant the sample volume/concentration. At the
equivalence point, the starting concentration of internal standard and target are equal.
Therefore, quantification is achieved by comparing the equivalence point at which the
amplicon from the competitor gives the same signal intensity of the target DNA on
stained agarose gels. The procedure assumes that the amplification reactions of the target
sequence and of the internal standard (i.e. the competitor) proceed with the same
efficiency in any phase of the reaction, including the plateau phase. It cannot be excluded
however that the efficiencies of the two reactions are instead different, because, for
instance, the competitor DNA is purified from plasmid DNA preparations that are of
higher quality than the plant-derived target DNA preparations. In systems such as the
QC-PCR method, the presence of PCR inhibitors will be noticed immediately because the
amplification of both internal standard and target DNA will be simultaneously affected.
In the QC-PCR, the competition between the amplification of internal standard DNA and
target DNA generally leads to loss of detection sensitivity.
c) Double QC-PCR
Double QC-PCR has two formats, the first one which was initially developed for
soybean, the concentration of soybean DNA in different samples is first normalized using
a QC PCR quantification of the soybean-specific lectin le1 gene. When the same samples
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are submitted to a second QC-PCR with a GM marker, relative quantification can be
established. However, since the generation of calibration curves is rather complex and the
accuracy depends on various factors, only one competitor concentration is being used,
equivalent to 1% GM soybean (Roundup Ready®). Therefore, the method could only
distinguish if a sample contained more or less GM material than the calibration
concentration of 1%. Within this determination, some degree of uncertainty could not be
avoided.
The second format of Double QC-PCR, method was initially developed for the
quantification of Bt maize. In this method multiple competitor concentrations are used
for the quantification of the amount of transgenic DNA, as well as for the quantification
of the total amount of amplifiable maize DNA. A good correlation can be obtained
between the actual and measured GMO concentrations. The use of Double QC-PCR
might reduce the inter- laboratory differences.
d) PCR-ELISA
PCR-ELISA uses the strategy like QC-PCR. The PCR will be quantitative also if PCR is
terminated before, a significant decrease in amplification efficiency occurs. PCR-ELISA
has been used to quantify relatively low amounts of PCR products. Despite the fact that
relative quantification using PCR-ELISA has been applied in different field tests and a
GMO detection kit using PCR- ELISA has been commercialised, this technique has not
been widely adopted for accurate GMO quantification purposes.
e) Multiplex PCR
With multiplex PCR-based methods several target DNA sequences can be screened for
and detected in a single reaction. Although in principle, standard PCR methods may be
combined in the same reaction, in practice this will often create an unacceptably high risk
of producing incorrect results from analyses of real samples. Firstly, each method may
require different reaction conditions, e.g. different temperature regimes or different
reagent concentrations. Secondly, the combination of primers from different methods
may increase the risk of amplifying DNA fragments other than the target fragments.
Thirdly, when more than one target fragment is being amplified in a PCR reaction, the
two fragments (amplicons) will compete for reagents etc. Normally, a fragment present
in a large number of starting copies will out compete another fragment that may be
present only in very few numbers. However, if two amplicons are amplified with
significantly different efficiency this may also have a severe impact on the final ratio of
the two amplicons, e.g. if the starting copy numbers were more or less the same for both.
Consequently, development of multiplex assays requires careful testing and validation.
After the PCR the resulting pool of amplified DNA fragments needs to be further
analysed to distinguish between the various amplicons. This may be done by the use of
specific hybridisation probes (possibly also during PCR in real-time assays), by gel
electrophoresis and comparison of fragment sizes or by the use of specifically labelled
primers. While several research groups are currently developing a number of multiplex
assays, hitherto only one paper has been published presenting a multiplex assay for
detection of five GM-maize.
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The advantage of multiplex methods is evidently that fewer reactions are needed to test a
sample for potential presence of GMO-derived DNA. In particular, if further quantitation
is needed, it may be of importance to know which GMO to quantify since a quantitative
PCR is relatively expensive. Identification of each specific GMO may also be helpful to
identify whether detected DNA is derived from approved or non-approved GMO.
f) Real-time PCR or Real-time Q-PCR
Another variation of quantitative PCR that improves accuracy, specificity and throughput
of quantitative PCR is “Real-time PCR”. To circumvent some of the problems of
conventional quantitative end-point PCR, a real-time Q-PCR was introduced. In theory,
production of PCR products should proceed exponentially. However, in practice it
reaches a plateau between 30 and 40 cycles because certain reaction components become
limited. In conventional PCR, products of the reaction are measured at a single point in
the reaction profile. Plotting the concentration of products present at this point as a
function of the initial amount of DNA present in each of those reactions shows that
proportionality between DNA concentration (dynamic range) and PCR products occurs
over a limited range of DNA concentrations, leading to loss of precision in quantitation.
However, it has been shown empirically that the concentration of DNA in real-time PCR
reaction is proportional to PCR cycle number during the exponential phase of PCR.
Therefore, if the number of cycles it takes for a sample to reach the same point in its
exponential growth curve is known, its precise initial DNA content can be determined.
Real-time PCR also allows for detection of low copy DNA number.
This technique was originally developed in 1992 and is rapidly gaining popularity due to
the introduction of several complete real-time PCR instruments and easy-to-use PCR
assays. A unique feature of this PCR technique is that the amplification of the target
DNA sequence can be followed during the whole reaction by indirect monitoring of the
product formation. Therefore, the conventional PCR reaction has to be adapted in order
to generate a constant measurable signal, whose intensity is directly related to the amount
of amplified product. Real time detection strategies rely on continuous measurements of
the increments in fluorescence generated during the PCR reaction. The number of PCR
cycles necessary to generate a signal that is significantly and statistically above noise
level is taken as a quantitative measure and is called cycle threshold (Ct). As long as the
Ct value is measured at the stage of the PCR where the efficiency is still constant, the Ct
value is inversely proportional to the log of the initial amount of target molecules.
Several commercially available real-time PCR thermal cyclers automate the analytical
procedure and allow cycle-by-cycle monitoring of reaction kinetics, permitting
calculation of the target sequence concentration. Several formats are used to estimate the
amount of PCR product: (1) the ds-DNA-binding dye SYBR Green I; (2) hydrolysis
probes (TaqMan® technology); (3) hybridization probes or fluorescence resonance
energy transfer (FRET) probes; (4) molecular beacons and (5) Scorpion™ probes. These
systems also permit differentiation between specific and nonspecific PCR products (such
as primer-dimer) by the probe hybridization or by melt curve analysis of PCR products,
because nonspecific products tend to melt at a much lower temperature than do the longer
specific products.
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The dsDNA dye SYBR® Green I stain (Molecular Probes, Eugene) has been employed
in place of Ethidium bromide as a double stranded DNA (dsDNA) dye in agarose gels to
reduce background and allow better real-time monitoring of product formation because
fluorescent signals of ds-DNA- binding dye, SYBR Green I increase when the PCR
products increase during PCR. This dye can successfully used for quantification of PCR
product, although it generates experimental artefacts due to the formation of undesirable
products such as primer dimers. However, such products can generally be recognized by
a melting curve analysis. On the amplification curve, plotting the increasing amount of
luminescent signals (Rn), the Rn part is selected where luminescent signals from both of
the standard solution for the standard curve and DNA sample solution amplify
exponentially, and a threshold line is drawn. The point, where the threshold line and
lumines-cent signal of the standard solution for standard curve cross, is used as the
threshold cycle (Ct). We can draw a standard curve and calculate the DNA concentration
of unknown sample by its Ct in real time PCR.
One of the most popular assays for real-time PCR is the Taqman® or 5’-exonuclease
assay, which employs a fluorogenic probe consisting of an oligonucleotide with both a
reporter and a quencher dye (FAM and TAMRA) attached. When the probe is intact the
reporter fluorescence is quenched by the proximity of the quencher dye. Due to its targetspecific sequence, the probe anneals specifically to the amplification product (target
DNA) between the forward and he reverse primers. If hybridisation has occurred, the 5’3’ exonuclease activity of the Taq polymerase cleaves the internal probe during the
extension step of amplification. The cleavage reduces the quenching effect and the
fluorescent signal of the reporter dye becomes a measure of the amount of amplification
product generated. Because the development of the fluorogenic reporter signal takes
place only if both the PCR primers and the TaqMan® probe anneal to the target DNA,
the specificity of real time PCR detection is considerably higher than that of conventional
PCR. The relative quantification of the target gene is made possible by preparing a
standard curve from known quantities of an additional endogenous gene and
extrapolation from the linear regression line.
The quantification of the GM marker and an endogenous reference gene could be
combined in a single tube by using a multiplex real time PCR approach. In this way, the
quantitative analysis of each sample is not affected by random differences in
experimental factors such as i.e. pipeting errors. In addition, it allows the utilisation of an
internal standard, which is a better control for false negative results. The use of multiplex
PCR for quantitative determination is made possible by the utilisation of different
reporter dyes, which can be detected separately in one reaction tube. Multiplex reactions
are economical and allow accurate relative quantification without previous estimation of
DNA quantity or copy numbers. With a multiplex reaction it can be established a direct
correlation between results of real-time PCR and % of GMO. This reduces the variation
and allows accurate data interpretation by simple statistical evaluation of the
quantification results. Due to the abovementioned advantages, multiplex real-time PCR is
increasingly applied in GMO detection. Multiplex real-time PCR assay has been
successfully applied to GM maize that employed zein as the endogenous reference gene
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and the P-35S promoter as a GMO marker for GM corn. A detection limit of 0.01 % GM
corn/non-GM corn was obtained.
In addition to the TaqMan® assay, various other techniques for the indirect monitoring of
the PCR products have been recently described. Other real-time PCR techniques that
make use of amplicon-specific probes are: fluorescence resonance energy transfer
(FRET) probes, molecular beacons and Scorpion™ probes. FRET probes give a high
level of specificity due to the use of two adjacent hybridization probes. Since FRET
probes are not cleaved during PCR cycling, melting curves can be applied as a
confirmatory test for the PCR. FRET probes are the technology of choice for sequencespecific quantification with the LightCycler™ (Roche Molecular Biosystems,
Indianapolis, IN). Since the introduction of the colour compensation feature in the
LightCycler™ software, FRET can be used in a multiplex PCR format for the
quantification of multiple targets in one reaction. A variant of the FRET approach has
been developed that uses a 3’-end primer labeled acceptor in combination with a 3’-end
donor labelled probe, which is designed to hybridise on the opposite strand of the labelled
primer. Since only one probe is used, this approach has a lower level of specificity.
Molecular beacons have been successfully employed in real-time PCR and for the
generation of MELTING CURVES, including the multiplex PCR format. Although
molecular beacons appear to be less popular than TaqMan® and FRET probes for
applications in quantitative PCR, they are widely used for discriminating single base pair
differences (SNP). Therefore they may be tailored for the detection and quantification of
new GM-crops that feature single nucleotide genetic modifications (e.g. produced by
techniques such as chimeraplasty).
Other advances in real-time PCR include self-probing amplicons that are generated
during the PCR reaction, with one of the PCR primers being replaced by a so-called
Scorpions™ primer. The unimolecular nature of the hybridisation event has its own
advantages over probe systems such as TaqMan®, FRET and molecular beacons,
therefore Scorpions™ can be used as an alternative technology in most of the real-time
assays.
With the increasing interest in real-time PCR, new fluorescent dyes, quenchers and
reporterquencher combinations are being investigated to explore the possibilities of
multiplying the number of targets, which can be measured within one reaction. The
growing number of commercially available real-time PCR thermocyclers is an indicator
of the success of this technology. Besides the possibility of accurate quantification, the
advantage of real-time PCR is the ability to increase the sample throughput in
comparison to other quantification techniques as post PCR analysis is reduced to data
treatment. Furthermore, with real-time PCR, the possibility of introducing variability and
false positives is reduced. Since both amplification and detection are combined in one
step carried out in a closed tube, the risk of cross contamination with PCR amplification
products is minimized. Presently, real-time PCR can be considered as the most powerful
tool for the detection and quantification of GMOs in a wide variety of agricultural and
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food products. While multiplex PCR formats with an endogenous reference gene will be
able to increase the accuracy, precision and throughput of this technique.
D) Other Methods For GMO Detection
I) Chromatography
Where the composition of GMO ingredients, e.g. fatty acids or triglycerides is altered,
conventional chemical methods based on chromatography can be applied for detection of
differences in the chemical profile. This has been demonstrated with oils deriving from
GM canola for which high performance liquid chromatography (HPLC) coupled with
atmospheric pressure chemical ionization mass spectrometry (APCI-MS) has been
applied to investigate the triglyceride patterns. The spectral identification was based on
the diacylglycerols fragments and on the protonated triglyceride molecular ions.
Quantification was performed with a flame ionization detector (FID). In comparing the
triglyceride patterns, it could be observed that the oils of the GM canola varieties had an
increased content of triacylglycerols, showing more oxidative stability for high stearic
acid canola oil as well for high lauric acid canola oil. This result is consistent with
previous oxidative stability studies on new varieties of soybean and high oleic acid
canola oils obtained by using HPLC-FID. In addition, the fatty acid compositions have
been measured by using gas chromatography (GC) coupled with FID to support the
HPLC results. However, it must be stressed that this methodology is only applicable
when significant changes occur in the composition of GM plants or derived products.
Moreover, it is a qualitative detection method rather than a quantitative method. Low
additions of, e.g., GM canola oil with an altered triglyceride composition to conventional
canola oil will most probably not be detected, also considering the natural variation of
ingredient patterns.
II) Near Infrared (NIR) Spectroscopy
Certain genetic modifications may alter the fiber structure in plants, whereas no
significant differences could be observed in the content of protein and oil (e.g. RR
soybeans). These could be detected by near infrared spectroscopy (NIR). Sample sets of
RR and non-RR soybeans have been used to develop discriminate analysis calibrations
for various models of near-infrared spectrometers. The results obtained by the three NIR
instruments vary slightly, but are promising in all cases. However, the capacity of NIR to
resolve small quantities of GMO varieties in non-GMO products is assumed to be low, as
is true for the chromatographic methods.
NIR transmittance spectroscopy has been used by grain handlers in elevators in most of
the world for nondestructive analysis of whole grains for the prediction of moisture,
protein, oil, fiber and starch. Recently, the technique has been used in attempts to
distinguish RRS from conventional soybean. In this study, spectral scans were taken from
three Infratec 1220 spectrometers where whole-grain samples flew through a fixed path
length. Locally Weighed Regression using a database of about 8000 samples was 93%
accurate for distinguishing RRS from unmodified soy. The advantages of this technique
are: (1) it is fast (less than 1 min), (2) sample preparation is not necessary because it uses
whole kernels (about 300 g), which are dropped into measurement cells or flow through
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the system, and (3) it is therefore cheap. The major disadvantage is that it does not
identify compounds, thereby necessitating a large set of samples to generate spectra. This
calibration dataset is then used to predict the GMO event. Thus, this method cannot be
more accurate than the reference method used to build the model. Moreover, a calibration
needs to be developed for each GMO to be predicted. Furthermore, although NIR is
sensitive to major organic compounds (e.g. vibration overtones of C–H, O–H and N–H),
its accuracy is limited. For example, with respect to GMOs, it does not detect a change in
DNA or a single protein, but much larger unknown structural changes, such as those
linked to the parietal portion of the seed (e.g. lignin or cellulose) that are introduced by
the presence of the new DNA.
III) Microfabricated Devices And Microchips For GMO Detection
One of the challenges that the GMO analyst will face in the near future is the rapid pace
of development of GM plants with new and multiple genes/genetic control elements. For
example, some approved GM crops would contain neither the 35S promoter nor the nos
terminator. The establishment of “gene registers” and the use of advanced bioinformatic
systems can help in obtaining prior knowledge of the possible types of genetic
modifications. Moreover, new technologies and instruments will be needed for the high
throughput and low cost detection of an increasing variety of genes.
New technologies resulting from the merger of chip-based Microsystems such as
microarrays and microfluidic systems appear to be a promising area for GMO analysis
applications. Microarrays have been successfully used for expression analysis,
polymorphism detection, DNA sequencing and genotyping. Microfluidic systems have
applications ranging from reactions to separations and analysis and may finally lead to
the development of micro Total Analysis Systems (mTAS) that perform the complete
analysis including sampling and sample pre-treatment. The microchip technology aims at
automating the complex work procedures of an analytical laboratory by transfering them
onto a small piece of glass or plastic, the so-called chip, as in a microarray-based system,
microscopic arrays of DNA molecules are immobilized upon a solid support. Based on
the principle of DNA hybridisation followed by monitoring, generally with fluorescence
measurements, simultaneous analysis of several thousand nucleic acids within the very
small area of a chip is possible. Therefore, the microarray system saves time and costs
while maintaining high precision and reproducibility.
While, in a microfluidic system it is possible to simulate pumps, valves, reaction tubes
and even analytical instruments by a clever transportation of liquids through miniature
channels (5-20 mm) arranged on a single chip. Among the advantages are increased
performance (e.g. more rapid cooling and heating times, more rapid diffusion across the
channels, improved speed of separation), valveless transport (electro-osmotic or
electrochemically driven flow), reduced consumption of reagents, portability, the
possibility of parallelisation of procedures and high sample throughput. Although several
authors have reported on PCR microsystems of different complexity, only few examples
of microchip applications to GMO analysis have been described so far. However, this
new technology may offer new perspectives in the field of GMO detection.
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The microfluidics technology is capable of accepting large volume biological fluid
samples, automatically extracting, purifying, and concentrating DNA or RNA, mixing the
target DNA/RNA with the appropriate amplification/detection reagents, and presenting
the final mixture to the amplification process. Besides applications in rapid pathogen
detection in foods or for point-of-care human diagnostics, the use of microPCR portable
systems can be envisioned in field detection of crop disease and point of use GMO
screening, e.g. in the early steps of the supply chain, without the need for an expensive
laboratory setting. It can also be envisioned that the integration of advanced diagnostic
micro instrumentation and Internet technology may be employed in the future to develop
wireless and mobile GMO detection instruments that can readily identify plant genetic
modifications on site.
Most of the reviewed microfabricated devices take advantage of the capability of PCR to
amplify low copy number targets from a background of non-specific nucleic acids.
However, the development of PCR protocols for GMO analysis is often affected by
misspriming due to polymerase errors that results in the extension of non-specifically
annealed sequences and the production of undesirable PCR artefacts. Moreover, in GMO
analysis of complex food matrices, inhibitors of Taq polymerase often co-purify with the
DNA template and lead to false negatives. Although PCR and its many variations and
formats still remain the most sensitive and widely used DNA diagnostic technology,
however, non-PCR detection methods and microfabricated devices have recently been
described which allow genetic analysis in conditions of low abundance of target
fragments.
One microtechnology that can be applied for both DNA and protein analysis is surface
plasmon resonance (SPR). In particular, SPR has emerged as a well-suited method to
study the real-time kinetics of biomolecular interactions between macromolecules in a
label-free fashion, e.g. antigen–antibody interactions, protein–DNA interactions and
receptor–ligand interactions. SPR stems from one of the basic principles of optics, the
one of total internal reflectance, and occurs when a thin conducting film is placed at the
interface between materials with differing refractive indices. If a ligand can be
conjugated to the surface of the biosensor chip, then the attachment of a free target
analyte present in solution can be measured as a function of mass increase. The change in
angle of reflected light (proportional to the difference in mass on the chip surface) before
and after incubation is recorded in a "sensorgram" and measured in relative units (R.U.).
Accordingly, changes in response levels in spiked samples can be correlated with known
analyte concentrations. Biosensor technologies including SPR can be used for screening
purposes in GMO analysis, because these have several advantages like fast time
responses, ease of use and low costs.
Another non-PCR detection micro technology called “scanometric DNA array
detection” has been developed. This technique uses DNA arrays and a signal
amplification method based on gold nanoparticle probes covered with 200
oligonucleotide strands that are complementary to a target DNA sequence immobilized
on a chip. When the nanoparticle probes bind to target DNA they polymerise and form
structures containing thousands of particles. The signal is amplified using a modified
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photographic developing solution that covers each gold nanoparticle with silver while the
network of particles grows in size, thus increasing the signal by a factor of 100,000. The
imaging of the detection reaction is performed with a simple flatbed scanner. With this
technology higher and sharper DNA temperature melting profiles can be obtained than
those found in conventional detection methods based on fluorescence technology, with
the ability of detecting as few as 60 DNA molecules without the need for a PCR
amplification step.
IV) Nanoscale GMO Detection
The enforcement of GMO regulations, including the recently introduced labelling and
threshold provisions, require the accurate determination of very low amounts of
transgenic DNA in a sample. Single cells isolated by flow cytometry or
micromanipulation can provide sufficient amounts of DNA to allow PCR-based
detection, although amplification of single-copy genes may be particularly challenging.
Single cell PCR enables to study the variations in gene expression between individual
cells in a population, in gene expression between different cell types at different
developmental stages, or can be used for diagnostics purposes including GMO detection.
Similarly single molecule detection (SMD) techniques like laser-induced fluorescence
spectroscopy (either on surfaces or on freely moving molecules in liquids) and magnetic
resonance, for monitoring the chemical and physical properties of biochemical reactions
of individual molecules in real- time, represent the most advanced frontier in analytical
technologies. A promising future perspective for SMD appears to be the detection and
direct quantification of trace amounts of DNA by counting single molecules in submicrolitre volumes of liquid, possibly leading to the development of “nanoscale GMO
detection” techniques.
Future Prospects and Challenges
In future, internationally recognized accreditation for GMO testing will be an important
tool to consider. At the moment, it is strongly recommended to choose laboratories that
perform well in proficiency programs and participate in check sample programs.
Different detection methods can be useful at different stages of the food/feed production
chain, provided that they are performed proficiently and appropriately, and are validated
according to commonly agreed validation procedures. International standardization and
validation of GMO analysis methods by harmonized and accepted protocols are still in
early phases. Standardization committees have been formed by the ISO, CENComité
Européen de Normalisation (CEN), and the Codex Alimentarius Commission which have
established preliminary guidelines for sampling strategies and GMO detection methods.
The objective of validation of an analytical PCR method is to demonstrate that the
successive procedures of sample extraction, preparation and analysis will yield accurate,
precise and reproducible results for a given analyte in a specified matrix. The process of
validation allows the independent use of methods and results, which are comparable
among each other.
If the GMO level is above the established threshold, the product is labeled, but if below
the threshold, the product needs not be labeled. Japan has set 5% GMO level while EU
regulations allow for the inadvertent co-mingling of GMOs to the extent of 1 % at the
NIBGE-FAO Workshop on GMO Detection
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single ingredient level. If a food product contains "gene stacked" GMO, then
quantification may become confusing. If for example, 0.75 % of the maize is from a
GMO derived from a cross between two different maize transformants, quantification
will report 1.5 % GMO maize. The current European regulations do not specify how this
type of case shall be handled. The high sensitivity and specificity of Q-PCR methods and
their flexibility with different food matrices make them suitable for detecting GMOs at
low thresholds in various foods. The greatest uncertainty of using DNA-based assays, as
for protein-based methods, is that not all products derived from GM foods (e.g. refined
oil) contain enough DNA. In addition, heating and other processes associated with food
production
can
degrade
DNA.
Keeping up with the growing complexity of the transgenic crops, GMO detection is a
long-term future challenge. Majority of the approved GM crop varieties can be detected
using a common 35S/NOS PCR-based detection method, but new transgenic crops are
likely to have different promoters and terminators, even with native promotor and
terminator, no selectable marker or reporter gene, or the selectable markers would be
eliminated by recombination, moreover, transgene homologous to the native gene using
antisense/sense suppression technology which will complicate GMO detection
approaches. As a result, new technologies need to be developed to cope with the
progressive complexity, providing accurate GMO testing results at a reasonable cost and
in a reasonable amount of time.
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Ahmed, F. E. 2004. Testing of genetically modified organisms in foods. The Haworth
Press, Inc. USA.
Anklam, E., F. Gadani, P. Heinze, H. Pijnenburg, G. V. D. Eede. 2002. Analytical
methods for detection and determination of genetically modified organisms in
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Anonymous 1998. Council regulation (EC) No. 1139/98, concerning the compulsory
indication of the labelling of certain foodstuffs produced from genetically modified
organisms. Official J. Eur. Communities: Legislation 159: 4–7.
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Anonymous. 2000. Foodstuffs–Detection and quantification of genetically modified
vegetal organisms and derived products–Part 1–Guidelines and requirements. Association
Française de Normalisation (AFNOR), Paris, France.
Brett, G. M. 1999. Design and development of immunoassays for detection of proteins.
Food Control 10: 401–406.
Byrdwell, W. C., Neff, W. E. 1996. Analysis of genetically modified canola varieties by
atmospheric pressure chemical ionization mass spectrometric and flame ionization
detection. J Liq Chromatogr Relat Technol 19: 2203–2225.
Chen, L. H. 2002. GMO regulation in Taiwan. Proceedings International Workshop on
Impacts and Biosafety of Genetically Modified Agricultural Products. (Pan, T. M. ed) pp.
61-68. FFTC/ASPAC.
Palma, A. 2001. Capillary electrophoresis. Genet. Eng. News 21: 21– 22.
Fagan, J. 2001. Performance assessment under field conditions of a rapid immunological
test for transgenic soybeans. Int. J. Food Sci. Technol. 36: 1– 11.
Feriotto, G., Gardenghi, S., Bianchi, N. and Gambari, R. 2003. Quantitation of Bt-176
Maize Genomic Sequences by Surface Plasmon Resonance-Based Biospecific Interaction
Analysis of Multiplex Polymearse Chain Reaction (PCR). J. Agric. Food Chem. 51:
4640-4646.
Gasch, A. 1997. Detection of genetically modified organisms with the polymerase chain
reaction: potential problems with food matrices. In Foods Produced by Modern Genetic
Engieering (Schreiber, F.A. and Bögl, K.W., eds) 2nd Status Report, pp. 90–79, BgVVhefte.
GeneScan Europe 2001. GMO Chip: Test Kit for the Detection of GMOs in Food
Products, Cat. No. 5321300105, Bremen, Germany.
Giles, J. 2004. Transgenic planting approved despite scepticism of UK public. Nature.
428:107.
González, I., García, T., Fernández, A., Sanz, B., Hernández, P. E., Marín, R. 1999.
Rapid enumeration of Escherichia coli in oysters by a quantitative PCRELISA. J Appl
Microbiol 66: 231–236.
Hagen, M. and Beneke, B. 2000. Detection of genetically modified soy (Roundup-Ready)
in processed food products. Berl. Munch. Tierarztl. Wochenschr. 113: 454–458.
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Hellebrand, M., Nagy, M., Mörsel, J. T. 1998. Determination of DNA traces in rapeseed
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Official Collection of Test Methods. 1999. Detection of a genetic modification of
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Stave, J. W. 1999. Detection of new or modified proteins in novel foods derived from
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Polymerase Chain Reaction
Shahid Mansoor, PhD
Principal Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Polymerase chain reaction (PCR) is a technique used to make numerous copies of a
specific segment of DNA quickly and accurately. The polymerase chain reaction enables
investigators to obtain the large quantities of DNA that are required for various
experiments and procedures in molecular biology, forensic analysis, evolutionary
biology, and medical diagnostics.
PCR was developed in 1983 by Karry B Mullis, an American biochemist who won the
Nobel Prize for Chemistry in 1993 for his invention. Before the development of PCR, the
methods used to amplify, or generate copies of, recombinant DNA fragments were timeconsuming and labour-intensive. In contrast, a machine designed to carry out PCR
reactions can complete many rounds of replication, producing billions of copies of a
DNA fragment, in only a few hours.
Mechanism of PCR
The polymerase chain reaction is a test tube system for DNA replication that allows a
"target" DNA sequence to be selectively amplified, or enriched, several million-fold in
just a few hours. Within a dividing cell, DNA replication involves a series of enzymemediated reactions, whose end result is a faithful copy of the entire genome. Within a test
tube, PCR uses just one indispensable enzyme - DNA polymerase - to amplify a specific
fraction of the genome.
During cellular DNA replication, enzymes first unwind and denature the DNA double
helix into single strands. Then, RNA polymerase synthesizes a short stretch of RNA
complementary to one of the DNA strands at the start site of replication. This DNA/RNA
heteroduplex acts as a "priming site" for the attachment of the DNA polymerase, which
then produces the complementary DNA strand. During PCR, high temperature is used to
separate the DNA molecules into single strands, and synthetic sequences of singlestranded DNA (20-30 nucleotides) serve as primers. Two different primer sequences are
used to bracket the target region to be amplified. One primer is complementary to one
DNA strand at the beginning of the target region; a second primer is complementary to a
sequence on the opposite DNA strand at the end of the target region.
PCR is a three-step process that is carried out in repeated cycles. The initial step is the
denaturation, or separation, of the two strands of the DNA molecule. This is
accomplished by heating the starting material to temperatures of about 95 C. Each strand
is a template on which a new strand is built. In the second step the temperature is reduced
to about 55 C so that the primers can anneal to the template. In the third step the
temperature is raised to about 72 C, and the DNA polymerase begins adding nucleotides
onto the ends of the annealed primers. At the end of the cycle, which lasts about five
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minutes, the temperature is raised and the process begins again. The number of copies
doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA.
In the original PCR procedure, one problem was that the DNA polymerase had to be
replenished after every cycle because it is not stable at the high temperatures needed for
denaturation. This problem was solved in 1987 with the discovery of a heat-stable DNA
polymerase called Taq, an enzyme isolated from the thermophilic bacterium Thermus
aquaticus, which inhabits hot springs. Taq polymerase also led to the invention of the
PCR machine.
To perform a PCR reaction, a small quantity of the target DNA is added to a test tube
with a buffered solution containing DNA polymerase, oligonucleotide primers, the four
deoxynucleotide building blocks of DNA, and the cofactor MgCl2. The PCR mixture is
taken through replication cycles consisting of:
1. one to several minutes at 94-96 C, during which the DNA is denatured into single
strands;
2. one to several minutes at 50-65 C, during which the primers hybridize or "anneal"
(by way of hydrogen bonds) to their complementary sequences on either side of
the target sequence; and
3. one to several minutes at 72 C, during which the polymerase binds and extends a
complementary DNA strand from each primer.
As amplification proceeds, the DNA sequence between the primers doubles after each
cycle. Following thirty such cycles, a theoretical amplification factor of one billion is
attained.
Two important innovations were responsible for automating PCR. First, a heat-stable
DNA polymerase was isolated from the bacterium Thermus aquaticus, which inhabits hot
springs. This enzyme, called the Taq polymerase, remains active despite repeated heating
during many cycles of amplification. Second, DNA thermal cyclers were invented that
use a computer to control the repetitive temperature changes required for PCR.
Following amplification, the PCR products are usually loaded into wells of an agarose gel
and electrophoresed. Since PCR amplifications can generate microgram quantities of
product, amplified fragments can be visualized easily following staining with a chemical
stain such as ethidium bromide. While such amplifications are impressive, the important
point to remember is that the amplification is selective - only the DNA sequence located
between the primers is amplified exponentially. The rest of the DNA in the genome is not
amplified and remains invisible in the gel.
Applications of PCR
Following the introduction of PCR the technique spread through the community of
molecular biologists like - well, a chain reaction. As more scientists became familiar with
PCR, they introduced modifications of their own and put the technique to new uses.
Almost overnight, PCR became a standard research technique and the practical
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applications soon followed. Not surprisingly, the first applications to leave the laboratory
dealt with detection of genetic mutations.
PCR can also be used to detect the presence of unwanted genetic material, as in the case
of a bacterial or viral infection. Conventional tests that involve the culture of
microorganisms or use of antibodies can take weeks to complete or be tedious to perform.
PCR offers a fast and simple alternative. For example, in the diagnosis of AIDS, PCR can
be used to detect the small percentage of cells infected by the human immunodeficiency
virus (HIV). DNA isolated from peripheral blood cells is added to a PCR reaction
containing primers complementary to DNA sequences specific to HIV. Following
amplification and gel electrophoresis, the presence of an appropriate-sized PCR product
indicates the presence of HIV sequence and therefore, HIV infection.
The sensitivity of PCR is so great that signals may be obtained from degraded DNA
samples and sometimes from individual cells. This ability and the inherent stability of
DNA have combined to permit DNA to be amplified from some unusual sources, such as
an extinct mammal called the quaga, an Egyptian mummy, and a three-million-year-old
termite trapped in amber. This situation has, almost overnight, transformed ignored
museum collections of biological specimens into treasure troves of genetic information.
Evolutionary biologists are using these specimens and PCR to explore the genetic
relatedness of organisms across species boundaries and now even across time.
Limitations of PCR
One of the major limitations of PCR is that the sequence of the nucleotides and the ends
of the target sequence must be known so that primers may be designed. However, the
target sequence may not be known when a novel gene has just been identified. One of the
potential problems of PCR is contamination. Because only a single molecule of target
DNA is required for amplification by PCR, it is possible to make multiple copies of
pieces of DNA that are not identical to the target DNA but that have sequences similar to
the primer binding sites (McMurry, 2000).
Further Reading
Mullis, K.B. 1990. The unusual origin of the polymerase chain reaction. Scientific
American 262:56-65.
Paabo, S., R.G. Higuchi, and A.C. Wilson. 1989. Ancient DNA and the polymerase chain
reaction. Journal of Biological Chemistry 264:9709-9712.
Saiki, R.K., et al. 1985. Enzymatic amplification of beta-globin sequences and restriction
site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.
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Real Time PCR for GMO Detection
Aftab Bashir, PhD
Principal Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
A genetically modified organism (GMO) is a living organism that has been altered by
means of gene technology. The genetic modification usually involves insertion of a piece
of DNA (the insert) or a synthetic combination of several smaller pieces of DNA into the
genome of the organism to be modified. This process is called transformation. These
smaller pieces of DNA are usually taken from other naturally occurring organisms.
A typical insert (gene construct) in a GMO is composed of three elements: 1) The
promoter element functions as an on/off switch for reading of the inserted/altered gene;
2) The gene that has been inserted/altered is coding for a specific selected feature; 3) The
terminator element functions as a stop signal for reading of the inserted/altered gene. In
addition several other elements can be present in a gene construct, and their function is
usually to control and stabilize the function of the gene, demonstrate the presence of the
construct in the GMO, or facilitate combination of the various elements of the
construct. A gene construct must be integrated in the genome of the organism to become
stably inherited. Therefore, the organisms own genome is also an important element.
Methods for GMO detection
Detection of a GMO or a derivative of a GMO can be done by detecting a molecule
(DNA, RNA or protein) that is specifically associated with or derived from the genetic
modification of interest. The majority of the methods that have been developed for
detection of GMO and GMO-derivatives focus on detecting DNA, while only a few
methods have been developed for detecting proteins or RNA for the following reasons.
1) DNA can be purified and multiplied in billions of copies in just a few hours with a
technique called PCR (polymerase chain reaction).
2) Multiplication of RNA and proteins is a more complicated and slow process.
3) DNA is a very stable molecule, while RNA is unstable.
4) The stability of a protein varies and depends on the type of protein.
There is normally a linear correlation between the quantity of GMO and DNA, if the
genetically modified DNA is nuclear, but not if it is extranuclear. However, there is
usually no such correlation between the quantity of GMO and protein/RNA. Finally, the
genetic modification itself is done at the DNA level. At present, the genetically modified
DNA is nuclear in all commercialized GMO.
DNA based methods are primarily based on multiplying a specific DNA with the PCR
technique. Two short pieces of synthetic DNA (primers) are needed, each complementary
to one end of the DNA to be multiplied. The first primer matches the start and the coding
strand of the DNA to be multiplied, while the second primer matches the end and the
non-coding (complementary) strand of the DNA to be multiplied. In a PCR the first step
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in a cycle involves separation of the two strands of the original DNA molecule. The
second step involves binding of the two primers to their complementary strands,
respectively. The third step involves making two perfect copies of the original double
stranded DNA molecule by adding the right nucleotides to the end of each primer, using
the complementary strands as templates. Once the cycle is completed, it can be repeated,
and for each cycle the number of copies is doubled, resulting in an exponential
amplification. After 20 cycles the copy number is approximately 1 million times higher
than at the beginning of the first cycle. However, after a number of cycles the amount of
amplification product will begin to inhibit further amplification, as will the reduction in
available nucleotides and primers for incorporation into new amplification products. This
effect is often referred to as the plateau effect.
One of the most commonly applied techniques for demonstration of the presence of a
DNA or RNA is gel electrophoresis, a technique that allows the amount and size of the
DNA/RNA to be estimated. This may eventually be coupled to digestion of the DNA
with restriction enzymes that e.g. are known to cut a PCR fragment into segments of
specified sizes. A more sophisticated technique involves determination of melting point
profiles, by means of e.g. SYBR Green 1, a dye that when intercalating double stranded
DNA emits fluorescent light. When the temperature is increased, the DNA strands begin
to separate. This leads to a corresponding reduction in fluorescence that can be measured
directly e.g. on a real-time thermal cycler (PCR machine). The melting point is more
characteristic of a specific DNA sequence than its size, but complete sequencing
(determination of the order of nucleotides) of the DNA/RNA allows for more specific
determination of the origin of the molecule. A fourth alternative is to use short synthetic
molecules (similar to primers but called probes) and allow these to bind (hybridize) to the
DNA/RNA. If appropriately designed, a probe is able to discriminate between the correct
molecule (sequence) and almost any other DNA/RNA molecule. Labeling of molecules
with fluorescence, radioactivity, antibodies or dyes facilitate detection of the present
molecules. For GMO analyses, gel electrophoresis and hybridization techniques are
currently the most commonly applied techniques.
PCR-based GMO analyses usually include testing for presence of DNA from the
particular species of interest, e.g. soybean DNA. Sometimes (in the absence of
amplifiable DNA from the particular plant species) GMO analyses also include testing
for presence of amplifiable (multicopy) DNA from plants or Eukaryotes, e.g. chloroplast
DNA or nuclear ribosomal small subunit genes (18S like). The promoter and terminator
elements used to transform most of the currently approved genetically modified plants are
the Cauliflower Mosaic Virus promoter (P-35S) and the Agrobacterium tumefaciens
nopaline synthase terminator (T-Nos). Although, other promoters and terminators have
also been used, almost all GM plants contain at least one copy of the P-35S, T-35S and/or
the T-Nos as a part of the gene construct integrated in its genome. Consequently,
methods detecting one of these elements are popular for screening purposes. One
problem with these methods is that the elements they detect are from naturally occurring
virus and bacteria which are often present in fresh vegetables or the environment in
which they are grown. Such elements therefore pose a significant risk of yielding false
positive results.
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The various genes inserted in a GMO may characterize a group of GMO, although they
may not identify the GMO. Detection of the synthetic specific gene coding for the
Bacillus thuringiensis endotoxin CryIA(b) demonstrates the presence of a genetically
modified maize, but the gene has been used in more than one GMO. Consequently,
methods like those that can detect the CryIA(b) gene can tell us more than the other
screening methods, but will not be suitable for identification of the specific GMO.
The synthetic CryIA(b) gene has been integrated with different specific regulatory
elements (promoters and terminators) in the various GMO containing the gene.
Currently, it is therefore possible to identify the GMO with PCR based detection methods
targeting the junctions where the gene and regulatory elements are fused. In principle,
PCR based quantification can be performed either after completion of the PCR (end-point
analysis), or during the PCR (real-time analysis). End-point analyses are usually based on
comparison of the final amount of amplified DNA of two DNA targets, the one to be
quantified and a competitor (an artificially constructed DNA that is added in a small and
known quantity prior to the PCR amplification and that is co-amplified with the target
that is to be quantified). This is called competitive quantitative PCR and it requires that
the two DNA targets are amplified with equal efficiency since the final amount of
product is not linearly correlated with the starting amount.
A dilution series of the DNA to be analyzed is prepared, and a constant amount of the
competitor is added. After completion of the PCR the resulting amplification products
are visualized through gel electrophoresis and when both DNA targets yield the same
amount of product it is assumed that the starting amount was also the same. By setting
up two competitive PCRs, one for the GMO (e.g. RoundupReady soybean) and one for
the species of interest (e.g. soybean), and including competitors in both, the quantity of
GMO relative to the species can be estimated by extrapolation from the degree of dilution
and concentration of the competitors. Such assays are referred to as quantification by
double competitive PCR.
In real-time analyses the amount of product synthesized during PCR is estimated directly
by measurement of fluorescence in the PCR reaction. Several types of hybridization
probes are available that will emit fluorescent light corresponding to the amount of
synthesized DNA. However, the amount of synthesized product can also be estimated
with fluorescent dyes, e.g. SYBR Green 1 that intercalates double-stranded DNA. With
the latter, it is not possible to distinguish between the specific product and non-specific
products, and consequently the use of specific hybridization probes is normally
preferred. As with double competitive PCR, the quantitative estimate is based on
extrapolation by comparison of the GMO sequence relative to the reference of interest.
The idea is that with the use of fluorescence it becomes possible to measure exactly the
number cycles that are needed to produce a certain amount of PCR product. This amount
corresponds to the amount producing a fluorescence signal clearly distinguishable from
the background signal and measured well before the plateau effect becomes a problem.
The number is called the Ct-value. Then by comparison of Ct-values for the GMO target
sequence, e.g. Roundup Ready soybean 3' integration junction, and the reference gene,
e.g. soybean lectin, it becomes possible to estimate the ratio of the GMO target sequence
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to the reference sequence in terms of difference in number of cycles needed to produce
the same quantity of product. Since one cycle corresponds to a doubling of the amount of
product, a simple formula can be presented to estimate the ratio in percent. While realtime PCR requires more sophisticated and expensive equipment than competitive PCR, it
is faster and more specific. Although, what is said above may give the impression that
quantification can be done directly from comparison of Ct-values, most laboratories now
adjust their estimates by comparison to a standard curve, i.e. a series of measurements
from a DNA series with known GMO content.
This lecture focuses on the real time quantitative PCR. The technology will be discussed
with reference to the detection of GMO’s. Although, it’s the most recent and effective
technology but its quite sophisticated and expensive. Scientists at NIBGE are well versed
to the knowledge and use of this technology. It is anticipated that we will acquire a real
time PCR machine very soon and establish the system not only in GMO detection but
also in health sciences and the study of comparative gene expression in developing cotton
fiber.
References:
Hardegger, M., P. Brodmann & A. Herrmann (1999). Quantitative detection of the 35S
promoter and the NOS terminator using quantitative competitive PCR. Eur. Food Res.
Technol. 209: 83-87.
Matsuoka, T., H. Kuribara, H. Akiyama, H. Miura, Y. Goda, Y. Kusakabe, K. Isshiki, M.
Toyoda & A. Hino (2001). A multiplex PCR method of detecting recombinant DNAs
from five lines of genetically modified maize. J. Food Hyg. Soc. Japan 42: 24-32.
Marta Hernández, Adolfo Rio, Teresa Esteve, Salomé Prat, Maria Pla (2001). A
rapeseed-specific gene, Acetyl-CoA carboxylase, can be used as a reference for
qualitative and real-time quantitative PCR detection of transgenes from mixed food
samples Journal of Agricultural and Food Chemistry 49(8): 3622-3627.
Isabel Taverniers, Pieter Windels, Erik Van Bockstaele, Marc De Loose (2001).
Use of cloned DNA fragments for event-specific quantification of genetically modified
organisms in pure and mixed food products European Food Research and Technology,
213: 417-424.
Catherine F. Terry, Neil Harris (2001). Event-specific detection of Roundup Ready Soya
using two different real time PCR detection chemistries European Food Research and
Technology, 213: 425-431.
Pieter Windels, Isabel Taverniers, Ann Depicker, Erik Van Bockstaele, Marc De Loose
(2001). Characterisation of the Roundup Ready soybean insert European Food Research
and Technology 213(2): 107-112
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Development of Biosafe Transgenic Plants
Muhammad Sarwar Khan, PhD
Senior Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant
genetics and continue to defy the mainstream approach to biotechnology, leading the
field in a biosafe direction. Recent success in engineering the chloroplast genome for
resistance to herbicides, insects, disease, drought, and for production of
biopharmaceuticals and industrial enzymes has opened the door to a new era in
biotechnology. This lecture summarizes the state of the art in chloroplast genetic
engineering and describes how chloroplast genetic engineering contributes to the
successful modification of plant genome to develop biosafe transgenic plants.
The plastid genome of higher plants is an attractive target for crop engineering because
proteins in chloroplasts may accumulate to high levels, multiple genes may be expressed
as polycistronic units and lack of pollen transmission in most crops results in transgene
containment (Daniell et al., 1998; Daniell and Khan, 2003). Plastid transformation is
accomplished through a multistep process (Figure 1), in which the transformation vectors
contain a selectable marker gene that may encode resistance to spectinomycin,
streptomycin, or kanamycin, and passenger gene(s) flanked by homologous plastid
targeting sequences (Figure 1, Khan and Maliga, 1999). These sequences are introduced
into plastids by biolistic DNA delivery (Svab and Maliga, 1993; Daniell et al., 2002) or
polyethylene glycol treatment (Golds et al., 1993; Koop et al., 1996; O’Neill et al.,
1993). The drugs used in the selection inhibit chlorophyll accumulation and shoot
formation on plant regeneration media, and resistance to these drugs is conferred by the
expression of chimeric aadA5 and neo (kan; Carrer et al., 1993) genes in plastids. Thus,
the transplastomic lines are identified by the ability to form green shoots on bleached
wild-type leaf sections (Daniell et al., 2002; Daniell and Khan, 2003).
Obtaining a genetically stable transplastomic line involves cultivation of the cells on a
selective medium, during which the cells divide at least 16–17 times (Moll et al., 1990).
During this time wild-type and transformed plastids and plastid genome copies gradually
sort out, yielding chimeric plants consisting of sectors of wild-type and transgenic cells1.
In the chimeric tissue, antibiotic resistance conferred by aadA or neo is not cell
autonomous; transplastomic and wild-type sectors are both green because of phenotypic
masking by the transgenic tissue. This chimerism necessitates a second cycle of plant
regeneration on selective medium. In the absence of a visual marker, this is an inefficient
process involving antibiotic selection and identification of transplastomic plants by PCR
or Southern probing. To facilitate this process, we describe here a method for obtaining
homoplastomic clones by visually identifying transformed sectors (Khan and Maliga,
1999). The method relies on using the Aequorea victoria green fluorescent protein (GFP)
as a visual marker, allowing direct imaging of the fluorescent gene product in living cells
without the need for prolonged and lethal histochemical staining procedures (Khan,
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2001). Its chromophore forms autocatalytically in the presence of oxygen and fluoresces
green when absorbing blue or ultraviolet (UV) light (Prasher, 1995).
Figure 1: Chloroplast transformation strategy. Left panel. Selection and regeneration of
drug-resistant plants. Right panel. Homologous recombination process for selectable
marker gene integration into the plastid genome using homologous sequences.
Selectable Marker and reporter Genes to transform plants
Several drugs like hygromycin, spectinomycin, streptomycin, kanamycin and
phosphinothricin (PPT) encoded by hph, aadA, nptII and bar genes (Chowdhury and
Vasil, 1992; Seki et al., 1995; Staub and Maliga, 1994), respectively, have been used to
select cells or plants carrying these genes during genetic engineering. During normal
selection procedure, marker gene recipient cells go through phases of embryogenesis and
organogenesis before regenerating to green shoots. During the time of embryogenesis and
organogenesis, wild-type and transformed cells gradually sort out. Such sorting out
mechanism is well defined for chloroplasts (Khan and Maliga, 1999) where extended
period of sorting yields chimeric plants consisting of sectors with wild-type and
transformed cells. In the chimeric tissue, antibiotic resistance conferred by marker gene
(s) is not cell autonomous: transgenic and wild type sectors are both green due to
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phenotypic masking by the transgenic tissue. Such chimarism necessitates a second cycle
of plant regeneration on a selective medium.
In addition to selectable marker genes, vital reporter genes undoubtedly contribute to the
development of transformation technology by serving as tools for visual monitoring of
transgene expression in transformed cells and tissues. A number of genes for example,
uidA, cat, nptII, ocs and luc have been used to study gene expression, in plants as well as
animals, as reporters (Gallo-Meagher and Irvine, 1993; 1996; Seki et al., 1995; Staub and
Maliga, 1994). However histochemical detection of GUS in plants needs prolonged
incubation because, chlorophyll bleaching is required to make GUS staining more effective
using either ethanol or chloral hydrate. Furthermore, chemicals and physical procedures
used in the staining disrupt cell ultrastructure (Baulcombe et al., 1995).
The use of a non-toxic marker to identify transgenic cells after transformation is an
effective procedure for discerning transformed cells and removing untransformed
cells/tissues. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, has
recently been used as a reporter gene in plants (Baulcombe et al., 1995; Chiu, et al.,
1996; Haseloffet al., 1997, Khan, 2000). The gfp provides an easily scored cellautonomous genetic marker in plants and has major uses in monitoring gene expression,
protein localization and screening of transformation events at high resolution. The green
fluorescent protein has successfully been expressed in E. coli and plants (Khan and
Maliga, 1999; Hibbred et al., 1998; Siderov et al., 1999, Khan, 2000).
Techniques to transform plants
In addition to having selectable and reporter genes as well as efficient tissue culture
system available, pre-requisites to transform plant genome are: a method to deliver
foreign DNA into the cell, and selection/screening strategies of the transgenic shoots. The
most commonly used protocols to transform plant cells include polyethylene glycol
(PEG) treatment, Agrobacterium-mediated transformation (Arencibia et al., 1998),
biolistic DNA delivery (Gallo-Meagher and Irvine, 1993; 1996; Christou et al., 1991) and
microinjection methods. Of these methods, only Agrobacterium mediated and biolistic
DNA delivery methods have yielded stable transformants. However, only biolistic DNA
delivery method is being used to establish plastid transformation in different plants
including dicots (Hibbred et al., 1998; Svab and Maliga, 1993) as well as monocots
(Khan and Maliga, 1999).
Salient examples of plastome manipulations
Plants resistant to Bacillus thuringiensis (Bt)-sensitive insects were generated by
integrating the cryIAc gene into the tobacco chloroplast genome (McBride et al., 1995).
Plants able to withstand even insects highly resistant to Bt were obtained by hyperexpression of the cry2A gene from engineered chloroplasts (Kota et al., 1999). In
addition, chloroplasts have also been engineered recently to generate plants tolerant to
bacterial and fungal diseases (DeGray et al., 2001), drought or herbicides (Daniell et al.,
1998; Iamtham and Day, 2000; Ye et al., 2001; Lutz et al., 2001).
Exceptionally high accumulation of foreign proteins (up to 46% of total soluble protein)
has been reported recently for chloroplast transgenes (DeCosa et al., 2001). This feature
NIBGE-FAO Workshop on GMO Detection
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should make the compartment ideal for low-cost production of biopharmaceuticals.
Several recent advances in plastid genome engineering like stable expression of GVGVP,
a protein based polymer having varied medical application (Guda et al., 2000) and
subsequently human somatotropin (Staub et al., 2000), pharmaceutical peptides (DeGray
et al., 2001), Human Serum Albumin (Fernandez-San Millan et al., 2001) and antigens
composed of functional oligomeric proteins with stable disulfide bridges will make
chloroplasts even more attractive as biopharmaceutical reactors. Moreover, industrial
enzymes like pHBs (Devine et al., 2004) and arabitol dehydrogenase (M.S. Khan,
unpublished)
References:
Arencibia, A.D., Carmona, E.R., Tellez, P., Chan, M-T., Yu, S-M., Trujillo, L.E. and
Oramas, P. An efficient protocol for sugarcane transformation mediated by
Agrobacterium tumefaciens. Transgenic Research 7, 213-222. (1998).
Baulcombe,D.C. Chapman, S. and Cruz, S.S. Jellyfish green fluorescent protein as a
reporter for virus infections. Plant J. 7, 1045-1053. (1995).
Carrer, H., Hockenberry, T.N., Svab, Z. & Maliga, P. Kanamycin resistance as a
selectable marker for plastid transformation in tobacco. Mol. Gen. Genet. 241, 49–56.
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Chiu, W-L., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H.and Sheen, J. (1996)
Engineered gfp as a vital reorterin plants. Curr. Biol. 6, 325-330.
Chowdhury, M.K.U and Vasil, I.K. Stably transformed herbicide resistant callus of
sugarcane via microprojectile bombardment of cell suspension cultutres and
electroporation of protoplasts. Plant Cell Rep. 11, 494-498. (1992).
Christou, P., Ford, T.L. and Kofron, M. Production of transgenic rice plants from
agronomically important Indica and Japonica varieties via electric discharge particle
acceleration of exogenous DNA into immature zygotic embryos. Biotechnol. 9, 957-962.
(1991).
Daniell, H. and M.S. Khan. Engineering the chloroplast genome for biotechnology
applications. N. Stewart ed. Transgenic Plants: Current Innovations and Future Trends,
pp.83-110, Horizon press UK. (2003).
Daniell, H., Datta, R., Varma, S., Gray, S. & Lee, S.B. Containment of herbicide
resistance through genetic engineering of the chloroplast genome. Nat.Biotechnol. 16,
345–348 (1998).
Daniell, H., Khan, M.S. and L. Allison. Milestones in chloroplast genetic engineering: an
environmentally friendly era in biotechnology. Trends in Plant Science 7, 84-91. (2002).
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Devine, A.L., Khan, M.S, Deuel, D.L., Van-Dyk, D.E., Viitanen, P.V. and Daniell, H.
Metabolic Engineering via the Chloroplast Genome to Produce 4-Hydroxybenzoic Acid a
Principle Monomer of Liquid Crystal Polymers. Plant Physiol. (2004, submitted).
DeCosa, B., Moar, W., Lee, S.B., Miller, M. and Daniell, H. Hyper-expression of the Bt
Cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat.
Biotechnol. 19, 71-74. (2001).
DeGray, G., Smith, F., Sanford, J. and Daniell, H. Hyper-expression of an antimicrobial
peptide via the chloroplast genome to confer resistance against phytopathogenic bacteria.
Plant Physiology. In Press (2001).
Fernandez-San Millan, A., Mingo-Castel, A. and Daniell, H. Manipulation of Gene
Regulation in Transgenic Chloroplasts Results in Hyper-expression of a Pharmaceutical
Protein Highly Susceptible to Proteolytic Degradation and Resolves Basic Questions on
RNA Processing. In review. (2001).
Gallo-Maegher, M. and Irvine, J.E. Effect of tissue type and promoter strength on
transient GUS expression in sugarcane following particle bombardment. Plant Cell Rep.
12, 666-670. (1993).
Gallo-Maegher, M. and Irvine, J.E. Herbicide resistant transgenic sugarcane plants
containing the bar gene. Crop Sci. 36, 1367-1374. (1996).
Golds, T., Maliga, P. & Koop, H.U. Stable plastid transformation in PEG-treated
protoplasts of Nicotiana tabaccum. Biotechnology 11, 95–97 (1993).
Guda, C., Lee, S.B. and Daniell, H. Stable expression of biodegradable protein based
polymer in tobacco chloroplasts. Plant Cell Rep. 19, 257-262 (2000).
Haseloff, J., K. R. Siemering, D. C. Prasher and S. Hodge. Removal of a cryptic intron
and subcellular localization of green fluorescent protein are required to mark Arabidopsis
plant brightly. Proc. Natl. Acad. Sci. USA 94, 2122-2127. (1997).
Hibberd, J.M. et al. Transient expression of green fluorescent protein in various plastid
types following microprojectile bombardment. Plant J. 16, 627-632. (1998).
Iamtham, S. and Day, A. Removal of antibiotic resistance genes from transgenic tobacco
plastids. Nat. Biotechnol. 18, 1172-1176. (2000)
Khan, M. S. and P. Maliga. Fluorescent antibiotic resistance marker to track plastid
transformation in higher plants. Nature Biotechnology 17, 910-915. (1999).
Khan, M.S. Utilizing heterologous promoters to express green fluorescent protein from
jellyfish in tobacco chloroplasts. Pak. J. Bot. 33, 43-52. (2001).
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Koop, H.U. et al. Integration of foreign sequences into the tobacco plastome via PEGmediated protoplast transformation. Planta 199, 193–101 (1996).
Kota, M., Daniell, H., Varma, S., Garczynski, F., Gould, F and Moar, W.J.
Overexpression of the Bacillus thuringiensis Cry2A protein in chloroplasts confers
resistance to plants against susceptible and Bt-resistant insects. Proc. Natl. Acad. Sci.
USA. 96, 1840-1845. (1999).
Lutz, K.A., Knapp, J. and Maliga, P. Expression of bar in the plastid genome confers
herbicide resistance. Plant Physiol. 125, 1585-1590. (2001).
Maliga, P. Towards plastid transformation in flowering plants. Trends Biotechnol.11,
101–107 (1993).
McBride, K. E., Svab, Z., Schaaf, D.J., Hoga, P.S., Stalker, D.M. and Maliga, P.
Amplification of a chimeric Bacillus gene in chloroplasts leads to an extraordinary level
of an insecticide protein in tobacco. Biotechnology 13: 362-365. (1995).
Moll, B., Posby, L. & Maliga, P. Streptomycin and lincomycin resistance are selective
plastid markers in cultured Nicotiana cells. Mol. Gen. Genet. 221, 245–250 (1990).
O’Neill, C., Horvath, G.V., Horvath, E., Dix, P.J. & Medgyesy, P. Chloroplast
transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an
alternative to biolistic delivery system. Plant J. 3, 729–738 (1993).
Prasher, D.C. Using GFP to see the light. Trends Genet. 11, 320–323 (1995).
Seki, M., Shigemoto, N., Sugita, M., Sugiura, M., Koop, H-U., Irifune, K. and Morikawa,
H. Transiant expression of beta-glucoronidase in plastids of various plnt cells and tissues
delivered by a pneumatic particle gun. J. Plant Res. 108, 235-240. (1995).
Sidorov, V.A. et al. Potato plastid transformation: high expression of green fluorescent
protein in chloroplasts. Plant J. 19, 209-216. (1999).
Staub, J.M. and Maliga, P. Translation of psbA mRNA is regulated by light via 5’untranslated region in tobacco plastids. Plant J. 547-553. (1994).
Staub, J.M., Gracia, B., Graves, J., Hajdukiewics, P.T.J., Hunter, P., Paradkar, V.,
Schlitter, M., Carrol, J.A., Spatola, L., Ward, D., Ye, G. and Russell, D.A. High-yield
production of a human therapeutic protein in tobacco chloroplasts. Nat. Biotechnol. 18,
333-338. (2000).
Svab, Z. and Maliga, P. High-frequency plastid transformation in tobacco by selection for
a chimeric aadA gene. Proc. Natl. Acad. Sci. USA 90, 913–917 (1993).
Ye, G-N et al., Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes
provide high-level glyphosate tolerance in tobacco. Plant J. 25, 261-270. (2001).
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Sampling for Detection of GMO
Muhammad Asif
Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Sample and sampling procedures
A consignment of grain lot has many unknown quality characteristics. Measuring these
characteristics on the entire lot can be costly. An experienced inspector must examine the
grain to determine whether a kernel is damaged or not. Time constraints and cost prohibit
an inspector from examining every kernel in a grain lot. Inspecting a small subset of the
lot is much less costly and time consuming than inspecting the whole lot. This subset of
the lot is called a sample.
Although, inspecting a sample is much less costly than inspecting the entire lot, but the
content of a sample does not always reflect the content of the lot. Fortunately, when
samples are properly taken, probability theory can assign some risk values to
measurements on samples. Sampling from a lot is one of the source of error while
estimating characteristic of a lot. Sources of error fall into three basic categories: (1)
sampling, (2) sample preparation, and (3) analytical method. Minimizing these errors is
necessary to assure better precision and accuracy in the final analytical result.
Buyers and sellers of a lot have to agree on the quality and price of the lot before a
transaction can take place. Basing the quality of a lot on a sample introduces risk to both
buyer and seller. Buyers and sellers want to control their risk where possible. Buyer and
seller should agree on a specific sampling and testing plan that best meets their mutual
needs. Using the information provided here, buyers and sellers would be able to make
informed and better decisions.
The sampling procedure determines the “representativity” of a result, whereas quality and
quantity of the analytes may vary depending on the sample preparation. Sampling and
sample preparation are thus crucial steps in the process of GMO detection. A sample is
simply a subset of a lot and we can estimate the risk for randomly selected samples using
probability theory. A random sample is one selected in a process in which every
possible sample from a lot has an equal chance of being selected. If every possible
sample from a lot could be measured, the average of the measurements would equal the
content of the lot. This means that, on average, a random sample produces an unbiased
estimate of the measurement of interest. In practice, a pure random sample is not always
easy to obtain from a lot. A sampling technique called systematic sampling has been
widely used to produce a sample that is a reasonable substitute for a random sample. In
grain inspection, variations of the systematic sampling process are used to select samples.
Risks can be estimated when random samples are taken. If the sampling procedure is not
random, or a close approximation, estimates can be biased.
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One sampling procedure could be to scoop a sample off the top of a lot using a can. If a
lot has been loaded and unloaded many times, the lot may be mixed sufficiently that it is
fairly uniform and scooping a sample may be adequate. However, some lots may be the
combinations of other lots and the resulting lot can be stratified. Scooping a sample off
the top may not be very representative of the lot. Grain sampling methods prescribed by
the USDA include methods for sampling moving grain streams and static grain lots. The
diverter type (DT) sampler is the most common sampling device for sampling from a
grain stream. The DT takes a classic systematic sample. The DT traverses a moving grain
stream and, per specific timer settings, diverts a small slice of the grain stream to the
inspector. The small slices are combined to obtain the sample for the lot.
A manual means of taking a sample from a grain stream, similar to the diverter type
sampler, is the pelican sampler. The pelican sampler is a leather bag on the end of a pole.
A person will pass the pelican through a falling grain stream at the end of spout, taking a
cut from the grain stream. The pelican is passed through the grain stream frequently. The
pelican is emptied between passes through the grain stream. The Ellis cup is another
manual sampling device for sampling from a conveyor belt. A person will frequently dip
the Ellis cup into the grain stream.
Various probing techniques are used to sample grain from static lots. Depending on the
size and shape of the container, multiple probes of the lot will be combined to obtain the
sample from the lot. Patterns for probing a lot are prescribed for various types of
containers. The individual probes are sufficiently close to effectively sample across any
stratification that may exist. To obtain the specified test sample size, a sub-sample of the
original grain sample must be obtained. Dividers such as the Boerner, cargo, and Gamet
have demonstrated the ability to subdivide an origin sample and have the resulting
samples conform to distributions expected from a random process.
GIPSA has instructions for taking samples from static lots - such as trucks, barges, and
railcars - and for taking samples from grain streams. Grain Inspection Handbook, Book 1,
Grain Sampling and Mechanical Sampling Systems Handbook contain these instructions
and can be obtained by accessing the web page of Grain Inspection, Packers and
Stockyards Administration (GIPSA) of the U.S. Department of Agriculture.
Eliminating Carry-Over of Biotech Grains:
Current testing technology for the detection of biotech grain can be very sensitive,
increasing the probability that cross-sample contamination could result in a false positive
detection. Furthermore, minor inadvertent commingling of biotech grain kernels with
non-biotech grain could result in a positive detection at very low concentrations.
Consequently, great care must be taken to ensure the integrity of the grain samples used
for testing and to avoid inadvertent commingling during grain handling processes.
Eliminating carry-over of biotech varieties to non-biotech varieties involves
understanding and controlling the critical points in the grain handling system. If grain
handlers choose to segregate biotech and non-biotech grains, the vehicles, tools, and
conveying equipment used in shipment, collection, and transportation of bulk grains
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throughout the distribution system must either be cleaned before loading non-biotech
crops, or dedicated to non-biotech crops. The complexity and cost related to such a
process has lead some companies to implement identity preservation systems, rather than
segregate non-biotech grain through the traditional marketing system.
The Impact of Sample Size on Risk Management:
The range of likely sample estimates decrease as the sample size increase. Several
assumptions underlie the sample size effects. One assumption is that every kernel in a
sample can be determined as biotech or non-biotech without error. The variability shown
in the sample estimates assumes only sampling variability. No allowance for error from
sample preparation or from analytical method has been incorporated into the estimate
ranges.
Measurements associated with grain lots are usually given as percent by weight. The
percent by kernel count and percent by weight would only be the same if the kernels were
all the same size, but kernels are not uniform. Percent by kernel count is, however,
usually a reasonable approximation to percent by weight. Kernel counts can be converted
to approximate weights by using average kernel weights observed from typical market
samples. The type of measurement is also a consideration in determining the sample size.
The analytical methods available for detecting biotech grains may be used to make
qualitative or quantitative tests on a sample. A qualitative test can be used to screen lots
by providing information on the presence or absence of biotech varieties. Quantitative
tests may quantify the total amount of biotech grain, the amount of individual varieties in
a sample, or the percentage of biotech or "non-native" DNA or protein present relative to
non-biotech grain.
When sampling is used in the measurement of some characteristics of a lot, the content of
the sample will likely deviate from the lot content. The buyer accepts some risk because
the sample may overestimate the quality of the lot. The buyer may assume that the quality
of the lot is better than it actually is. Likewise, the seller accepts some risk that the
sample may underestimate the quality of the lot. In this case, the seller is delivering better
quality than the sample reflects. Ideally, buyers and sellers would agree to use a sampling
plan that provides acceptable risk management. A contract may specify a certain quality
level. However, due to sampling variation, a seller may have to provide better quality to
have grain lots accepted most of the time. Sellers should choose a quality level that they
want to have accepted most of the time, say 90% or 95%. This level is sometimes called
the acceptable quality level (AQL). Likewise, due to sampling variation, the buyer may
sometimes have to accept lower quality than the contract specifies. Buyers should choose
a lower quality level (LQL) that they want to accept infrequently. This LQL may be
acceptable 5% or 10% of the time (Fig. 1). The ideal sample plan would meet both the
AQL and the LQL. A single sample with a qualitative test gives little flexibility for
choosing an AQL. Quantitative tests, when available, and multiple sample plans provide
more flexibility to choose both an AQL and LQL.
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Fig. 1: Probability of Accepting Lots.
Sample Size – Qualitative:
When the desired concentration of a specific trait is zero, a single qualitative test would
be an adequate testing plan. The sample plans assume that a single kernel can be detected
in a sample, regardless of the size of the sample. When sampling is used to estimate the
concentration in a lot, sampling error will always be present. A negative result or test
does not guarantee that some concentration doesn’t exist in the lot. Probability can be
used to describe the risks associated with accepting lots with certain concentrations in the
lot.
In reality, all analytical methods have limits of detection. A qualitative test will detect the
presence of a single kernel in a sample, regardless of the size of the sample. A positive
result does not tell how many biotech kernels are in the sample; only that at least one
biotech kernel is present in the sample. To choose a sample size, the acceptable and
unacceptable concentrations must be decided upon. Since samples are subject to sampling
error, acceptable lots may be rejected, and unacceptable lots may be accepted just by
chance. Buyers and sellers must agree upon acceptable risk. The chart (Fig. 2) shows
probabilities for sample sizes of 60, 120, 200, and 400 kernels. If the desired
concentration on the lot is not to exceed 5.0 %, a sample size of as little as 60 kernels
may be satisfactory. Based on a 60 kernel sample, there is a 95 % chance of rejecting a
lot at a 5 % concentration. If 1.0 % is the desired maximum concentration in the lot, a
sample size of 400 kernels would be more appropriate. Larger sample sizes are used only
when low concentrations of biotech kernels are acceptable.
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Fig. 2: Probability of Acceptance.
Sample Size – Quantitative:
Quantification of the percent biotech grain in a lot is much more problematic than with
qualitative testing. The currently available technologies employed for GMO detection are
mostly PCR and ELISA. Both methods present significant challenges in converting the
amount of DNA or the amount of expressed protein into the percent of biotech grain by
weight. The overall variability of quantitative results, therefore, will be affected by
analytical methods as well as sample size. Sample size typically will have little influence
on the sample preparation or analytical method. Sample preparation and analytical
method are significant sources of error, and increasing the sample size will not reduce the
overall variability in measurements as much as expected.
The probability of accepting an unacceptable lot, may be called "buyers risk" because this
is the chance that the buyer will get an unacceptable lot. The chance of rejecting an
acceptable lot may be called the "sellers risk" because this is the chance that the seller
will have an acceptable lot rejected. The ideal sampling plan will minimize both the
buyers and sellers risk. Unfortunately, no one sampling plan will produce both objectives.
Increasing the sample size can reduce both buyer risk and seller risk. Theoretically, the
only limiting factor on the sample size is the lot size. Sample size is often determined by
a compromise between the seller and buyer risks and the cost of taking and processing a
sample. The following graph (Fig. 3) gives sampling plans that allow a maximum of 1.0
% biotech kernels in the sample. If the desired concentration on the lot is not to exceed
5.0 %, a sample size of as little as 400 kernels may be satisfactory. Again, increasing the
sample size will reduce both the buyer and seller risks.
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Fig. 3: Probability of Acceptance of a lot.
Since the size of sample is important, so to detect GM DNA present in beans or kernels at
a 0.1% level (1 bean in 1000), an initial sample of 5,000-10,000 (~2.5kg) beans or
kernels would need to be sampled. In the case of mixed processed products (starches,
animal feed, ready -meals…), a 500g sample (or two sample packets) is sufficient to
perform a test at a 0.1% sensitivity level. For oils a 500ml sample should be, whereas for
lecithin a 100ml sample or 500g is sufficient.
Sample Preparation: Obtaining A Portion for Analysis:
Samples are typically collections of kernels from a lot where each kernel in the sample
would be measured independently, but the major GMO analytical technologies (PCR and
ELISA), usually do not process individual kernels but rather make a measurement on a
preparation from the sample.
Sampling variability is only one source of error in measurements while, Sample
preparation and analytical methods are two other significant sources of error. Reducing a
sample to a portion for analysis is often necessary to meet method limitations. For
accurate analysis, the sample portion analyzed must be representative of the sample
submitted from a lot. Preparation of a sample for analysis must include grinding and
mixing of the grain prior to subdivision. Grinding will produce more uniform subsamples
for analysis. For example, a representative three pound (1360 grams) composite sample
would contain 8500 soybeans. If this composite has 1% biotech content that would mean
about 85 biotech soybeans would be present. If a standard divider is used to reduce the
unground sample to an analytical portion of 60-70 beans, not all of the resulting 128
possible sample divisions would have a biotech soybean. If a hypothetical distribution of
one soybean per subsample were assumed, then only 85 of the 128 possible "cuts" would
test positive for the presence of biotech grain. Forty-three subsamples would test
negative.
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Thorough grinding and mixing would give a much better and different distribution of the
analyte, producing more consistent results from subsample to subsample. The analyses to
be performed must be considered and may be the determining factor with respect to
particle size. In a plant seed, the DNA and much of the protein is concentrated in the
embryo, and the embryo may only be 10% of the weight of a seed. In the 1360 gram (3
lb.) sample above, the embryonic material from genetically modified soybean seeds will
be only one-tenth of 1%. For PCR analysis, where analytical sample sizes are routinely
only one gram, research studies have shown that a particle size of less than 200
produces a homogenous sample. Therefore, grinding a representative composite sample
to an appropriate particle size, followed by thorough mixing, will minimize sample
preparation errors. Analytical results on subsamples of this homogenous sample will be
much closer to the actual content (1%).
Each 1gram subsample should contain about 250,000 soybean particles. Although one
percent of these particles would be from biotech soybeans, only about 250 of these would
actually contain biotech DNA. Using a normal approximation, 97 % of the analytical
results would be in the range of 0.86 to 1.14 %. ELISA testing, which routinely uses
larger sample sizes, may not need as fine a particle size as PCR testing.
Cleanliness in Sample Preparation:
Carry over of materials from one sample to another takes on an even greater significance
during sample preparation prior to analysis. Due to the sensitivity seen with many
methods for detection of biotech grains, care must be taken to avoid transferring materials
to subsequent samples. Whole grains, dust and residual matter must be removed from all
equipment. Grinders should be cleaned through vacuuming of dust, washing with soap
and water or solvents, or a combination of appropriate cleaning methods for the specific
grinder in use. Sample dividers and mixers must also be thoroughly cleaned. Analysts
should verify the equipment cleaning process is appropriate to prevent cross
contamination. Many of the analytical techniques practiced for detection of biotech crops
today can detect levels lower than 0.1%. Physical separation of sample preparation
operations from analytical operations is also highly recommended to avoid contamination
of sample extracts.
Selecting a Sampling Protocol to Minimize Risk:
Sample size, theoretically, is selected to best meet the needs of the buyer and seller.
Selecting a sample size often involves a compromise between precision and cost of
analysis. In measurement systems where kernels are processed individually, the cost of
processing a sample increases in proportion to increases in sample size. For these
systems, selecting the smallest sample size that provides acceptable precision is the most
cost effective sample size. Many measurement systems process and measure bulk
samples. In these systems, the cost of processing a sample may not increase in proportion
to increases in the sample size. Processing a large sample may cost only slightly more
than processing a small sample. Under these circumstances, processing the largest sample
the technology will handle may be the best sample size.
79
NIBGE-FAO Workshop on GMO Detection
For single sample qualitative testing, sample sizes can be determined with a relatively
simple formula. Given the desired lot concentration and probability of detection, a sample
size is computed with the following formula:
n = log(1-(G/100))/log(1-(P/100))
n is the sample size (number of kernels), G is the probability (in percent) of rejecting a lot
concentration, and P is percent concentration in the lot.
The following table-1 provides recommended sample sizes for qualitative testing based
upon this formula.
Biotech
Concentration
Number of
Kernels
0.1
Approximate Weight in Grams
Corn
Soybeans
2995
881
474
0.5
598
176
95
1.0
299
88
48
2.0
149
44
24
3.0
99
30
16
4.0
74
22
12
5.0
59
18
10
(Note: Sample Sizes such that Lots Containing the Given Concentration Levels Are
Rejected 95% of the Time.)
The sample size should amount to 299 kernels or beans in order to obtain a 95%
probability of rejecting a lot with 1% concentration of GMO, i.e. a “buyer’s risk” of 5%
to accept a lot with more than 1% GMO content. If the threshold limit was set at 0.5%
GMO at a 95% probability of rejection, the size of the field sample would need to be
increased up to 598 kernels. However, at a sample size of 299 kernels the “seller’s risk”
of having a lot rejected, which contains only 0.5% GMO, is still about 78%. Therefore, in
order to provide means of controlling marketing risks for both buyer and seller, multiple
sampling plans for qualitative analytical testing have been developed. A multiple
sampling plan is defined by the number of samples needed and tested, by the maximum
number of positive results allowed for the lot to be acceptable, and by the number of
kernels in each sample. Buyer and seller have to agree on these three values, and thereby
determine the marketing risk both of them are willing to take.
For very low values of lot concentration, the sample size may become very large.
Suppose someone wants to detect a 0.01 percent lot concentration with a 99%
probability. The required sample size would then be 46,050 kernels. Such a large sample,
however, may not be appropriate for use with all testing methods.
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(Note: Main portion of this article has been taken from “USDA-Grain Inspection, Packers
and Stockyards Administration GIPSA (2000) Sampling for the Detection of Biotech
Grains”.). Further details on the sampling of GM grains and other food stuffs can be
found at (http://www.usda.gov/gipsa/biotech/sample1.htm), (http://europa.eu.int/eurlex/en/lif/dat/1998/en_398L0053.html), web pages of European Center for Normalization
(CEN), International Seed Trade Association (ISTA), Codex Alimentarius Commission
of the UN and International Standards Organization (ISO).
Suggested readings:
Ahmed, F. E. 2004. Testing of genetically modified organisms in foods. The Haworth
Press, Inc. USA.
Anonymous. 2000. Sampling for the detection of Biotech grains.
http://www.usda.gov/gipsa/biotech/sample1.htm
Bould, A. 1986. Handbook for Seed Sampling, International seed Testing.
Codex Alimentarius Commission. 2003. ALINORM 03/23 Report of the 24 th session of
the CODEX Committee on methods of analysis and sampling. Appendix IV Proposed
draft general guidelines on sampling (at step 5 of the procedure). Joint FAO/WHO Food
Standards Programme, 32-101.
International Standards Organisation (ISO). 1999. ISO 13690. Cereals, pulses and milled
products – sampling of static batches. 18 pp.
International Seed Trade Association (ISTA) Handbook, 1st edition, 64 pp. ISBN 3906549-02-X, USA.
Kay, S. and Paoletti, C. 2001. Sampling strategies for GMO detection and/or
quantification. European Commission Directorate General, JRC. 16 pp.
http://biotech.jrc.it/doc/EuroReport_sampling_strategies.pdf
Laura, B., H. Petra, K. Simon and V. E. Guy. 2001. Review of gmo detection and
quantification techniques. EC JRC,Institute for Health and Consumer Protection, Food
Products and Consumer Goods Unit, I-21020 Ispra, Italy.
Pan, T. 2002. Current status and detection of genetically modified organism. J. Food &
Drug Analy. 10 (4): 229-241.
Paoletti, C., Donatelli, M., Kay, S., van den Ede G. 2002. Simulating kernel lot sampling;
the effect of heterogeneity on the detection of GMO contaminations. Seed Science and
Technology. http://biotech.jrc.it/doc/SeedScienceAndTechnology.pdf
Reed, P. 2002. Protocol for bulk grain sampling and testing for GM presence. Draft SOP
produced on behalf of European Enforcement Project. CSL, York, UK. 8pp
USDA, GIPSA (Grain Inspection, Packers and Stockyards Administration) Grain
inspection handbook – Book 1 Sampling. http://www.cliiltd.com/grain_inspection.htm
NIBGE-FAO Workshop on GMO Detection
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Development of IR-Cotton: A Case Study
Mehboob-ur-Rahman, PhD
Senior Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Globally, cotton and other crop plants require an intensive use of pesticides to inhibit
insect pests population. In Pakistan, over the past 30 years, pesticides are being used to
protect cotton crop against sucking and chewing insects. Among the chewing,
lepidopteran are the primary pests causing 20-30% annual yield losses in the country.
Different approaches have been used to develop inbuilt resistance against the bollworms.
So far, the only successful approach to engineering crops for insect tolerance/resistance is
the use of addition of Bt toxin, a family of toxins originally derived from soil bacteria.
Bacillus thuringiensis (Bt) is a gram-positive, aerobic, spore- forming bacteria that
is found in soil, plant surfaces and in grain storage dust. There are almost eighty
different serotypes of Bt, which are capable of producing many different toxins, including
endotoxins, exotoxins and enterotoxins. Toxins produced by Bt are known as "Bt toxins".
The Bt toxins consist of two main types, Cry (crystal) toxins (cry genes) and Cyt
(Cytolytic) toxins. The Cry proteins are effective against different insect orders, being the
most effective against lepidoptera (caterpillars), coleoptera (beetles) and diptera (small
flies and mosquitoes).
Bollgard, the first transgenic Bt cotton, has been grown globally on more than 32 million
acres since commercial introduction in 1996. The advantages of Bt cotton have been
resulted in the form of reduced insecticide use, improved control of target insect pests,
improved yield, reduced production costs, and improved profitability.
At the moment, Monsanto launched Bollgard II by combining Cry2Ab2 and Cry1Ac
proteins in a single product provides an additional tool to delay the development of insect
resistance to Cry proteins in cotton. It provides increased control of cotton bollworm, as
well as certain secondary insect pests of cotton, including armyworm.
Safety Assessment of Bt-Cotton/IR-Cotton
Environmental Assessment
Cotton belongs to genus Gossypium, consisting of four species of agronomic importance:
the two diploid Asiatic species, G. arboreum and G. herbaceum, and the twoallotetraploid New World species, G. barbadense and G. hirsutum. Cotton is normally
considered a self-pollinating crop but can be cross-pollinated by certain insects.
However, outcrossing of the cry1Ac gene from Bt cotton to other Gossypium species or to
other Malvaceous genera is extremely unlikely for the following reasons:
• cultivated cotton is incompatible with cultivated or wild diploid cotton species;
therefore, it cannot cross and produce fertile offspring.
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• although outcrossing to wild or feral allotetraploid Gossypium species can occur, cotton
production generally does not occur in the same geographical locations as the wild
relatives. For example, outcrossing to G. tomentosum in Hawaii is possible, but cotton is
not grown commercially in Hawaii.
• there are no known plant species other than those of the genus Gossypium that are
sexually compatible with cultivated cotton.
Crossing of insect protection genes to other cultivated cotton genotypes is
possible should the plants be in close proximity; however, studies have shown that this
occurs at a very low frequency and is not considered to be a concern as it is unlikely to
cause any adverse impact to the environment.
Assessment of Agronomic Performance
The IR-cotton has been grown and observed at multiple locations for weediness, plant
growth characteristics, susceptibility to insects, and disease infection. Based on results of
the field monitoring programs, there were no significant differences in agronomic
characteristics between IR-cotton and the parental variety. IR-Cotton meets all
morphological, yield, and quality characteristics of cotton varieties produced in the
country.
Cotton is not considered to have weedy characteristics as an annual plant grown in the
United States. It does not possess any of the attributes commonly associated with weeds
such as seed dormancy, long soil persistence, germination under diverse environmental
conditions, rapid vegetative growth, a short life cycle, high seed output, high seed
dispersal, or long distance dispersal of seeds. Multiple genes typically control these
characteristics of weeds.
Wild populations of cotton are rare, widely dispersed and confined to beach strands or to
small islands. Cotton appears to be somewhat opportunistic towards disturbed land and is
not especially effective in invading established ecosystems.
There is little probability that Bt-Cotton or any Gossypium species with Bt cotton could
become a weed. All wild and feral relatives of cotton are tropical, woody, perennial
shrubs, other than a few herbaceous perennials in northwest Australia. With the exception
of G. thurberi and G. sturtianum in Australia, these cannot naturally exist even in the
milder temperate regions.
Bt cotton does not have any different weediness characteristics than other conventional
cotton varieties. Bt cotton does not exhibit different agronomic or morphological traits
compared to controls, which would confer a competitive advantage over other species in
the ecosystem in which it is grown. Based on these mechanistic arguments and field
experience, there is no indication that insertion of the cry1Ac gene into the cotton genome
would have any effect on the weediness traits of the cotton plant.
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Assessment of Effect to Non-Target Organisms
There is extensive information about microbial preparations of Bacillus thuringiensis
subsp. kurstaki (B.t.k) containing Cry proteins that demonstrate that these proteins are
nontoxic to non-target organisms. It is documented that the Cry proteins are extremely
selective for lepidopteran insects, bind specifically to receptors on the mid-gut of
lepidopteran insects, and have no deleterious effect on beneficial/non-target insects.
The non-target organism species included larval and adult honey bee (Apis mellifera L.),
a beneficial insect pollinator; green lacewing larvae (Chrysopa carnea), a beneficial
predaceous insect commonly found on cotton and other cultivated crops; parasitic
Hymenoptera (Nasonia vitripennis), a beneficial parasitic wasp of the housefly; the
ladybird beetle (Hippodamia convergens), a beneficial predacious insect which feeds on
aphids and other plant bugs commonly found on stems and foliage of weeds and
cultivated plants; Collembola (Folsomia candida) and earthworm (Eisemia fetida)
nontarget soil organisms; and northern bobwhite quail.
No adverse effects were observed at the maximum expected environmental
concentrations to which these non-target organisms would be exposed. In most studies,
the NOEC exceeded the maximum predicted environmental concentration by 10- to over
100-fold, demonstrating a wide margin of safety for these organisms.
In summary, Cry proteins exhibit a high degree of specificity and therefore do not pose a
significant hazard to non-target animals such as mammals, birds, fish, water fleas,
earthworms, and beneficial insects. Although several endangered lepidopteran and
dipteran species may potentially be susceptible to Cry proteins, no exposure is predicted
because of their feeding habit or because the habitats of these endangered species in
cottongrowing areas do not overlap with cotton fields.
Assessment of Genetic Stability
The cry1Ac gene conferring insect protection in Bt cotton was demonstrated as stably
integrated into the chromosome. This conclusion is based on molecular analyses, data on
phenotypic expression, and inheritance patterns.
Insect Resistance Management
Effective insect resistance management (IRM) programs for B.t. crops are a vital part of
responsible product stewardship and should be instituted based on the best available
knowledge, employing what is known about the trait, the mode of action, the targeted
insects and the environment in which the product is introduced, while being properly
respectful of uncertainties so as to make B.t. technologies available to growers as an
additional pest management tool.
Allergenicity
The potential allergenicity of newly introduced proteins in genetically modified
foods is a major safety concern. This is true in particular for genetic material obtained
from sources with an unknown allergenic history, such as the soil bacterium B.
thuringiensis. An illustrative case of a genetically modified food for which the allergenic
risk has to be assessed is cotton.
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To evaluate the biosafety of Bt protein expressed in Bt transgenic cotton plants,
they did numbers of toxicity tests including acute tests, chronic tests, gene toxicity tests
and so on. The results showed that there was no acute toxicity, chronic toxicity or gene
toxicity to each kind of animals mentioned above.
Comet Assay
DNA based test called Comet assay could provide an important
indication of genotoxic potential. Principally, it based on the DNA
damage of the cell under study, which appeared as tail after conducting
electrophoresis.
Concerns regarding safety evaluation
The safety evaluation may focus on different levels of food crop, for example, the whole
crop; crop tissues; or purified products, depending on the scope of the application.
Risk assessment studies have been carried out by different researchers all
over the world. For example studied the risk assessment of Bt cotton by
feeding rats for 28 days. They included the parameters, as body weight,
feed conversion, histopathology of the organs and blood chemistry.
In addition to the feeding studies described above, studies have been
performed on domestic animals fed genetically modified crops to establish
performance. It is apparent that no harmonized design exists yet for
feeding trials in animals to test the safety of genetically modified foods.
Extensive testing of Bt-protected crops has been conducted which
establishes the safety of these products to humans, animals, and the
environment. Food and feed derived from Bt-protected crops which have
been fully approved by regulatory agencies, have been shown to be
substantially equivalent to the food and feed derived from conventional
crops. Because the Cry protein is contained within the plant (in
microgram quantities), the potential for exposure to farm workers and
non-target organisms is extremely low. The Cry proteins produced in Btprotected crops have been shown to rapidly degrade when crop residue is
incorporated into the soil. Thus the environmental impact of these crops is
negligible. Examples of feeding studies with whole genetically modified
foods are summarized in Table 1.
Pakistan has started modern biotechnology research in early 1990s in crop plants
especially cotton. These investments were accelerated in late 1990s by the Govt of
Pakistan. Unlike biotechnology research in most other countries of the world, the private
sector has not played a major role in biotechnology research in Pakistan.
Insect pests, particularly the cotton bollworm (Helicoverpa armigera), have been a major
problem for cotton production in Pakistan. Pakistan's farmers have learned to combat
these pests using pesticides. Initially, farmers used chlorinated hydrocarbons (such as
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DDT) until they were banned for environmental and health reasons in the early 1980s. In
the mid-1980s, farmers began to use organophosphates, but in the case of cotton, pests
developed resistance. In the early 1990s, farmers began to use pyrethroids, which were
more effective and safer than organophosphates. However, as in the case of other
pesticides, Pakistan's bollworms rapidly began to develop resistance to pyrethroids in the
mid-1990s.
At this time, farmers resorted to cocktails of organophosphates, pyrethroids and whatever
else they could obtain (including DDT, although the use of cholorinated hydrocarbons is
illegal) ± with less and less impact on the pests.
With rising pest pressure and increasingly ineffective pesticides, the use of pesticides by
cotton farmers in Pakistan has risen sharply. Farmers use more pesticide per hectare on
cotton than on any other field crop.
Pakistan's pest problems have led the nation's scientists to seek new pesticides, to breed
cotton varieties for resistance to pests, and to develop integrated pest management
programmes to control the pests. Consequently, when the possibility of incorporating
genes for resistance to the pests came closer to reality, Pakistan's scientists started
working on the problem. With funding primarily from government, Bt cotton varieties
naming IR-Cotton (Insect Resistant Cotton) were developed using a Bt gene. The gene
was transformed into major cotton cultivars. Its hazardous impact was deduced using
rabbit as experimental animal and it was found safe.
Conclusions
Bt cotton is gaining popularity in different cotton growing countries. The evidence from
around 10 years' experience with Bt cotton in America and other counties like China is
extremely valuable to increase yield per ha, and reduce pesticide costs, the time spent
spraying dangerous pesticides, and the number of incidences of pesticide poisoning.
China is a developing country like us, farmers decisions to adopt Bt cotton based on their
assessment of the cost benefit ratio. They find it profitable, and so we would expect
cotton growers in Pakistan to achieve similar gains. Similarly, in India, where cotton
growers face the same bollworm pressures, farmers are likely to benefit greatly from this
technology.
The lesson from development of GM crops in China is the importance of indigenous
biotechnology research. Bt cotton (IR-Cotton-local varieties) was developed by public
sector in Pakistan. Commercial release in the country is hampered due to the absence of
Biosatey regulations.
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Table 1. Toxicity studies performed with genetically modified food crops
Suggested Readings
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NIBGE-FAO Workshop on GMO Detection
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Barriere Y., Vérité R., Surault F., and Emile J.C. 2001.Feeding value of corn silage
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Capacity Building in Biosafety of GM Crops in Pakistan
Ahmad M. Khalid and Yusuf Zafar
National Institute for Biotechnology and Genetic Engineering
(NIBGE), P.O. Box 577, Faisalabad, Pakistan
Tel:(+92-41)651471; Fax:(+92-41)651472; Email: [email protected]
Abstract
Agriculture plays an important role in the national economy of Pakistan, where
most of the rapidly increasing population resides in rural areas and depends on
agriculture for subsistence. Biotechnology has considerable potential for promoting the
efficiency of crop improvement, food production and poverty reduction. Use of modern
biotechnology started in Pakistan since 1985. Currently there are 26 biotech
centres/institute in the country. Few centres have excellent physical facilities and trained
manpower to develop GM crops. Most of the activities have been on rice and cotton
which are among the top five crops of Pakistan. Biotic (virus/bacterial/ insect) and abiotic
(salt) resistant and quality (male sterility) genes have already been incorporated in some
crop plants. Despite local production of transgenic plants, field evaluation is blocked due
to absence of legislation on Biosafety Guideline although draft document under
UNEP/GEF was prepared in January, 1999. No GM crops either produced locally or
imported have been released in the country. Pakistan is signatory of WTO, CBD and
Cartagena protocols. Several legislations under TRIPS agreement has been promulgated
in the country however, National Biosafety Guidelines, Plant Breeders Rights Act 2002
and Geographical Indication for Goods are still passing through discussion, evaluation
and analysis phases. Meanwhile, illegal GM crop (cotton) already sneaked into farmer’s
field. Concerted and coordinated efforts are needed among various ministries for
implementation of regulation and capacity building for import/export and local handling
of GM crops. Pakistan could easily be benefited with the experience of Asian countries
where conditions are similar and agriculture sector like of Pakistan, so the exchange of
information and experiences is important among these nations.
Overview of Agricultural Biotechnology in Pakistan
Agriculture sector being the lynchpin of the country’s economy continues to be
the single largest sector and a dominant driving force for growth and development of the
national economy. It accounts for 24 percent of the GDP and employs 48.4 percent of the
total work force. Agriculture contributes to growth as a supplier of raw materials to
industry as well as a market for industrial products and also contributes substantially to
Pakistan’s exports earnings. Almost 67.5 percent of country’s population are living in
rural areas and are directly or indirectly linked with agriculture for their livelihood. Any
Improvement in agriculture will not only help country’s economic growth to rise at a
faster rate but will also benefit a large segment of the country’s population. Agriculture
sector has grown at an average rate of 4.5 percent per annum during the decade of the
1990s. The growth, however, has fluctuated widely rising by as high as 11.7 percent and
declining by 5.3 percent. The total population as estimated on 1 January 2003 is 140
million peoples. Out of the country’s total geographic area of 79.61 million hectares; only
about 22.27 m. ha are cultivated.
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The major crops grown are wheat, rice, cotton, sugarcane and maize. Gram and
other pulses, oil seeds and fodder crops are also grown in different parts of the country on
sizeable areas. In Pakistan the average yields of crops despite rapid increase in Green
Revolution era are still low as compared to, other countries. A large gap exists between
the potential and realized yield for almost all the major crops. The average yield of most
of the crops are either stagnant or even declined during the last decade while inputs cost
and amounts of fertilizers, pesticides, etc. are continue to increase.
Agriculture, being, the mainstay of the nation’s economy and also provides food
security needs immediate attention if it is to be developed sustainably. The agricultural
production system in the country can operate on sound scientific lines and on a stable
basis only if farm technology is kept in tune with the changing environmental and socioeconomic conditions through an efficient and dynamic agricultural research system
(ARS).
Agriculture plays an important role in the national economy of Pakistan, where
most of the rapidly increasing population resides in rural areas and depends on
agriculture for subsistence. A sustained increase in farm productivity is vital for the
region, particularly with the limited availability of extra arable land. In this respect,
biotechnology plays a remarkable role by promoting the efficiency of agro-processing.
Although, biotechnology presents considerable potential for food production and poverty
reduction. However, it also provokes concerns related to health and environment risks,
ethics and democracy in decision-making. In the mean time, as the impact of
biotechnology on human health and the environment remain unknown, biosafety has
become a primary issue.
Research on Biotechnology, Genetic Engineering and Tissue Culture are now
integral part of Agriculture sector of 9th five-year plan. Biotechnology has been viewed
by government functionaries, political leaders and leading scientists, a priority for over
two decades. Presently excellent physical facilities and trained manpower are available at
few centers (CEMB, NIBGE) while many new centers are coming up in all parts of the
country.
Traditional biotech activities particularly related to plant tissue culture have been
carried out in few academic and research institutes since 1970s. An exclusive national
Center of Excellence in Molecular Biology (CEMB) was established in 1983-4 in
University of the Punjab, Lahore. In 1986, Government of Pakistan approved building of
National Institute for Biotechnology and Genetic Engineering (NIBGE) in Faisalabad,
which was formally inaugurated in 1994 at a cost of US$ 1.2 million. Except these two
major inputs, all other activities remained peripheral. However, during the last 3-4 years,
there is renewed interest in establishing Biotech centers in major cities. Provincial
Governments also initiated some programmes during the same period. There are now 26
centres who claimed to be conducting Biotech research of various levels.Despite all these
developments, there is yet no coherent national policy regarding biotechnology in general
and agriculture biotechnology in particular. Lack of clear national objectives/priorities,
resulted in duplication of work and dilution of efforts. In contrast to developed world, the
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biotech research in the country is exclusively carried out in public sector research centers.
Multinational companies (MNCs) are conducting business but R&D activities of these
MNCs in Pakistan are negligible, if any.
The principle aim of the present paper is to provide an over view of GM crops
research in the country and various related policy and legislatives issues which are
essential to realize the vast potential of this new emerging technology for agriculture
sector in Pakistan. For background information and national status of agricultural
biotechnology in Pakistan, readers are requested to consult already published reviews on
this topic (Broerse, 1990; Masood, 1995; Desalvia, 1997; Khan, 1997; Khan & Afzal,
1997; Zafar, 1997 and Zafar, 2002, Khalid, et al 2003).
The vision of national biotech. Policy is to harness the potential of Biotechnology
as a key contributor to the development of Pakistan. In this context, Agricultural
Biotechnology strategies have been proposed under several focus areas. Ministry of
Science & Technology has prepared an action plan for promotion of biotechnology
research and development. Biotechnology has been declared among the top priority areas.
The projects have been launched to improve the existing research in agriculture,
livestock, and medical sectors at universities and R&D organizations. Active scientists
will be provided funds for taking part in innovative aspects of research in biotechnology
and to utilize the revolutionary findings in this field to strengthen existing technologies.
GM for Crops in Pakistan
For the improvement of agronomically important traits, plant breeding generally
recombines characters present in different parental lines of cultivated species or their wild
relatives. Conventional breeding programmes generally reach this goal by generating a
segregating population after a cross and then screening the phenotypes of pooled or
individual plants for the presence of the desirable trait. This is followed by a time
consuming and costly process of repeated backcrossing, selfing and testing.
Biotechnology plays a remarkable role by promoting the efficiency of crop improvement
and it presents a considerable potential for food production and poverty reduction.
In Pakistan, crop improvement efforts using modern technology started as early as
1985 at CEMB, Punjab University, Lahore. Later on, genetic engineering of plants was
also initiated in Plant Biotechnology Division of NIBGE in 1992. Except these two
centers genetic engineering is not done anywhere in the country. Most of the activities on
rice and cotton which are among the top five crops of Pakistan. Tomato and potato have
recently been taken up. Research on rice is mainly due to generous support by
Rockefeller Foundation Programme on Rice Biotechnology while cotton was in focus by
US$ 5 million loan of Asian Development Bank to ministry of food,agriculture and live
stock (MINFAL,GOP) for cotton sector. Although transgenic plants have been obtained
by these two centers ( Table1), Although facility for field evaluation is there but not fully
exploited due to absence of biosafety rules. Further, delay and uncertainty is expected
due to actual performance of genetically engineered crop in the field and difficulties to
protect it from further use by various seed agencies (public and private). The present
research efforts and its potential contribution are hard to assess. Even according to the
95
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most optimistic estimates, it will be at least 2-3 years before plants with desired traits can
be produced and used in breeding programmes. Moreover, no transgenic plant is so far
released in the country either developed through local efforts or imported from developed
countries.
Policy and Legislative Framework of Biosafety of GM Crops
Biotechnology has been viewed by many political leaders. Policy-makers and
leading scientists in developing countries as a priority for nearly a decade. Nevertheless,
the development of biosafety regulations has been slow. Pakistan still lacks regulatory
mechanisms today. In Pakistan a draft has been prepared by NIBGE and submitted to the
Ministry of Environment, Government of Pakistan. There is an urgent need t develop
biosafety capacity and regulatory mechanisms and to have these in place.
Table:1 Status of transgenic crop development in Pakistan
Crop
Cotton
Trait
Insect Resistance with Bt gene
Virus (CLCuV) resistance with Tr AC 1 gene
Virus (CLCuV) resistance with RNA interference (RNAi)
Rice
Bacterial blight resistance with Xa21 gene
Salt tolerance with Yeast and Arabidopsis Na+/H+
antiporter genes
Tomato
Virus (TLCV) resistance through RNAi
Male sterility through RNAi
Sugarcane Insect resistance with Cry gene
Tobacco
Insect (Helicoverpa armigera and Heliothis vericens)
resistance with synthetic spider venom gene
Salt tolerance with Yeast and Arabidopsis Na+/H+
antiporter genes
Salt tolerance with ArDH chloroplast transformation
Chickpea Drought and Salt tolerance with Yeast and Arabidopsis
Na+/H+ antiporter genes
Status
Field Trials
Field Trials
Experimental
Experimental
Experimental
Experimental
Experimental
Experimental
Experimental
Experimental
Experimental
Experimental
The 18 countries including Pakistan that participated in the UNEP project to develop
national biosafety frameworks were chosen because of their different sizes, geography
and geographical locations, and level of socioeconomic development. These countries
were at very different stages in the development of biotechnology applications. They
were expected to start in April 1998 to prepare a national biosafety framework using the
UNEP International Technical Guidelines for biosafety as a guide. Organizers hoped that
this would result in a harmonized approach to risk assessment and risk management of
LMOs within both the individual countries and within the regions. Pakistan was unable to
start the program and eventually withdrew. Pakistan is not included in the second round
of UNDP/GEF programme of capacity building.
Pakistan is signatory to WTO, CBD and cartagena Protocol. However, several ministries/
organization are dealing with various issues with n apparent coordination. Under TRIPS
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agreement all developing countries must have to provide intellectual Property Rights by
January 1, 2000 for implementation. From the date developing countries have an
additional five years to extend patent protection over products in those areas of
technology for which they offered no protection when the WTO Agreement entered into
force. The least developed counties have a total of ten years within which they must
implement TRIPS.
Following legislation under trips agreement have been promulgated in Pakistan:
1. Patents ordinance, 2000: Ministry of Industries & Production, GOP
2. Trademarks ordinance, 2001: Ministry of Commerce, GOP
3. The Copyrights ordinance 2000: Ministry of Education, GOP
4. Industrial designs ordinance, 2000: Ministry of Industries & Production, GOP
5. Registration of layout designs of integrated circuits ordinance, 2000: Ministry of
Industries & Production, GOP
Following legislation under trips agreement are passing though discussion, evaluation
and analysis etc.
a) Plant breeders rights act 2002: Ministry of Food, Agriculture & Livestock
b) Geographical indications for Goods: Ministry of Commerce.
Implementation of the Biosafety Framework/ Procedures
At present various ministries in Pakistan are handling issues of WTO (ministry of
commerce) Geographical Indications (M/o Commerce), TRIPS (Pakistan Patent Office
M/o Industries), Copyrights (M/o of Education) CBD, Biosafety Guidelines, Cartagena
Protocols (M/o Environment), and Plant Breeder Rights (M/o Food, Agriculture &
Livestock. Several R&D organizations and universities have established “WTO Cells”.
Many NGOs (Action Aid, Oxfam SDIP, SUNGI etc)) are also actively involved in
raising several issues related to biotechnology, GM crops and globalization.
Except Biosafety Guideline and Plant Breeders Rights almost all the policy/legislative
issues have been implemented. National Biosafety Guideline have been prepared by
national/ international experts, NGOs and UNEP/GEF consultant (Dr. Julian Kinderlerer,
UK). This document was presented to ministry of Environment in January’ 2000.
Enactment of these biosafety guideline is still pending. There is not yet coherent national
policy or plan to co-ordinate all these efforts
Training Needs
There are 44 public sector universities including 4 Agriculture universities in the country.
Several universities are awarding B.Sc (hons), M.Sc, M.Phil and Ph.D degrees in
Biotechnology. Currently, 26 institutes/units of biotechnology are operative in the
country. There are variations in terms of infrastructure, expertise and resources. Few of
them (CEMB, NIBGE, KIBGE, HEJ) are very well equipped and conducting R & D
work of international standard.
Present emphasis is on R & D activities for the development GMOs. However, following
areas are completely ignored:
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• Gene escape/ contamination-Field studies
• Evaluation of GM crops for Biosafety Guideline (toxicology, allergy tests, gene escape
etc).
There is an urgent need to organize training workshops for policy makers, legislatiors and
lawyers about WTO, CBD and Cartagena protocols.
Pakistan is a participatory country of FAO project for Capacity building on biosafety of
GM crops in Asia, through this project a scientist has also been trained in GMO testing.
Pakistan has developed a good facility of GMO testing at NIBGE. This institute provides
the facility of GMO tests through ELISA/ Immunostrip Test and Qualitative Detection
through PCR for 35S promotor, Nos terminator, Npt11, Hygromycin, GFP, BAR, Cry
1Ab, Cry 1Ac and Cry 2Ab. Types of samples tested for GMOs include wheat grains,
cotton seed, maize starch. Morover, NIBGE is ISO-9001-2000 certified institute. NIBGE
has a plan for national training cources for capacity building, public awarenessrisk,
assessment and risk management. Currently for the detection of GMOs NIBGE-FAO
Training course/ workshop is being arranged at NIBGE in Pakistan, in April 2004, under
FAO project for Capacity Building in Biosafety of GM Crops in Asia
(GCP/RAS/185/JPN).
Research Needs:
R& D activities in Pakistan reached to the level of production of GM crops of cotton,
rice, tomato,potato and sugarcane for various traits (Biotic/Abiotic resistance). Excellent
facilities are available in few centres and foreign qualified and well experienced
expertise, though limited but available in these centres. Thus development of GM crops
has become almost a routine activity particularly for cotton, rice potato and tomato. Now
facilities are to develop for GMO testing from different food, highly processed food
material and feed through quantitative real time PCR.
There is a strong need to conduct research in the following areas.
1.
Functional genomics for search of new genes
2.
Availability of novel genes is one of the biggest impediments of progress
of genetic engineering. The delivery of any gene promoters or any other factor to
develop novel plant transformation system are blocked due to patents or are
available at high cost. Various strict legal issues are also involved. A national
depository be made as research in genetic engineering is being done in the country
exclusively in public sector. Success in this area largely depends on the national
developments in Genomics.
3.
Chloroplast transformation is a recent emerging area which is biosafe (can
not be transferred through pollens), site directed and highly efficient in terms of
quantity of desired product. This technology may be given priority status
4. Biopharming is another upcoming area where drugs and therapeutic vaccines have
been planned to be developed in plants through genetic engineering. This is
another priority area for developing countries like of Pakistan.
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Asia Bionet
For enhancing coordination and information flow among various stake holders a regular
and effective coordination forum is essential. Establishment of “Asia Bionet” will allow
developing countries like Pakistan to benefit from the experiences of those countries who
have already implemented the legislation/ guidelines related to IPR, TRIPS, CBD and
Biosafety guideline.
1. Pakistan can send trainees to member countries of Asia Bionet to learn
policy/legislative/ research issues related to GM crops.
2. Asia Bionet can act as a focal point for Capacity Building in member states.
3. Experts from the member states of Asia Bionet could visit Pakistan for awareness
campaign, training course /workshop and expert assignments.
Pakistan is struggling for improving the existing conditions of GM crops in the country
through the collaborative research work and projects with other countries in Asia through
FAO project, capacity building in biosafety of GM crops in Asia.
Bibliography
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Da Silva, E.A (1997) Biotechnology in the Islamic World. Nature/Biotechnology
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Khalid, A. M., Y. Zafar, M. A. Ghauri and M. D. Idrees. 2003. Activity report, National
Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Khan, M.K (1997) Science and Technology for National Development: Fifty Years of
Science and Technology in Pakistan. Pakistan Science Foundation, (PSF), Islamabad.
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Masood, E (1995) Science in Pakistan: Imprisoning the Beams of the Sun. Nature 376
(24th August) : 631-638
Zafar, Y. (1997) Status of Biotechnology in Agriculture. In Expert Group Meeting on
Strategies for the Development and Application of Biotechnology for Economic Growth.
Nov.12-13, 1997, Pakistan Council for Science & Technology (PCST), Islamabad. Pp:
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Plant DNA Extraction
Muhammad Asif
Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
Basic principle of DNA extraction
The basic principle of DNA extraction consists of releasing the DNA present in the
matrix removal of polysaccharides (pectin, cellulose, hemi-cellulose, starch, etc.), RNA
and proteins. Further, concurrently or subsequently, purifying the DNA from PCR
inhibitions. Purification can be achieved, by fractionated precipitation, using solvents
such as phenol, chloroform, ethanol, isopropanol, and/or by adsorption on solid matrices
(anion exchange resin, silica or glass gel, diatomaceous earth, membranes, etc.). In order
to obtain a good quality and purified DNA, it is advised, where relevant, to remove:
Polysaccharides (pectin, celluose, hemi-cellulose, starch, etc.) with depolymerases
(pectinase, cellulase, hemi-cellulase, a-amylase, etc) before precipitation of the DNA;
RNA and/or proteins by an approprite treatment, such as enzymatic treatment; Lipid
fractions by initial treatment with enzyme solutions or solvents (e.g. n-hexane) and Salts
(e.g. from the extraction/lysis buffer, from the precipitation step) otherwise these interfere
with the subsequent analysis.
DNA is resuspended in water in a buffer solution, which prevents DNA from
degradation. The DNA extracted shall be stored under such conditions that the stability is
ensured to perform the subsequent analyses. Repeated freezing and thawing of DNA
solutions should be avoided.
DNA Extraction Methods
In order to perform DNA based GMO detection, DNA must be extracted first. Several
methods of DNA extraction are available and many of them have been evaluated for their
applicability to GMO detection in plant material and plant derived foods. But only two
are widely used: the CTAB-method and the Wizard method, employing DNA-binding
silica resins/columns (various commercially available kits). Although the use of these
methods often results in rather low yields, the quality and purity of the DNA is
satisfactory in comparison to that obtained with other methods, e.g. alkali, Chelex100, or
ROSE which yield larger amounts of low quality DNA. Both cost effective methods
produce satisfactory DNA isolation without unacceptable DNA degradation for PCR
analysis.
Breakage of cell walls, which is usually achieved by grinding the tissue in dry ice or
liquid nitrogen; Disruption of cell membranes by a detergent (e.g. CTAB or SDS) which
is (besides EDTA and a buffering salt like Tris-HCl) a necessary component of any DNA
extraction buffer; Inactivation of endogenous nucleases by the addition of detergents and
EDTA, a chelator of Mg2+, which is an obligatory co- factor of many enzymes; Addition
of proteinase K for inactivation and degradation of proteins, particularly in protocols
using DNA-binding silica columns; Separation of inhibitory polysaccharides from DNA
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through differential solubilisation in solutions containing CTAB; Separation of
hydrophobic cell constituents from DNA, e.g. lipids and polyphenols by extraction with
an organic solvent like chloroform; Separation from the detergent and concentration of
DNA by alcohol/salt precipitation. Alternatively, the separation of DNA from other cell
components can be achieved via purification on a DNA-binding silica column.
The CTAB method was originally described by Murray and Thompson (1980). They
were able to extract from plants pure DNA of high molecular weight, using CTAB as a
detergent in the DNA extraction buffer. It appears to be an efficient method for a wide
range of plant materials and plant- derived foods and it provides a good separation of
DNA from polysaccharides.
DNA-binding silica columns have proven to be suitable for extraction of good quality
DNA. However, it has been reported that polysaccharides tend to bind to silica columns
affecting the efficiency of the separation.
Extraction of DNA from complex matrixes or food products is not always successful.
Failures in extracting detectable DNA levels have so far been reported for soybean sauce,
refined soybean oil and distilled ethanol produced from GM potatoes. Pauli and coworkers (2000) were able to extract detectable DNA levels from a large variety of food
products and processing stages, but did not succeed in extracting it from refined sugar
and oil.
CTAB Method For DNA Extraction
The method consists of a lysis step (thermal lysis in the presence of CTAB) followed by
several extraction steps in order to remove contaminants such as polysaccharides and
proteins.
For some matrices it is helpful to perform different enzymatic steps. So, alpha-amylase is
added to the lysis buffer to digest the starches in case of amylaceous matrices. Treatment
of samples with proteinase K is necessary in a variety of matrices to eliminate proteins.
Also treatment with RNase is usually recommended for those matrices where RNA coprecipitation may disturb the subsequent analytical test.
The salt concentration during the extraction steps is very important for the removal of the
contaminants, since a CTAB-nucleic precipitate will occur if the salt concentration drops
below about 0.5 mol/l at room temperature and / or if the temperature drops below 16oC.
By increasing the salt concentration (e.g. addition of sodium chloride) the removal of
denatured proteins and polysaccharides complexed to CTAB is achieved, while the
nucleic acids are solubilised. Chloroform is used to further separate the nucleic acids
from CTAB and polysaccharides/proteins complexes. Finally isopropanol precipitation
and ethanol washing purify the nucleic acids.
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The following is the procedure (modified CTAB method) for DNA isolation from
different crop plants and foodstuffs:
1. Turn on the water bath and set at 65oC and preheat 2XCTAB with 1% 2mercaptoethanol.
2. Pre-cool the autoclaved pestel and mortar with liquid nitrogen.
3. Cut four to five young leaves, wash with distilled water, blot dry and grind into a
very fine powder in liquid nitrogen.
4. Transfer this powder to a 50 ml falcon tube.
5. Add 15 ml of hot (65oC) 2XCTAB to the tube before the frozen powder start
thawing.
6. Mix gently by inverting the tube for several times and incubate at 65oC for half an
hour.
7. Add 15 ml of chloroform/isoamylalcohol (24:1). Mix gently by inverting the tube
to form an emulsion.
8. Centrifuge for 10 minutes at 9000 rpm.
9. Transfer the supernatant solution (the top aqueous phase) to a new 50 ml falcon
tube and discard the remaining chloroform phase.
10. Repeat the steps 7 to 9.
11. Add 0.6 volumes chilled 2-propanol to precipitate the DNA.
12. Centrifuge at 9000 rpm for 5 minutes and discard the supernatant solution.
13. Take the pellet and wash it twice or thrice with 70% ethanol
14. Air dry the pellet and resuspend in 0.5 ml 0.1X TE buffer or dH2O.
15. Transfer the suspension into an eppendorf and then add 5 ul of RNase and
incubate for one hour at 37oC.
16. Add equal volume of chloroform/isoamylalcohol (24:1) and mix gently.
17. Spin for 10 minutes at 13000 rpm in the microcentrifuge and take the supernatant
in a new eppendorf.
18. Add 1/10th 3M NaCl and mix gently.
19. Precipitate the DNA with chilled absolute ethanol (2 volumes).
20. Spin at 13000 rpm for 10 minutes, discard the supernatant and wash the pellet
with 70% ethanol.
21. Air dry the pellet and resuspend in 0.1X TE buffer or dH2O.
22. Measure the concentration of the DNA by loading in a 0.8% agarose gel with
DNA standards or by DyNA Quant 200 fluorometer.
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DNA Quantification
Concentration and Quality of DNA
Estimate the quantity and quality of DNA extraction by spectrophotometer and agarose
gel electrophoresis method. Measurement of DNA concentration can be done by
comparing DNA with the standard DNA on 0.8% agarose gel electrophoresis and
estimation of the absorbance of DNA by spectrophotometer at 260 nm. The quality of
DNA can be estimated by A260/A280 ratio. The A260/280 higher than 2.0 generally
indicates RNA contamination. A260/A280 ratio lower than 1.8 normally indicates protein
contamination during extraction process. Good quality DNA should give the A260/A280
in the range of 1.8-2.0. Concentration of DNA can be quantified by fluorometer DyNA
QuantTM 200 (SanFrancisco, USA).
Solutions for DyNA Quant TM 200 Flourometer:
Hoechest dye stock solution:
(10ml, 1 mg/ml Hoechest H 33258)
Add 10 ml distilled water to 10 mg H 33258. Do not filter. Store at 4C for up to six
months in an amber bottle.
10X TNE (Tris-NaCl-EDTA) buffer:
(1000 mL, buffer stock solution)
012.11g
003.72g
116.89g
Tris
EDTA Na, 2H2O
NaCl
100mM
10mM
0.2M
Dissolve in about 800 mL distilled water. Adjust pH to 7.4 with concentrated HCl. Add
distilled water to 1000 mL. Filter before use (0.45uM). Store at 4C for up to three
months.
Assay solution high range B
(100 to 5000 ng/mL final concentration)
1µg/mL H 33258 in 1x TNE
(0.2 M NaCl, 10mM Tris-Cl, 1mM EDTA pH 7.4)
H 33258 stock solution
100.0 µ L
10X TNE buffer
10.0 mL
Distilled filtered water
90.0 mL
Keep assay solution B at room temperature. Prepare fresh daily. Do not filter once dye in
added.
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Agarose Gel Electrophoresis
Check the quality and concentration of DNA on 0.8% (w/v) agarose gel by comparing
them with standard DNA.
a)
Preparing the gel
Add 0.8gm of agarose in flask containing 100ml electrophoresis buffer (TBE / TAE
buffer).
Melt the agarose in a microwave oven for 5 minutes and swirl to ensure even mixing.
Cool down the melted agarose under room temperature or by keeping it under tap water
with constant shaking.
Add 2µl ethidium bromide (0.5 µg/ml) to flask containing melted agarose.
Seal the open ends of gel casting tray with adhesive tape.
Pour in the melted agarose and insert the gel comb, making sure that no bubbles are
trapped underneath the comb and all bubbles on the surface of the agarose are removed
using microtip before the gel sets.
Keep it under room temperature for 15-20 minutes for solidification.
b)
Loading and running the gel
Place the gel casting tray containing solidified gel in the electrophoresis tank.
Add sufficient electrophoresis buffer to cover the gel. Make sure no air bubbles are
trapped within the wells.
Add 5µl 6X loading dye (Bromophenol Blue) in DNA samples.
Load the samples into wells with a micropipet.
Use 1 Kb ladder (GiBCO BRL) as a size marker.
Set the voltage to the desired level to start the electrophoresis and turn off the power
supply when the Bromophenol Blue dye has migrated well for the separation of DNA
fragments.
C)
Gel documentation
Visualize the amplification products by placing gel on a transilluminator and take
photograph using Eagle still video system.
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Buffers and Reagents:
2X CTAB (Hexadecyle trimethyle ammonium bromide):
2% CTAB, (Sigma Cat. # C-0636).
100mM Tris base (M.W. 121.1, pH 8.0)
20mM EDTA (M.W. 372.24, pH 8.0)
1.4 M NaCl (M.W.58.44)
1% PVP (Polyvinylpyrrolidone)
0.1X TE buffer:
1.0 mM Tris base (pH 8.0)
0.1 mM EDTA (pH 8.0).
5X TBE (Tris Borate EDTA for Gel Electrophoresis):
Tris base
54.0 gm
Boric acid
27.5 gm
0.5 M EDTA
20.0 ml
dH2O
X ml to make total 1000 ml buffer
RNase Stock solution:
The solid enzyme (RNase A) is dissolved in 10 mM Tris. Cl (pH 7.5) and 15mM NaCl.
The solution should be heated at or near boiling (in a water bath) for at least 15 minutes
to get rid of DNases and then cooled slowly to room temperature (Sambrook et al., 1989).
6X Loading dye:
Sucrose
40g
Bromophenol blue
0.25g
5mM EDTA
1ml
10mM Tris (pH 7.4) 1ml
Make the volume up to 100ml by adding distilled water.
1Kb DNA Ladder
1Kb ladder (stock solution)
Loading dye (6X)
dH2O
10 µl
15 µl
200µl
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Basic silica method for DNA Extraction
The method consists of a lysis step (thermal lysis in presence of sodium dodecyl sulfate
in a buffered solution) following by a purification step using silica resin, in the presence
of the chaotropic reagent guanidine-hydrochloride. The principle of the method is binding
of nucleic acids to silica under low water activity due to entrophic effect. Contaminants
are washed away form the resin by isopropanol, while DNA stays attached. A final
elution step with a low salt buffer solution permits DNA recovery.
Extraction procedure (silica method)
1. Weigh 200 mg to 300 mg of milled or crushed material into a vial.
2. Add 2 ml of extraction buffer and 20 ul proteinase-K solution.
3. Incubate for 1 h to 5h in the oven at oC. During incubation time, shake the
samples vigorously.
4. Centrifuge for 15min at 2,000 g
5. Transfer 550 ul of supernatant into a new tube.
6. Add 2 ul Rnase solution for min at 37 oC (this hydrolyzing RNA step is advised
before the silica binding otherwise the hydrolyses RNA and the resulting
nucleotides may interfere with subsequent UV=spectrometry measurements.
7. Add 55 ul of the Guanidine HCl 5 mol/l solution plus 100 ul of the silica
suspension to the supernatant.
8. Mix carefully several times.
9. Leave the tuve on the bench for approximately 1 min.
10. Centrifuge for 2 min at approximately 800g. Discard the supernatant and add 500
ul of the isopropanol solution, close the tube and mix, possibly with the aid or the
vortex mixer, to completely resuspend the pellet.
11. Centrifuge for 2 min at approximately 1500 g. Discard the supernatant and add
dry the pellet
12. Add 100 ul of TE-buffer solution. Mix carefully in order to dissolve the pellet.
13. Incubate the samples at 60 oC for 5 min
14. Centrifuge for 5 min at 2000g
15. Transfer 80% of the supernatant amount to a new tube. Be careful not to transfer
any silica particles. because of their inhibiting activity an enzymes (e.g. Taq
polymerase)
16. Treat the transfer supernatant with 2 ul of Rnase solution for 1 h at 37 oCor
overnight at room temperature.
Reagent for Silica method:
TNE-SDS extraction buffer: NaCL= 0.150 mol/l, Tris =0.002 EDTA (disodium
slat)=0.002 mol/l SDS=10g/l. Adjust pH to 8.0 with HCl or NaOH and autoclave before
adding SDS. PBS-buffer solution: Nacl=0.157 mol/l, KCl=0.0027mol/l, Na2HpO5 =
0.0018 mol/l. Adjust pH to 7.5 with HCl. Rnase-A solution: 10 mg/ml. Store in aliquots
at –20 oC, avoid repeated freezing and thawing.
Proteinase-K solution: Proteinase-K=20 mg/ml dissolved in sterile water. Do not
autoclave. Store at 20 0C, avoid repeated freezing and thawing.
Guanidine-HCl solution: guanidine hydrochloride = 5mol/l
Guanidine-HCl solution: guanidine hydrochloride = 6mol/l
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Silica suspension: Weigh 5 g of silica into 50 ml PVS-buffer. Mix well and let everything
to settle down for 2h. Remove the supernatant by aspiration with a pipette. Add another
50 ml of the PBS-buffer, mix well and let to settle sown again for 2 h. Remove the
supernatant by aspiratio. Centrifuge 2 min at 2,000g. Disgard the remaining supernatant.
Resuspend the pellet up to 50 ml in Gaunidine-HCl solution 6 molar Use within 2-5
months. Mix well before use.
Isopropanol solution: isopropanol 80 %
TE-buffer solution: Tris = 0.01 mol/l, EDTA (disodium salt) = 0.001 mol/ll Adjust the
pH to 8.0 with HCl or NaOH.
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DNA Extraction using GENESCAN Kit
DNA extraction
The extraction of DNA form the sample is a crucial step and in order to guarantee
optimal quantity and purity a variety of different methods are used depending on the
sample matrix. For most products the DNA can be extracted by using the “standard
method”.
However, in order to extract DNA form lecithin (or fats, fatty acids, oil etc.) a special
lysis buffer is also available and in order to extract DNA from foodstuff containing cocoa
a modified version of the standard method is used. Furthermore we offer the GENESpin
DNA extraction kit with silica membrane spin columns, which was specially developed
for food and feed applications and which provides DNA at high yields and free of PCR
inhibitors. GENESpin DNA is suitable for most PCR applications including real-time
PCR.
Each sample should be analyzed in duplicate. We recommend and additional extraction
control for each analysis, i.e. a complete DNA extraction and PCR without sample
material. Contamination of regents or equipment with amplicons can be detected this
way.
Standard method of DNA Extraction:
1. Homogenize the sample mechanically (use appropriate blender depending on the
type of sample). For analysis of beans or whole meal homogenize at least 100 g of
material.
2. Place 2 g of the homogenized sample into a 15 ml reaction tube and mix with 10
ml of the lysis buffer (if the solution is too viscous increase volume of lysis buffer
to 20 ml) and add 10 µl Proteinase K solution).
3. Incubate for 2 h at 60 oC under constant shaking.
4. Centrifuge for 10 min at >4000 x g.
5. Transfer 800 ul from the spernatant into a fresh 1,5 ml reaction tube.
6. Add 600 ul chloroform an mix by vortexing.
7. Centrifuge for 15 min at >10,000 x g to separate phases.
8. Transfer 600 µl from the aqueous supernatant into a fresh 1,5 ml reaction tube.
9. (Extraction control: add 2 µl glycogen solution in order to precipitate any trace of
DNA)
10. Add 480 µl isopropanol and mix thoroughly. Incubate for 30 min (in order to
precipitate the DNA).
11. Centrifuge for 15 min at > 10000 x g to pellet DNA.
12. Discard the supernatant.
13. Add 500 µl75% EtOH to the DNA pellet and mix by vortexing.
14. Centrifuge for 5 min at > 10000 x g.
15. Discard the wuprnatant completely.
16. Centrifuge again for 1 min at > 10000 x g. Carefully remove all of the remaining
supernatant using a micropipette and discard.
17. Dissolve the DNA pellet (may not always be visible) in 100 µl ddh2O (sterile).
Take 10 µl of the DNA solution for the estimation of the DNA amount by gel
electrophoresis (see 5.4).
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for PCR reactions 10-50 ng DNA should sufficient. If no DNA is visible in the agarose
gel (less than 5 ng/µl), take 5 µl of the extracted DNA solution for the PCR without
further dilution.
Suggested Readings:
Ahmed, F. E. 2004. Testing of genetically modified organisms in foods. The Haworth
Press, Inc. USA.
Anklam E, Gadani F, Heinze P, Pijnenburg H, Van den Eede G. 2002. Eur Food Res
Technol 214:
3-26
Anonymous. 2004. A recommended procedure for DNA extraction from plant tissues.
Monsanto Biotech. Regulatory Sci. 1-5.
Anonymous. 2003. Genetically modified organisms (GMOs) detection of food samples.
FAO regional training workshop on GMO detection. 27-31 Oct. Thailand.
Bonfini L., P. Heinze, S. Kay, E. G. Van. 2001. Review of GMO detection and
quantification techniques. E C, JRC, Institute for Health and Consumer Protection, Food
Products and Consumer Goods Unit, I-21020 Ispra, Italy.
Boyle, J.S. an A.M. Lew. 1995. Trends Genet. 1: p.8.
Gasch, A. 1997. Detection of genetically modified organisms with the polymerase chain
reaction: potential problems with food matrices. In Foods Produced by Modern Genetic
Engieering (Schreiber, F.A. and Bögl, K.W., eds) 2nd Status Report, pp. 90–79, BgVVhefte.
Hellebrand, M., Nagy, M., Mörsel, J. T. 1998. Determination of DNA traces in rapeseed
oil. Z Lebensm Unters Forsch A 206: 237–242.
Hupfer C, J. Schmitz-Winnenthal and K H Engel. 1999. Lebensmittelchemie 53: 2–20
Kim, S.S., C.H. Le, J.S. Shin, Y.S. Chung and N.I. Hyung. 1997. A simple and rapid
method for isolation of high quality genomic DNA form fruit trees and conifers using
PVP. Nucleic Acid Research, Vol. 25, No. 5, 1085-1086
Melzak KA, Sherwood CS, Turner RFB, Haynes CA. 1996. J Colloid Interface Sci 181:
635–644
Meyer, R. 1994. Detection of pork in heated meat products by polymerase chain reaction.
J. AOAC Int. 77: 617–622.
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Meyer R, Jaccaud E. 1997. Detection of genetically modified soya in processed food
products: Development and validation of PCR assay for the specific detection of
glyphosate-tolerant soybeans.
In: Amado R, Battaglia R (eds) Proceedings 9th European Conference on Food
Chemistry. Authenticity and adulteration of food – The analytical approach, vol 1.
Interlaken, Switzerland 24– 26 September 1997, pp 23–28
Murray HG, Thompson WF. 1980. Nucleic Acids Res 8: 4321–4325
Pan, T. 2002. Current status and detection of genetically modified organism. J. Food
Drug Analy. 10 (4): 229-241.
Pauli, U., Liniger, M., Zimmermann, A. 1998. Detection of DNA in soybean oil. Z
Lebensm Unters Forsch 207: 264–267.
Pauli, U., Liniger, M., Zimmermann, A. and Schrott, M. 2000. Extraction and
Amplification of DNA from 55 Foodstuffs. Mitt. Lebensm. Hyg. 91: 491-501.
Pauli, U., Liniger, M. and Schrott, M. 2001. Quantitative Detection of Genetically
Modified Soybean and Maize: Method Evaluation in a Swiss Ring Trial. . Mitt. Lebensm.
Hyg. 92: 145-158.
Regers, S.O., and A.J. Bendich. 1988. Extraction of DNA from plant tissues. Plant
Molecular Biology Mannual A6: 1-10
Rogers, S. O. and Bendich, A. J. 1988. Extraction of DNA from plant tissues. Plant
Molecular Biology Manual A6: 1-10.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A Laboratory
Manual. Third ed. Cold Spring Harbor Laboratory Press, USA.
Spoth B, Strauss E. 1999. Promega Notes Magazine 73: 23–25
Tinker, N. A. 1993. Random amplified polymeric DNA and pedigree relationship in
spring barley. Theor. Appl. Genet. 85: 976–984.
Zimmermann, A., Lüthy, J., Pauli, U. 1998. Quantitative and qualitative evaluation of
nine different extraction methods for nucleic acids on soya bean food samples. Z
Lebensm Unters Forsch 207: 81–90.
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GMO Detection Methods
Muhammad Asif
Scientific Officer
National Institute for Biotechnology & Genetic Engineering (NIBGE)
P.O. Box-577, Jhang Road, Faisalabad-Pakistan
GMO Detection with ELISA Immunostrips
Mostly, different kits (agdia and Envirologix of USA) of ELISA Immunostrips are used
to determine the presence of the Bt-Cry1Ab or Bt-Cry1Ac protein in seed and leaves of
corn, cotton, and other crops. The expression of Bt-Cry1Ab or Bt-cry1Ac transgenic
protein in plants results in insect resistance. This test is suitable for testing both leaf tissue
and seed of different crops. Kit for Cry1Ab/Ac Leaf and Seed is designed to extract and
detect the presence of the Cry1Ab and Cry1Ac Bt endotoxins at the levels typically
expressed in genetically modified corn and cotton plant tissues.
How the kit works:
Each Strip has an absorbent pad at each end. The protective tape with the arrow indicates
the end of the strip to insert into the extraction tube. The sample will travel up the
membrane strip and be absorbed into the larger pad at the top of the strip. The portion of
the strip between the protective tape and the absorbent pad at the top of the strip is used
to view the reactions and results.
To extract leaf tissue:
Sandwich a section of leaf tissue between the cap and body of the Disposable Tissue
Extractor tube; snap two circular tissue punches by closing the cap. Push the leaf punches
down into the tapered bottom of the tube with the pestle. Sample identification should be
marked on the tube with a waterproof marker. Insert the pestle into the tube and grind the
tissue by rotating the pestle against the sides of the tube with twisting motions. Continue
this process for 20 to 30 seconds, or until the leaf tissue is well ground. Carefully squeeze
10 drops (0.25 ml) of Buffer into the tube containing corn leaf, or 15 drops (0.35 ml) of
Buffer tubes containing cotton leaf. Repeat the grinding step to mix tissue with Extraction
Buffer. Dispose of the pestles (do not re-use pestles on more than one sample).
To extract seed:
Crush a single corn or cotton seed (use pliers with seed in resealable bag). Transfer to an
extraction tube marked with sample identification. Carefully squeeze 20 drops (0.5 ml) of
Buffer into the tube. Alternatively, remove the dropper tip from the bottle and dispense
500 microliters of buffer into the tube, using a pipette. Close the tube cap securely and
shake the tube vigorously for 20 to 30 seconds. Allow the solid material to settle to the
bottom of the tube. Repeat the protocol for each sample to be tested, using a new tube
and pestle for each. Use caution to prevent sample-to-sample cross-contamination with
plant tissue, fluids, or disposables.
NIBGE-FAO Workshop on GMO Detection
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Interpretation of the Results:
Development of the Control Line within a few minutes indicates that the strip has
functioned properly. Any strip that does not develop a Control Line should be discarded
and the sample re-tested using another strip. If the sample extract contains Cry1Ab or
Cry1Ac endotoxin, a second line (Test Line) will develop on the membrane strip between
the Control Line and the protective tape, within 10 minutes of sample addition. The
results should be interpreted as positive for Cry1Ab or Cry1Ac endotoxin expression. If
no Test Line is observed, the results should be interpreted as negative for Cry1Ab or
Cry1Ac endotoxin.
Qualitative Detection of GMOs through PCR
PCR is an exponential reaction. In theory the detection of one single DNA target should
be possible. In practice 10 to 100 template molecules have to be present. The extreme
sensitivity requires special precautions for handling and equipment. After a successful
amplification several billion amplicons are present in the reaction tube. Each of them
might lead to a false positive result by contamination of sample materials, e.g. by
spreading in aerosols. The most important rule to avoid false positive results Separate the
different procedures spatially. Use separate rooms for sample preparation, amplification
and analysis of amplicons. Never open PCR tubes with amplicons in the sample
preparation room. Mixing of PCR components should be done in a separate room, best on
a clean bench. A simple method for decontamination is UV radiation. A further room is
necessary to analyze amplicons by gel electrophoresis. Here PCR tubes are opened and
the danger of contamination through aerosols is very high. Use filter tips for
micropipettes and Wear disposable gloves.
1)
Control Reactions
I)
Amplification of plant chloroplast-DNA
The amplification of plant chloroplast DNA indicates that DNA of sufficient quantity and
quality has been isolated from the sample. A specific DNA sequence from the plant like
cotton chloroplast-gene is amplified both form conventional plant DNA and from
genetically modified plant DNA. If both chloroplast-DNA control reactions (“double
classification”) show a positive result, DNA of sufficient quantity and quality has been
isolated. If both reactions show a negative result either no or highly degraded or damaged
DNA has been isolated. In this case reactions should be repeated with a larger amount of
DNA.
Another possible explanation for a negative result of the chloroplast-DNA control
reaction would be the presence of inhibitory substances in the matrix, which could not be
removed during DNA isolation. This can be clarified by a spiking experiment. If no
inhibitory substances are present the result will be positive. Sometimes further cleaning
steps can remove inhibitory substances. Higher dilution of the sample DNA also may
solve the problem. But there are always cases where PCR cannot be performed. In the
case of an unclear result a very small amount of extracted DNA may be the explanation.
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NIBGE-FAO Workshop on GMO Detection
This is particularly true for mixed foods, which only contain tiny amounts of plant DNA.
In these cases spiking experiments are recommended.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (CP-F and CP-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
200nM each
1U
50-100ng
PCR Profile
94oC
05
min
1 cycle
94oC
60oC
72oC
30
30
60
sec
sec
sec
40 cycles
72oC
10
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
II)
Amplification of Soybean derived components
This method describes a routine procedure for the detection of a species specific, single
copy gene sequence occurring in soybean (Glycine max). This method can be used to
assess the quality of DNA from soybean derived products and to determine that there is
sufficient DNA present for GMO analysis. A specific fragment from the soybean lectin
gene is amplified by PCR and separated by agarose gel electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (LE-F and LE-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
200nM each
1U
50-100ng
PCR Profile
94oC
05
min
1 cycle
94oC
60oC
72oC
30
30
60
sec
sec
sec
40 cycles
72oC
10
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
III)
Amplification of Maize derived components
This method describes a routine procedure for the detection of a single copy species
specific, of an endogenous gene that is zein gene sequence in maize (Zea mays). This
method can be used to assess the quality of DNA from maize derived products and to
determine whether there is sufficient DNA present for GMO analysis. Amplification of
the maize and maize derived ingredients is performed by PCR and products are separated
by agarose gel electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (ZE-F and ZE-R)
Taq DNA polymerase
Template DNA
PCR water
PCR Profile
95oC
10X
25mM
100mM each
200nM each
1U
50-100ng
3
min
1 cycle
95oC
60oC
72oC
60
60
60
sec
sec
sec
40 cycles
72oC
7
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
IV)
Amplification of cotton derived components
This is the method for the specific amplification of a cotton-specific sequence with a
76bp fragment of acp1, an endogenous cotton gene encoding a cotton fiber-specific acy1
carrier protein, using a pair of acp1 gene-specific primers. This method can be used to
assess the quality of DNA from cotton derived products and to determine whether there is
sufficient cotton DNA present for GMO analysis. Product amplified is by PCR and
separated by agarose gel electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
DNTP (dATP, dCTP, dTTP, dGTP)
Primer (acp1-F & acp1-R)
Taq polymerase
Template DNA
PCR water
PCR Profile:
95oC
1X
5mM
200µM each
150nM each
1U
200ng
5
min
1 cycle
95oC
60oC
72oC
15
60
60
sec
sec
sec
45 cycle
72oC
7
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
2)
Qualitative GMO Detection
I)
GMO Detection based on 35S Promoter
Promoters are recognition or binding sequences for RNA polymerase, which are
responsible for the expression of genes. The constitutive 35S promoter from Cauliflower
Mosaic Virus (CaMV) is frequently used in genetically modified plants so due to the
presence of 35S promoter in many genetically modified plants, this method can be
successfully used to screen for the presence of GM plant derived DNA. A specific DNA
fragment from the CaMV 35S promoter sequence is amplified by PCR and detected after
separation by agarose gel electrophoresis. For identification of the PCR product, a
verification step shall be performed.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (35S-F and 35S-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
200nM each
1U
50-100ng
PCR Profile
94oC
05
min
1 cycle
94oC
60oC
72oC
30
30
60
sec
sec
sec
40 cycles
72oC
10
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
II)
GMO Detection based on NOS Terminator
This is the method for detection of a variable copy number DNA sequence from the
Agrobacterium tumefaciens nopaline synthase (nos) terminator. Due to the presence of
nos terminator in many genetically modified plants, this method can be used to screen for
the presence of GM plants and their derived components. A specific DNA fragment from
the nos terminator sequence is amplified by PCR and identified after agarose gel
electrophoresis. Confirmation of the results by restriction analysis or sequencing is also
performed.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (nos-F and nos-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
200nM each
1U
50-100ng
PCR Profile
94oC
05
min
1 cycle
94oC
60oC
72oC
30
30
60
sec
sec
sec
40 cycles
72oC
10
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
III) GMO Detection based on npt11 gene
This is the procedure for detection of npt11 gene sequences. Due to the presence of npt11
gene for kanamycine resistance as a selectable marker in many genetically modified
plants, this method can be used to screen for the presence of GM plants and their derived
components. A specific DNA fragment from the npt11 gene sequence is amplified by
PCR and identified after agarose gel electrophoresis. Confirmation of the results by
restriction analysis or sequencing is also performed.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (kana-F and kana-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
200nM each
1U
50-100ng
PCR Profile
94oC
05
min
1 cycle
94oC
60oC
72oC
30
30
60
sec
sec
sec
40 cycles
72oC
7
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
IV)
Detection of Roundup ReadyTM soybean (RRS)
This is the method for the detection of genetically modified glyphosate resistant Roundup
ReadyTM soybean (Monsanto Co., USA) in raw or processed materials, by amplification
of a single copy DNA segment from the junction region between the CaMV 35S
promoter and the Petunia hybrida chloroplast targeting signal preceding the
Agrobacterium EPSPS sequence. A DNA fragment of the RRS-specific transgene is
amplified using two RRS-specific primers, PCR products identified after separation by
agarose gel electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (RR-F and RR-R)
Taq DNA polymerase
Template DNA
PCR water
10X
25mM
100mM each
600nM each
1.25U
200ng
PCR Profile
95oC
10
min
1 cycle
95oC
60oC
72oC
45
30
60
sec
sec
sec
40 cycles
72oC
7
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
V)
Detection of Bt-176 Maximizer Maize
This is the method for the specific amplification of a synthetic CryIA(b) gene for
qualitative or quantitative detection of Bt 176 Maximizer maize (Novartis). After
amplification reaction, PCR products are separated by agarose gel electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (CRY1Ab-F and CRY1Ab-R)
Taq DNA polymerase
Template DNA
PCR water
PCR Profile
95oC
10X
25mM
100mM each
500nM each
2.5U
200ng
3
min
1 cycle
95oC
60oC
72oC
30
60
60
sec
sec
sec
40 cycles
72oC
3
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
VI)
Detection of Bt Cotton
This is the method for the detection of genetically modified insect resistant cotton in raw
or processed materials, by amplification of a specific DNA segment from Bt Cry1Ac
gene. A DNA product from Cry1Ac gene is amplified using two specific primers. The
PCR products are identified and confirmed after separation by agarose gel
electrophoresis.
Chemicals
PCR reagents, Agarose, 0.5X TBE buffer, Ethidium bromide, 6X Loading dye, DNA
molecular weight markers, DH2O
Material and Apparatus
Thermal cycler, Micropipettes (set), filter Tips (1 - 200 ul), reaction tubes, permanent
marker, Balance, Flask, Cylinder, Microwave, Gel casting platform and combs,
Horizontal gel electrophoresis apparatus, Power supply, Gel documentation system,
Thermal paper
Polymerase Chain Reaction (PCR)
Total volume of each reaction mixture is 25µl consisting of following reagents:
PCR Buffer
MgC12
dNTPs (dATP, dCTP, dTTP, dGTP)
Primers (Cry1Ac-F and Cry1Ac-R)
Taq DNA polymerase
Template DNA
PCR water
PCR Profile
94oC
10X
25mM
100mM each
200nM each
1U
50ng
5
min
1 cycle
95oC
60oC
72oC
60
45
60
sec
sec
sec
40 cycles
72oC
10
min
1 cycle
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NIBGE-FAO Workshop on GMO Detection
Qualitative GMO Detection using GMOScreen kit (GeneScanGermany)
Preparation of the PREMaster buffer
Complete PREMaster buffer by adding Taq polymerase.
CP/A-Mater-Mix (control PCR):
PCR Reagents
CP/A PREMaster
Taq Plymerase
35S- / NOS-Master-Mix:
PCR Reagents
PREMaster 35-S-P/C
or PREMaster Nos-T/C
Taq Polymerase
1X
19,9 µl
0,1 µl (0,5 Units)
1X
19,9 µl
0,1 µl (0,5 Units)
PCR
Label all PCR reaction tubes.
Add 20 µl of the master mix
Add 5 µl of the DNA solution (10-50 ng).
Add 25 µl oil if not using thermocyclers with a heated lid.
The PCR is carried out according to the following profile.
Temperature
94 oC
94 oC
62 oC
72 oC
72 oC
Time
10 min
25 sec
30 sec 50 cycles
45 sec
3 min
The same temperature profile is used for the specific and the control reaction. It may be
necessary to optimize the PCR parameters depending on specifics of thermocycler.
Verification of the PCR amplicons
Electrophorese 20 µl of each PCR sample on a 1.2% agarose gel. Run DNA-length
standard (H.A. DNA standard III, included in the kit) in parallel. If PCR results are
positive then additional verification is done restriction analysis using EcoRV and Hinf1
for 35S and NOS respectively.
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NIBGE-FAO Workshop on GMO Detection
Quantitative Detection of GMOs using Real Time PCR
I)
Quantitative Detection of 35S CaMV promoter
This method is for the detection of a variable copy number DNA sequence form
Cauliflower Mosaic Virus (CaMV) 35S promoter in processed food matrices. Due to the
presence of CaMV 35S promoter in many genetically modified plants, this method can be
used to screen for GM plant derived DNA. Quantification of 35S CaMV promoter is
done by real-time PCR amplification and hybridization of specific internal probe
(TaqMan probe) to 35S promoter sequences.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe :
: 35S-F
: 35S-R
35S-TMP
(labeled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
5mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (35S-F & 35S-R)
300nM each
Probe (35S-TMP)
100nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
45 cycle
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NIBGE-FAO Workshop on GMO Detection
II)
Quantitative Detection of NOS Terminator
This procedure is for the detection of a variable copy number DNA sequence form the
Agrobacterium tumefaciens nopaline synthase (nos) terminator. Due to the presence of
nos terminator in many genetically modified plants, this method can be used to screen for
GM plant derived DNA. Quantification of the nos terminator is performed by real-time
PCR amplification and hybridization of a specific internal probe (TaqMan probe) to nos
terminator specific sequences.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe :
:NOS-F
:NOS-R
NOS-P (labeled with TET and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
4mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (NOS-F & NOS-R)
200nM each
Probe (NOS-P)
200nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
55oC
15
45
sec
sec
45 cycle
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NIBGE-FAO Workshop on GMO Detection
III)
Quantitative Detection of Soybean derive components
This is the method for specific amplification of an endogenous (housekeeping) soybean
gene (lectin) for quantitative detection of the soy ingredient in foodstuffs. Quantification
of the soybean and soybean derived ingredients is done by PCR amplification along with
real-time hybridization of specific internal probe (TaqMan probe) to an endogenous gene
mostly using thermal cycler ABI Prism 7700 SDS (PE Biosystems).
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
: Lectin-F
: Lectin-R
: Lectin-TMP (labelled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
5mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (Lectin-F
& Lectin-R)
900nM each
Probe (Lectin-TMP)
100nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
45 cycle
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NIBGE-FAO Workshop on GMO Detection
IV)
Quantitative Detection of Roundup Ready® soybean
This procedure is for the detection of Roundup ReadyTM specific gene construct
introduced in the soybean genome in order to quantify the relative amount of roundup
ReadyTM soy (RRS) DNA in soy ingredients of different foodstuff. The measured
fluorescence signal trespasses a set threshold value after a certain number of cycles. This
cycle number is called Ct-value. For quantification of the amount of RRS DNA is soya
DNA the RRS-specific and soya reference gene specific Ct-value are measured. The
standard curve method is used to calculate the number of RRS DNA copies relative to the
copies of the soya reference gene.
DNA fragment of the RRS-specific trans-gene is amplified using two RRS-specific
primers PCR products are measured during each cycle (real time) by means of a target
specific oligonucleotide probe labeled with two fluorescent dyes, FAM as reporter dye
and TAMRA as quencher. The 5’-nuclease activity the Taq-DNA polymerase is exploited
which results in the specific cleavage of the probe leading to increased fluorescence that
is monitored.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
: RRS-F
: RRS-R
: RRS-TMP
(labeled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
5mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (RRS-F & RRS-R)
900nM each
Probe (RRS-TMP)
100nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
45 cycle
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NIBGE-FAO Workshop on GMO Detection
V)
Quantitative Detection of Maize derived components
This method is for the specific amplification of an endogenous (housekeeping) maize
gene (zein) for quantitative detection of the ingredient maize in foodstuffs. Quantification
of the maize and maize derived ingredients is achieved by PCR amplification and realtime hybridization to specific internal probe (TaqMan probe) of an endogenous gene.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
: Zetm-F
: Zetm-R
: Zetmp-TMP (labelled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
2.5U
PCR Buffer `
1X
MgC12
6.5mM
DNTP (dATP, dCTP, dTTP, dGTP)
400µM each
Primer (Zetm-F & Zetm-R)
25nM each
Probe (Zetmp-TMP)
200nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
50 cycle
129
NIBGE-FAO Workshop on GMO Detection
VI)
Quantitative Detection of Bt176 Maximizer maize
This is the method for the specific amplification of a synthetic CryIAb gene for
quantitative detection of Bt176 Maximizer maize in foodstuff. Quantification is
performed by PCR amplification and ral-time hybridization of a specific internal probe to
the synthetic CryIAb gene, inserted in transgenic Bt176 Maximizer maize.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
:Crytm-F
:Crytm-R
:Crytm–TMP (labeled with TET and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
6.5mM
DNTP (dATP, dCTP, dTTP, dGTP)
400µM each
Primer (Crym-F & Crym-R)
100nM each
Probe (Crym-TMP)
200nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
50 cycle
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NIBGE-FAO Workshop on GMO Detection
VII) Quantitative Detection of cotton derived components
This is the method for the specific amplification of a cotton-specific sequence with a 76bp fragment of acp1, an endogenous cotton gene encoding a cotton fiber-specific acy1
carrier protein, using a pair of acp1 gene-specific primers and an acp1 gene-specific
probe labeled with FAM and TAMRA for real time quantification.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
:acp1-F
:acp1-R
:acp1–P
(labeled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1U
PCR Buffer `
1X
MgC12
5mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (acp1-F & acp1-R)
150nM each
Probe (acp1-P)
50nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
60oC
15
60
sec
sec
45 cycle
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NIBGE-FAO Workshop on GMO Detection
VII) Quantitative Detection of Boallgard® cotton 531
This method describes an event-specific real-time quantitative TaqMan® PCR procedure
for determination of the content of Boallgard® cotton 531 DNA to total cotton DNA in a
sample. For specific detection of 531 cotton genomic DNA, a 72-bp fragment of the
region that spans the 5’ insert-to plant junction in cotton 531 is amplified using two
specific primers. PCR products are measured during each cycle (real-time) by means of a
target-specific oligonucleotide probe labeled with two fluorescent dyes: FAM (6caroxy1-fluorescein) as a reporter dye at its 5’-end and TAMRA (tetramethy1-6carboxyrhodamine) as a quencher dye at its 3’-end. The 5’-nuclease activity of the Taq
DNA polymerase is exploited which results in the specific cleavage of the probe, leading
to increased fluorescence, which is then monitored.
Oligonucleotides
Forward primer
Reverse primer
TaqMan prpbe
:531-F
:531-R
:531–P (labeled with FAM and TAMRA)
Amplification reaction mixture per reaction tube:
Template DNA
200ng
Taq polymerase
1.25U
PCR Buffer `
1X
MgC12
5mM
DNTP (dATP, dCTP, dTTP, dGTP)
200µM each
Primer (531-F & 531-R)
150nM each
Probe (531-P)
50nM
PCR water
Reaction condition
Optimized for ABI Prism 7700 Sequence Detection System (PE Applied Biosystems)
95oC
10
min
1 cycle
95oC
55oC
15
60
sec
sec
45 cycle
NIBGE-FAO Workshop on GMO Detection
132
Suggested Readings:
Ahmed, F. E. 2004. Testing of genetically modified organisms in foods. The Haworth
Press, Inc. USA.
Anonymous. 2004. A recommended procedure for real time quantitative TaqMan PCR
for bollgard cotton 531. Monsanto Biotech. Regulatory Sci. 1-7.
Anonymous. 2003. Genetically modified organisms (GMOs) detection of food samples.
FAO regional training workshop on GMO detection. 27-31 Oct. Thailand.
Carolyn, D. H., K. Angus and I. J. Bruce. 1999. PCR detection for genetically modified
soya and maize in foodstuffs. Molecular Breeding 5: 579-586.
Chiueh L., Y. Chen, D. Yang-Chih and Shih. 2002. Study on the Detection Method of Six
Varieties of Genetically Modified maize and Processed Foods. J. Food & Drug Analy.
Vol. 10(1): 25-33.
Codex Ad Hoc Intergovernmental Task Force Of Foods Derived From Biotechnology:
Germany Federal Institute for Health Protection of Consumers, European commission
Joint Research Centre (JRC).
Meyer, R. and Jaccaud, E. 1997. Detection of genetically modified soya in processed
food products: development and validation of a PCR assay for the specific detection of
Glyphosate-Tolerant Soybeans. Euro Food Chem. 1: 23-28.
Pan, T. 2002. Current status and detection of genetically modified organism. J. Food &
Drug Analy. 10: 229-241.
Pauli, U., Liniger, M., Zimmermann, A. and Schrott, M. 2000. Extraction and
Amplification of DNA from 55 Foodstuffs. Mitt. Lebensm. Hyg. 91: 491-501.
Pauli, U., Liniger, M. and Schrott, M. 2001. Quantitative Detection of Genetically
Modified Soybean and Maize: Method Evaluation in a Swiss Ring Trial. . Mitt. Lebensm.
Hyg. 92: 145-158.
Shirai, Momma, K., Ozawa, S., Hashimoto, W., Kito, M., Utsumi and Murata, K. 1998.
Safety Assessment of Genetically Engineered Food: Detection and Monitoring
Glyphosate-Tolerant Soybeans. Biotechnolo. Biochem.. 62(7): 1461-1464.
Vollenhofer, S., Burg, K., Schmidt, J. and Kroath, H. 1999. Genetically Modified
Organisms in Food-Screening and Specific Detection by Polymerase Chain Reaction. J.
Agric. Food Chem. 47: 5038-5043.
Wurz, A., Bluth, A., Zeltz, P., Pfeifer, C. and Willmund R. 1999. Quantitative analysis of
genetically modified organisms (GMO) in processed food by PCR-based methods. Food
Control. 10: 385-389.
NIBGE-FAO Workshop on GMO Detection
CARTAGENA
PROTOCOL
ON
BIOSAFETY
TO THE
CONVENTION
ON
BIOLOGICAL
DIVERSITY
TEXT AND ANNEXES
133
NIBGE-FAO Workshop on GMO Detection
134
Introduction
The Convention on Biological Diversity was finalized in Nairobi in May 1992 and opened for
signature at the United Nations Conference on Environment and Development (UNCED) in
Rio de Janeiro on 5 June 1992. It entered into force on 29 December 1993. Today, the
Convention is the main international instrument for addressing biodiversity issues. It provides
a comprehensive and holistic approach to the conservation of biological diversity, the
sustainable use of natural resources and the fair and equitable sharing of benefits deriving from
the use of genetic resources.
Biosafety is one of the issues addressed by the Convention. This concept refers to the need to
protect human health and the environment from the possible adverse effects of the products of
modern biotechnology. At the same time, modern biotechnology is recognized as having a
great potential for the promotion of human well-being, particularly in meeting critical needs for
food, agriculture and health care. The Convention clearly recognizes these twin aspects of
modern biotechnology. On the one hand, it provides for the access to and transfer of
technologies, including biotechnology, that are relevant to the conservation and sustainable use
of biological diversity (for example, in Article 16, paragragh 1, and Article 19, paragraphs 1
and 2). On the other hand, Articles 8(g) and 19, paragraph 3, seek to ensure the development of
appropriate procedures to enhance the safety of biotechnology in the context of the
Convention's overall goal of reducing all potential threats to biological diversity, taking also
into account the risks to human health. Article 8(g) deals with measures that Parties should
take at national level, while Article 19, paragraph 3, sets the stage for the development of an
international legally binding instrument to address the issue of biosafety.
At its second meeting, held in November 1995, the Conference of the Parties to the Convention
established an Open-ended Ad Hoc Working Group on Biosafety to develop a draft protocol on
biosafety, focusing specifically on transboundary movement of any living modified organism
resulting from modern biotechnology that may have adverse effect on the conservation and
sustainable use of biological diversity. After several years of negotiations, the Protocol, known
as the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, was
finalized and adopted in Montreal on 29 January 2000 at an extraordinary meeting of the
Conference of the Parties.
The conclusion of the Biosafety Protocol has been hailed as a significant step forward in that it
provides an international regulatory framework to reconcile the respective needs of trade and
environmental protection with respect to a rapidly growing global industry, the biotechnology
industry. The Protocol thus creates an enabling environment for the environmentally sound
application of biotechnology, making it possible to derive maximum benefit from the potential
that biotechnology has to offer, while minimizing the possible risks to the environment and to
human health.
NIBGE-FAO Workshop on GMO Detection
135
CARTAGENA PROTOCOL ON BIOSAFETY TO THE
CONVENTION ON BIOLOGICAL DIVERSITY
The Parties to this Protocol,
Being Parties to the Convention on Biological Diversity, hereinafter referred to as "the
Convention",
Recalling Article 19, paragraphs 3 and 4, and Articles 8 (g) and 17 of the Convention,
Recalling also decision 11/5 of 17 November 1995 of the Conference of the Parties to the
Convention to develop a Protocol on biosafety, specifically focusing on transboundary
movement of any living modified organism resulting from modern biotechnology that may
have adverse effect on the conservation and suslainable use of biological diversity,
setting out for consideration, in particular, appropriate procedures for advance informed
agreement,
Reaffirming the precautionary approach contained in Principle 15 of the Rio Declaration
on Environment and Development,
Aware o/'the rapid expansion of modern biotechnology and the growing public concern
over its potential adverse effects on biological diversity, taking also into account risks to
human health,
Recognizing that modern biotechnology has great potential for human well-being if
developed and used with adequate safety measures for the environment and human
health,
Recognizing also the crucial importance to humankind of centres of origin and centres of
genetic diversity,
Taking into account the limited capabilities of many countries, particularly developing
countries, to cope with the nature and scale of known and potential risks associated with
living modified organisms,
Recognizing that trade and environment agreements should be mutually supportive with
a view lo achieving sustainable development,
Emphasizing that this Protocol shall not be interpreted as implying a change in the rights
and obligations of a Party under any existing international agreements,
Understanding that the above recital is not intended
subordinate this Protocol to other international agreements,
Have agreed as follows:
to
NIBGE-FAO Workshop on GMO Detection
136
Article 1
OBJECTIVE
In accordance with the precautionary approach contained in Principle 15 of the Rio
Declaration on Environment and Development, the objective of this Protocol is to contribute
to ensuring an adequate level of protection in the field of the safe transfer, handling and use
of living modified organisms resulting from modern biotechnology that may have adverse
effects on the conservation and sustainable use of biological diversity, taking also into
account risks to human health, and specifically focusing on transboundary movements.
Article 2
GENERAL PROVISIONS
1. Each Party shall take necessary and appropriate legal, administrative and other measures
to implement its obligations under this Protocol.
2. The Parties shall ensure that the development, handling, transport, use, transfer and
release of any living modified organisms are undertaken in a manner that prevents or
reduces the risks to biological diversity, taking also into account risks to human health.
3. Nothing in this Protocol shall affect in any way the sovereignty of States over their
territorial sea established in accordance with international law, and the sovereign rights and
the jurisdiction which States have in their exclusive economic /ones and their continental
shelves in accordance with international law, and the exercise by ships and aircraft of all
States of navigational rights and freedoms as provided for in international law and as
reflected in relevant international instruments.
4. Nothing in this Protocol shall be interpreted as restricting the right of a Party to take
action that is more protective of the conservation and sustainable use of biological diversity
than that called for in this Protocol, provided that such action is consistent with the objective
and the provisions of this Protocol and is in accordance with that Party's other obligations
under international law.
5. The Parties are encouraged to take into account, as appropriate, available expertise,
instruments and work undertaken in international forums with competence in the area of
risks to human health..
NIBGE-FAO Workshop on GMO Detection
137
Article 3
USE OF TERMS
For the purposes of this Protocol:
(a) "Conference of the Parties" means the Conference of the Parties to the Convention;
(b) "Contained use" means any operation, undertaken within a facility, installation or
other physical structure, which involves living modified organisms that are controlled
by specific measures that effectively limit their contact with, and their impact on, the
external environment;
(c) "Export" means intentional transboundary movement from one Party to another
Parly;
(d) "Exporter" means any legal or natural person, under the jurisdiction of the Party of
export, who arranges for a living modified organism to be exported;
(e) "Import" means intentional transboundary movement into one Party from another
Party;
(f) "Importer" means any legal or natural person, under the jurisdiction of the Party of
import, who arranges for a living modified organism to be imported;
(g) "Living modified organism" means any living organism that possesses a novel
combination of genetic material obtained through the use of modern biotechnology;
(h) "Living organism" means any biological entity capable of transferring or replicating
genetic material, including sterile organisms, viruses and viroids;
(i) "Modern biotechnology" means the application of:
a. In vitro nucleic acid techniques, including recombinant
deoxyribonucleic acid (DNA) and direct injection of nucleic acid into cells or
organelles, or
b. Fusion of cells beyond the taxonomic family,
that overcome natural physiological reproductive or recombination barriers and that are
not techniques used in traditional breeding and selection;
(j) "Regional economic integration organization" means an organization constituted
by sovereign States of a given region, to which its member States have transferred
competence in respect of matters governed by this Protocol and which has been duly
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authorized, in accordance with its internal procedures, to sign, ratify, accept, approve or
accede to it;
(k) "Transboundary movement" means the movement of a living modified organism
from one Party to another Party, save that for the purposes of Articles 17 and 24
transboundary movement extends to movement between Parties and non-Parties.
Article 4
SCOPE
This Protocol shall apply to the transboundary movement, transit, handling and use of all
living modified organisms that may have adverse effects on the conservation and
sustainable use of biological diversity, taking also into account risks to human health.
Article 5
PHARMACEUTICALS
Notwithstanding Article 4 and without prejudice to any right of a Party to subject all
living modified organisms to risk assessment prior to the making of decisions on import,
this Protocol shall not apply to the transboundary movement of living modified
organisms which are pharmaceuticals for humans that are addressed by other relevant
international agreements or organisations.
Article 6
TRANSIT AND CONTAINED USE
1. Notwithstanding Article 4 and without prejudice to any right of a Party of transit to
regulate the transport of living modified organisms through its territory and make
available to the Biosafety Clearing-House, any decision of that Party, subject to Article 2,
paragraph 3, regarding the transit through its territory of a specific living modified
organism, the provisions of this Protocol with respect to the advance informed agreement
procedure shall not apply to living modified organisms in transit.
2. Notwithstanding Article 4 and without prejudice to any right of a Party to subject all
living modified organisms to risk assessment prior to decisions on import and to set
standards for contained use within its jurisdiction, the provisions of this Protocol with
respect to the advance informed agreement procedure shall not apply to the transboundary
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movement of living modified organisms destined for contained use undertaken in
accordance with the standards of the Party of import.
Article 7
APPLICATION OF THE ADVANCE INFORMED
AGREEMENT PROCEDURE
1. Subject to Articles 5 and 6, the advance informed agreement procedure in Articles 8 to
10 and 12 shall apply prior to the first intentional transboundary movement of living
modified organisms for intentional introduction into the environment of the Party of import.
2. "Intentional introduction into the environment" in paragraph 1 above, does not refer to
living modified organisms intended for direct use as food or feed, or for processing.
3. Article 11 shall apply prior to the first transboundary movement of living modified
organisms intended for direct use as food or feed, or for processing.
4.
The advance informed agreement procedure shall not apply to the intentional
transboundary movement of living modified organisms identified in a decision of the
Conference of the Parties serving as the meeting of the Parties to this Protocol as being not
likely to have adverse effects on the conservation and sustainable use of biological diversity,
taking also into account risks to human health.
Article 8
NOTIFICATION
1. The Party of export shall notify, or require the exporter to ensure notification to, in
writing, the competent national authority of the Party of import prior to the intentional
transboundary movement of a living modified organism that falls within the scope of Article
7, paragraph 1. The notification shall contain, at a minimum, the information specified in
Annex 1.
2. The Party of export shall ensure that there is a legal requirement for the accuracy of
information provided by the exporter.
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Article 9
ACKNOWLEDGEMENT OF RECEIPT OF NOTIFICATION
1. The Party of import shall acknowledge receipt of the notification, in writing, to the
notifier within ninety days of its receipt.
2. The acknowledgement shall state:
(a) The date of receipt of the notification;
(b) Whether the notification, prima facie, contains the information referred to in Article 8;
(c) Whether to proceed according to the domestic regulatory framework of the Party of
import or according to the procedure specified in Article 10.
3. The domestic regulatory framework referred to in paragraph 2 (c) above, shall be
consistent with this Protocol.
4. A failure by the Party of import to acknowledge receipt of a notification shall not
imply its consent to an intentional transboundary movement.
Article 10
DECISION PROCEDURE
1. Decisions taken by the Party of import shall be in accordance with Article 15.
2. The Party of import shall, within the period of time referred to in Article 9, inform
the notifier, in writing, whether the intentional transboundary movement may proceed:
(a) Only after the Party of import has given its written consent; or
(b) After no less than ninety days without a subsequent written consent.
3. Within two hundred and seventy days of the date of receipt of notification, the Party
of import shall communicate, in writing, to the nolifier and to the Biosafety ClearingHouse the decision referred to in paragraph 2 (a) above:
(a) Approving the import, with or without conditions, including how the decision will
apply to subsequent imports of the same living modified organism;
(b) Prohibiting the import;
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(c) Requesting additional relevant information in accordance with its domestic
regulatory framework or Annex I; in calculating the time within which the Party of
import is to respond, the number of days it has to wait for additional relevant information
shall not be taken into account; or
(d) Informing the notifier that the period specified in this paragraph is extended by a
defined period of time.
4. Except in a case in which consent is unconditional, a decision under paragraph 3 above,
shall set out the reasons on which it is based.
5. A failure by the Party of import to communicate its decision within two hundred and
seventy days of the date of receipt of the notification shall not imply its consent to an
intentional transboundary movement.
6. Lack of scientific certainty due to insufficient relevant scientific information and
knowledge regarding the extent of the potential adverse effects of a living modified
organism on the conservation and sustainable use of biological diversity in the Party of
import, taking also into account risks to human health, shall not prevent that Party from
taking a decision, as appropriate, with regard to the import of the living modified organism
in question as referred to in paragraph 3 above, in order to avoid or minimize such potential
adverse effects.
7. The Conference of the Parties serving as the meeting of the Parties shall, at its first
meeting, decide upon appropriate procedures and mechanisms to facilitate decision-making
by Parties of import.
Article 11
PROCEDURE FOR LIVING MODIFIED ORGANISMS
INTENDED FOR DIRECT USE AS FOOD OR FEED,
OR FOR PROCESSING
1. A Party that makes a final decision regarding domestic use. including placing on the
market, of a living modified organism that may be subject to transboundary movement for
direct use as food or feed, or for processing shall, within fifteen days of making that decision,
inform the Parties through the Biosafety Clearing-House. This information shall contain, at a
minimum, the information specified in Annex II. The Party shall provide a copy of the
information, in writing, to the national focal point of each Party that informs the Secretarial in
advance that it does not have access to the Biosafety Clearing-House. This provision shall not
apply to decisions regarding field trials.
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2. The Party making a decision under paragraph I above, shall ensure that there is a legal
requirement for the accuracy of information provided by the applicant.
3. Any Party may request additional information from the authority identified in paragraph
(b) of Annex II.
4. A Party may take a decision on the import of living modified organisms intended for
direct use as food or feed, or for processing, under its domestic regulatory framework that is
consistent with the objective of this Protocol.
5. Each Party shall make available to the Biosafety Clearing-House copies of any national
laws, regulations and guidelines applicable to the import of living modified organisms
intended for direct use as food or feed, or for processing, if available.
6. A developing country Party or a Party with an economy in transition may, in the absence
of the domestic regulatory framework referred to in paragraph 4 above, and in exercise of its
domestic jurisdiction, declare through the Biosafety Clearing-House that its decision prior to
the first import of a living modified organism intended for direct use as food or feed, or for
processing, on which information has been provided under paragraph 1 above, will be taken
according to the following:
(a) A risk assessment undertaken in accordance with Annex III; and
(b) A decision made within a predictable timeframe, not exceeding two hundred and seventy
days.
7. Failure by a Party to communicate its decision according to paragraph 6 above, shall not
imply its consent or refusal to the import of a living modified organism intended for direct
use as food or feed, or for processing, unless otherwise specified by the Party.
8. Lack of scientific certainty due to insufficient relevant scientific information and
knowledge regarding the extent of the potential adverse effects of a living modified
organism on the conservation and sustainable use of biological diversity in the Party of
import, taking also into account risks to human health, shall not prevent that Party from
taking a decision, as appropriate, with regard to the import of that living modified organism
intended for direct use as food or feed, or for processing, in order to avoid or minimize such
potential adverse effects.
9. A Party may indicate its needs for financial and technical assistance and capacitybuilding with respect to living modified organisms intended for direct use as food or feed,
or for processing. Parties shall cooperate to meet these needs in accordance with Articles 22
and 28.
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Article-12
REVIEW OF DECISIONS
1. A Party of import may, at any lime, in light of new scientific information on potential
adverse effects on the conservation and sustainablc use of biological diversity, taking also
into account the risks to human health, review and change a decision regarding an intentional
transboundary movement. In such case, the Party shall, within thirty days, inform any
notifier that has previously notified movements of the living modified organism referred to
in such decision, as well as the Biosafety Clearing-House, and shall set out the reasons for its
decision.
2. A Party of export or a notifier may request the Party of import to review a decision it has
made in respect of it under Article It) where the Party of export or the notifier considers
that:
(a) A change in circumstances has occurred that may influence the outcome of the risk
assessment upon which the decision was based; or
(b) Additional relevant scientific or technical information has become available.
3. The Party of import shall respond in writing to such a request within ninety days and set
out the reasons for its decision.
4. The Party of import may, al its discretion, require a risk assessment for subsequent
imports.
Article 13
SIMPLIFIED PROCEDURE
I. A Party of import may, provided that adequate measures are applied to ensure the safe
intentional movement of living modified organisms in accordance with the objective of this
Protocol, specify in advance to the Biosafety Clearing-House:
(a) Cases in which intentional transboundary movement to it may take place at the same time
as the movement is notified to the Parly of import; and
(b) Imports of living modified organisms to it to be exempted from the advance informed
agreement procedure.
Notifications under subparagraph (a) above, may apply to subsequenl similar movements to
the same Party.
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2. The information relating to an intentional transboundary movement that is to be
provided in the notifications referred to in paragraph 1 (a) above, shall be the information
specified in Annex I.
Article 14
BILATERAL, REGIONAL AND MULTILATERAL
AGREEMENTS AND ARRANGEMENTS
1. Parties may enter into bilateral, regional and multilateral agreements and arrangements
regarding intentional transboundary movements of living modified organisms, consistent
with the objective of this Protocol and provided that such agreements and arrangements do
not result in a lower level of protection than that provided for by the Protocol.
2. The Parties shall inform each other, through the Biosafety Clearing-House, of any such
bilateral, regional and multilateral agreements and arrangements that they have entered into
before or after the date of entry into force of this Protocol.
3. The provisions of this Protocol shall not affect intentional transboundary movements that
take place pursuant to such agreements and arrangements as between the parlies to those
agreements or arrangements.
4. Any Party may determine that its domestic regulations shall apply with respect to
specific imports to it and shall notify the Biosafety Clearing-House of its decision.
Article15
RISK ASSESSMENT
I.
Risk assessments undertaken pursuant to this Protocol shall be carried out in a
scientifically sound manner, in accordance with Annex 111 and taking into account
recognized risk assessment techniques. Such risk assessments shall be based, at a minimum,
on information provided in accordance with Article 8 and other available scientific
evidence in order to identify and evaluate the possible adverse effects of living modified
organisms on the conservation and sustainable use of biological diversity, taking also into
account risks to human health.
2. The Party of import shall ensure that risk assessments are carried out for decisions
taken under Article H). It may require the exporter to carry out the risk assessment.
3. The cost of risk assessment shall be borne by the notifier if the Party of import so
requires.
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Article 16
RISK MANAGEMENT
1. The Parties shall, taking into account Article 8 (g) of the Convention, establish and
maintain appropriate mechanisms, measures and strategies to regulate, manage and
control risks identified in the risk assessment provisions of this Protocol associated with
the use, handling and transboundary movement of living modified organisms.
2. Measures based on risk assessment shall be imposed to the extent necessary to
prevent adverse effects of the living modified organism on the conservation and
sustainable use of biological diversity, taking also into account risks to human health,
within the territory of the Party of import.
3. Each Party shall take appropriate measures to prevent unintentional transboundary
movements of living modified organisms, including such measures as requiring a risk
assessment to be carried out prior to the first release of a living modified organism.
4. Without prejudice to paragraph 2 above, each Party shall endeavour to ensure that
any living modified organism, whether imported or locally developed, has undergone an
appropriate period of observation that is commensurate with its life-cycle or generation
time before it is put to its intended use.
5. Parties shall cooperate with a view to:
(a) Identifying living modified organisms or specific traits of living modified organisms
that may have adverse effects on the conservation and sustainable use of biological
diversity, taking also into account risks to human health; and
(b) Taking appropriate measures regarding the treatment of such living modified
organisms or specific traits.
Article 17
UNINTENTIONAL TRANSBOUNDARY MOVEMENTS
AND EMERGENCY MEASURES
1. Each Party shall take appropriate measures to notify affected or potentially affected
States, the Biosafety Clearing-House and, where appropriate, relevant international
organizations, when it knows of an occurrence under its jurisdiction resulting in a
release that leads, or may lead, to an unintentional transboundary movement of a living
modified organism that is likely to have significant adverse effects on the conservation
and sustainable use of biological diversity, taking also into account risks to human
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health in such States. The notification shall be provided as soon as the Party knows of
the above situation.
2. Each Party shall, no later than the dale of entry into force of this Protocol for it,
make available to the Biosafety Clearing-House the relevant details setting out its point
of contact for the purposes of receiving notifications under this Article.
3. Any notification arising from paragraph I above, should include:
(a) Available relevant information on the estimated quantities and relevant
characteristics and/or traits of the living modified organism;
(b) Information on the circumstances and estimated date of the release, and on the use
of the living modified organism in the originating Party;
(c) Any available information about the possible adverse effects on the conservation
and sustainable use of biological diversity, taking also into account risks to human
health, as well as available information about possible risk management measures;
(d) Any other relevant information; and
(e) A point of contact for further information.
4.
In order to minimize any significant adverse effects on the conservation and
sustainable use of biological diversity, taking also into account risks to human health,
each Party, under whose jurisdiction the release of the living modified organism referred
to in paragraph I above, occurs, shall immediately consult the affected or potentially
affected States to enable them to determine appropriate responses and initiate necessary
action, including emergency measures.
Article 18
HNADLINGTRANSPORT, PACKAGING
AND IDENTIFICATION
1. In order to avoid adverse effects on the conservation and sustainable use of biological
diversity, taking also into account risks to human health, each Party shall take necessary
measures to require that living modified organisms that are subject to intentional
transboundary movement within the scope of this Protocol are handled, packaged and
transported under conditions of safety, taking into consideration relevant international rules
and standards.
2. Each Party shall take measures to require that documentation accompanying:
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(a) Living modified organisms that arc intended for direct use as food or feed, or for
processing, clearly identifies that they '"may contain" living modified organisms and are not
intended for intentional introduction into the environment, as well as a contact point for
further information. The Conference of the Parties serving as the meeting of the Parties to
this Protocol shall take a decision on the detailed requirements for this purpose, including
specification of their identity and any unique identification, no later than two years after the
date of entry into force of this Protocol;
(b) Living modified organisms that are destined for contained use clearly identifies them as
living modified organisms; and specifies any requirements for the safe handling, storage,
transport and use, the contact point for further information, including the name and address
of the individual and institution to whom the living modified organisms are consigned; and
(c) Living modified organisms that are intended for intentional introduction into the
environment of the Party of import and any other living modified organisms within the
scope of the Protocol, clearly identifies them as living modified organisms; specifies the
identity and relevant traits and/or characteristics, any requirements for the safe handling,
storage, transport and use, the contact point for further information and, as appropriate, the
name and address of the importer and exporter; and contains a declaration that the
movement is in conformity with the requirements of this Protocol applicable to the exporter.
3. The Conference of the Parties serving as the meeting of the Parties to this Protocol shall
consider the need for and modalities of developing standards with regard to identification,
handling, packaging and transport practices, in consultation with other relevant international
bodies.
Article 19
COMPETENT NATIONAL AUTHORITIES
AND NATIONAL FOCAL POINTS
1. Each Party shall designate one national focal point to be responsible on its behalf for
liaison with the Secretarial. Each Party shall also designate one or more competent
national authorities, which shall be responsible for performing the administrative
functions required by this Protocol and which shall be authorized to act on its behalf with
respect to those functions. A Party may designate a single entity to fulfil the functions of
both focal point and competent national authority.
2. Each Party shall, no later than the date of entry into force of this Protocol for it,
notify the Secretarial of the names and addresses of its focal point and ils competent
national authority or authorities. Where a Party designates more than one competent
national aulhorily, il shall convey to the Secretariat, with its notification thereof, relevanl
information on the respective responsibilities of those authorities. Where applicable,
such information shall, at a minimum, specify which competent authority is responsible
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for which type of living modified organism. Each Party shall forthwith notify Ihe
Secretariat of any changes in the designation of its nalional focal point or in Ihe name
and address or responsibilities of its competent national authority or authorities.
3. The Secretariat shall forthwith inform the Parties of the notifications it receives
under paragraph 2 above, and shall also make such information available through the
Biosafety Clearing-House.
Article 20
INFORMATION SHARING AND THE
BIOSAFETY CLEARING-HOUSE
I.
A Biosafety Clearing-House is hereby established as part of the clearinghouse
mechanism under Article 18, paragraph 3, of the Convention, in order to:
(a) Facilitate the exchange of scientific, technical, environmental and legal information
on. and experience with, living modified organisms; and
(b) Assist Parties to implement Ihe Protocol, taking into account the special needs of
developing country Parties, in particular the least developed and small island developing
States among them, and countries with economies in transition as well as countries thai are
centres of origin and centres of genetic diversity.
2. The Biosafety Clearing-House shall serve as a means through which information is
made available for the purposes of paragraph 1 above. It shall provide access to information
made available by the Parties relevant to the implementation of the Protocol. It shall also
provide access, where possible, to other international biosafely information exchange
mechanisms.
3. Without prejudice to the protection of confidential information, each Party shall make
available to the Biosafely Clearing-House any information required to be made available to
the Biosafety Clearing-House under this Protocol, and:
(a) Any existing laws, regulations and guidelines for implementation of the Protocol, as
well as information required by the Parties for the advance informed agreement procedure;
(b) Any bilateral, regional and multilateral agreements and arrangements;
(c) Summaries of its risk assessments or environmental reviews of living modified
organisms generated by its regulatory process, and carried out in accordance with Article 15,
including, where appropriate, relevant information regarding products thereof, namely,
processed materials that are of living modified organism origin, containing detectable novel
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combinations of replieable genetic material obtained through the use of modern
biotechnology;
(d) Its final decisions regarding the importation or release of living modified organisms; and
(c) Reports submitted by it pursuant to Article 33, including those on implementation of the
advance informed agreement procedure.
4. The modalities of the operation of the Biosafety Clearing-House, including reports on its
activities, shall be considered and decided upon by the Conference of the Parties serving as
the meeting of the Parties to this Protocol at its first meeting, and kept under review
thereafter.
Article 21
CONFIDENTIAL INFORMATION
1. The Party of import shall permit the notifier to identify information submitted under the
procedures of this Protocol or required by the Party of import as part of the advance
informed agreement procedure of the Protocol that is to be treated as confidential.
Justification shall be given in such eases upon request.
2.
The Party of import shall consult the notifier if it decides that
information identified by the notifier as confidential does not qualify for such
treatment and shall, prior to any disclosure, inform the notifier of its decision,
providing reasons on request, as well as an opportunity for consultation and for an
internal review of the decision prior to disclosure.
3.
Each Party shall protect confidential information received under this Protocol,
including any confidential information received in the context of the advance informed
agreement procedure of the Protocol. Each Party shall ensure that it has procedures to
protect such information and shall protect the confidentiality of such information in a
manner no less favourable than its treatment of confidential information in connection with
domestically produced living modified organisms.
4. The Party of import shall not use such information for a commercial purpose, except
with the written consent of the notifier.
5. If a notifier withdraws or has withdrawn a notification, the Party of import shall respect
the confidentiality of commercial and industrial information, including research and
development information as well as information on which the Party and the notifier disagree
as to its confidentiality.
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6.
Without prejudice to paragraph 5 above, the following information shall not be
considered confidential:
(a) The name and address of the notifier;
(b) A general description of the living modified organism or organisms;
(c) A summary of the risk assessment of the effects on the conservation and sustainable use
of biological diversity, taking also into account risks to human health; and
(d) Any methods and plans for emergency response.
Article 22
CAPACITY-BUILDING
1. The Parties shall cooperate in the development and/or strengthening of human resources
and institutional capacities in biosafety, including biotechnology to the extent that it is
required for biosafety, for the purpose of the effective implementation of this Protocol, in
developing country Parties, in particular the least developed and small island developing
States among them, and in Parties with economies in transition, including through existing
global, regional, subregional and national institutions and organizations and, as appropriate,
through facilitating private sector involvement.
2. For the purposes of implementing paragraph 1 above, in relation to cooperation, the
needs of developing country Parties, in particular the least developed and small island
developing States among them, for financial resources and access to and transfer of
technology and know-how in accordance with the relevant provisions ot the Convention,
shall be taken fully into account for capacity-building in biosafety. Cooperation in capacitybuilding shall, subject to the different situation, capabilities and requirements of each Party,
include scientific and technical training in the proper and safe management of
biotechnology, and in the use of risk assessment and risk management for biosafety, and the
enhancement of technological and institutional capacities in biosafety. The needs of Parties
with economies in transition shall also be taken fully into account for such capacity-building
in biosafety.
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Article 23
PUBLIC AWARENESS AND PARTICIPATION
1. The Parties shall:
(a) Promote and facilitate public awareness, education and participation concerning the safe
transfer, handling and use of living modified organisms in relation to the conservation and
sustainablc use of biological diversity, taking also into account risks to human health. In
doing so, the Parties shall cooperate, as appropriate, with other States and international
bodies;
(b) Endeavour to ensure that public awareness and education encompass access to
information on living modified organisms identified in accordance with this Protocol that
may be imported.
2. The Parties shall, in accordance with their respective laws and regulations, consult the
public in the decision-making process regarding living modified organisms and shall make
the results of such decisions available to the public, while respecting confidential
information in accordance with Article 21.
3. Each Party shall endeavour to inform its public about the means of public access to the
Biosafety Clearing-House.
Article 24
NON-PARTIES
1. Transboundary movements of living modified organisms between Parties and nonParlies shall be consistent with the objective of this Protocol. The Parties may enter into
bilateral, regional and multilateral agreements and arrangements with non-Parties regarding
such transboundary movements.
2. The Parties shall encourage non-Parties to adhere to this Protocol and to contribute
appropriate information to the Biosafety Clearing-House on living modified organisms
released in, or moved into or out of, areas within their national jurisdictions.
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Article 25
ILLEGAL TRANSBOUNDARY MOVEMENTS
1. Each Party shall adopt appropriate domestic measures aimed at preventing and, if
appropriate, penalizing transboundary movements of living modified organisms carried
out in contravention of its domestic measures to implement this Protocol. Such
movements shall be deemed illegal transboundary movements.
2. In the case of an illegal transboundary movement, the affected Party may request the
Party of origin to dispose, at its own expense, of the living modified organism in
question by repatriation or destruction, as appropriate.
3.
Each Party shall make available to the Biosafety Clearing-House information
concerning cases of illegal transboundary movements pertaining to it.
Article26
SOCIO-ECONOMIC CONSIDERATIONS
1. The Parties, in reaching a decision on import under this Protocol or under its
domestic measures implementing the Protocol, may take into account, consistent with
their international obligations, socio-economic considerations arising from the impact of
living modified organisms on the conservation and sustainablc use of biological diversity,
especially with regard to the value of biological diversity to indigenous and local
communities.
2. The Parties are encouraged to cooperate on research and information exchange on any
socio-economic impacts of living modified organisms, especially on indigenous and
local communities.
Article 27
LIABILITY AND REDRESS
The Conference of the Parties serving as the meeting of the Parties to this Protocol shall,
at its first meeting, adopt a process with respect to the appropriate elaboration of
international rules and procedures in the field of liability and redress for damage resulting
from transboundary movements of living modified organisms, analysing and taking due
account of the ongoing processes in international law on these matters, and shall
endeavour to complete this process within four years.
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Article 28
FINANCIAL MECHANISM AND RESOURCES
1. In considering financial resources for the implementation of this Protocol, the
Parties shall take into account the provisions of Article 20 of the Convention.
2. The financial mechanism established in Article 21 of the Convention shall, through
the institutional structure entrusted with its operation, be the financial mechanism for
this Protocol.
3. Regarding the capacity-building referred to in Article 22 of this Protocol, the
Conference of the Parties serving as the meeting of the Parties to this Protocol, in
providing guidance with respect to the financial mechanism referred to in paragraph 2
above, for consideration by the Conference of the Parties, shall take into account the
need for financial resources by developing country Parties, in particular the least
developed and the small island developing States among them.
4. In the context of paragraph 1 above, the Parties shall also take into account the needs
of the developing country Parties, in particular the least developed and the small island
developing States among them, and of the Parties with economies in transition, in their
efforts to identify and implement their capacity-building requirements for the purposes of
the implementation of this Protocol.
5. The guidance to the financial mechanism of the Convenf/on in relevant decisions of
the Conference of the Parties, including those agreed before the adoption of this
Protocol, shall
apply, mutatis mutandis, to the provisions of this Article.
6. The developed country Parties may also provide, and the developing country Parties
and the Parties with economies in transition avail themselves of, financial and
technological resources for the implementation of the provisions of this Protocol through
bilateral, regional and multilateral channels.
Article 29
CONFERENCE OF THE PARTIES SERVING AS THE
MEETING OF THE PARTIES TO THIS PROTOCOL
1. The Conference of the Parties shall serve as (he meeting of the Parties to this
Protocol.
2. Parties to the Convention that are not Parties to this Protocol may participate as
observers in the proceedings of any meeting of the Conference of the Parties serving as
the meeting of the Parties to this Protocol. When the Conference of the Parties serves as
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the meeting of the Parties to this Protocol, decisions under this Protocol shall be lakcn
only by those that are Parties to it.
3. When the Conference of the Parties serves as the meeting of the Parties to this
Protocol, any member of the bureau of the Conference of the Parlies representing a Party
to the Convention but, at that time, not a Party to this Protocol, shall be substituted by a
member 10 be elected by and from among the Parties to this Protocol.
4. The Conference of the Parties serving as the meeting of the Parties to this Protocol
shall keep under regular review the implementation of this Protocol and shall make,
within its mandate, the decisions necessary to promote its effective implementation. It
shall perform the functions assigned to it by this Protocol and shall:
(a) Make recommendations on any matters necessary for the implementation of this
Protocol:
(b) Establish such subsidiary bodies as are deemed necessary for the implementation of
this Protocol;
(c) Seek and utilize, where appropriate, the services and cooperation of, and information
provided by, competent international organizations and intergovernmental and nongovernmental bodies;
(d) Establish the form and the intervals for transmitting the information to be submitted
in accordance with Article 33 of this Protocol and consider such information as well as
reports submitted by any subsidiary body;
(e) Consider and adopt, as required, amendments to this Protocol and its annexes, as
well as any additional annexes to this Protocol, that are deemed necessary for the
implementation of this Protocol; and
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(f) Exercise such other functions as may be required for the implementation of this
Protocol.
5. The rules of procedure of the Conference of the Parties and financial rules of the
Convention shall be applied, mutatis mutandis, under this Protocol, except as may be
otherwise decided by consensus by the Conference of the Parties serving as the meeting of
the Parties to this Protocol.
6. The first meeting of the Conference of the Parties serving as the meeting of the Parties to
this Protocol shall be convened by the Secretariat in conjunction with the first meeting of the
Conference of the Parties that is scheduled after the date of the entry into force of this
Protocol. Subsequent ordinary meetings of the Conference of the Parties serving as the
meeting of the Parties to this Protocol shall be held in conjunction with ordinary meetings of
the Conference of the Parties, unless otherwise decided by the Conference of the Parties
serving as the meeting of the Parties to this Protocol.
7. Extraordinary meetings of the Conference of the Parties serving as the meeting of the
Parties to this Protocol shall be held at such other times as may be deemed necessary by the
Conference of the Parties serving as the meeting of the Parties to this Protocol, or at the
written request of any Party, provided that, within six months of the request being
communicated to the Parties by the Secretariat, it is supported by at least one third of the
Parties.
8. The United Nations, its specialized agencies and the International Atomic Energy
Agency, as well as any State member thereof or observers thereto not party to the
Convention, may be represented as observers at meetings of the Conference of the Parties
serving as the meeting of the Parties to this Protocol. Any body or agency, whether national
or international, governmental or nongovernmental, that is qualified in matters covered by
this Protocol and that has informed the Secretariat of its wish to be represented at a meeting
of the Conference of the Parties serving as a meeting of the Parties to this Protocol as an
observer, may be so admitted, unless at least one third of the Parties present object. Except
as otherwise provided in this Article, the admission and participation of observers shall be
subject to the rules of procedure, as referred to in paragraph 5 above.
Article 30
SUBSIDIARY BODIES
1. Any subsidiary body established by or under the Convention may, upon a decision by
the Conference of the Parties serving as the meeting of the Parties to this Protocol, serve
the Protocol, in which case the meeting of the Parties shall specify which functions that
body shall exercise.
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2. Parties to the Convention that are not Parties to this Protocol may participate as
observers in the proceedings of any meeting of any such subsidiary bodies. When a
subsidiary body of the Convention serves as a subsidiary body to this Protocol, decisions
under the Protocol shall be taken only by the Parties to the Protocol.
3. When a subsidiary body of the Convention exercises its functions with regard to
matters concerning this Protocol, any member of the bureau of that subsidiary body
representing a Party to the Convention but, at that time, not a Party to the Protocol, shall
be substituted by a member to be elected by and from among the Parties to the Protocol.
Article 31
SECRETARIAT
1. The Secretariat established by Article 24 of the Convention shall serve as the
secretariat to this Protocol.
2. Article 24, paragraph I, of the Convention on the functions of the Secretariat shall
apply, mutatis mutandis, to this Protocol.
3. To the extent that they are distinct, the costs of the secretariat services for this Protocol
shall be met by the Parties hereto. The Conference of the Parties serving as the meeting
of the Parties to this Protocol shall, at its first meeting, decide on the necessary budgetary
arrangements to this end.
Article 32
RELATIONSHIP WITH THE CONVENTION
Except as otherwise provided in this Protocol, the provisions of the Convention relating to
its protocols shall apply to this Protocol.
Article 33
MONITORING AND REPORTING
Eaeh Party shall monitor the implementation of its obligations under this Protocol, and
shall, at intervals to be determined by the Conference of the Parties serving as the
meeting of the Parties to this Protocol, report to the Conference of the Parlies serving as
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the meeting of the Parties to this Protocol on measures that it has taken to implement the
Protocol.
Article 34
COMPLIANCE
The Conference of the Parties serving as the meeting of the Parties to this Protocol
shall, at its first meeting, consider and approve cooperative procedures and institutional
mechanisms to promote compliance with the provisions of this Protocol and to address
cases of non-compliance. These procedures and mechanisms shall include provisions
to offer advice or assistance, where appropriate. They shall be separate from, and
without prejudice to, the dispute settlement procedures and mechanisms established by
Article 27 of the Convention.
Article 35
ASSESSMENT AND REVIEW
The Conference of the Parties serving as the meeting of (he Parlies to this Protocol shall
undertake, five years after the entry into force of this Protocol and at least every five
years thereafter, an evaluation ol'lhe effectiveness of the Protocol, including an
assessment of its procedures and annexes.
Article 36
SIGNATURE
This Protocol shall be open for signature at the United Nations Office at Nairobi by
States and regional economic integration organizations from 15 to 26 May 2000, and at
United Nations Headquarters in New York from 5 June 2000 to 4 June 2001.
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Article 37
ENTRY INTO FORCE
1. This Protocol shall enter into force on the ninetieth day after the date of deposit of the
fiftieth instrument of ratification, acceptance, approval or accession by Stales or regional
economic integration organizations that are Parties to the Convention.
2. This Protocol shall enter into force for a State or regional economic integration
organization that ratifies, accepts or approves this Protocol or accedes thereto after its
entry into force pursuant to paragraph 1 above, on the ninetieth day after the date on
which that State or regional economic integration organization deposits its instrument of
ratification, acceptance, approval or accession, or on the date on which the Convention
enters into force for that State or regional economic integration organization, whichever
shall be the later.
3. For the purposes of paragraphs 1 and 2 above, any instrument deposited by a regional
economic integration organization shall not be counted as additional to those deposited
by member States of such organization.
Article 38
RESERVATIONS
No reservations may be made to this Protocol.
Article 39
WITHDRAWAL
1. At any time after two years from the date on which this Protocol has entered into
force for a Party, that Party may withdraw from the Protocol by giving written
notification to the Depositary.
2. Any such withdrawal shall take place upon expiry of one year after the date of its
receipt by the Depositary, or on such later date as may be specified in the notification of
the withdrawal.
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Article 40
AUTHENTIC TEXTS
The original of this Protocol, of which the Arabic, Chinese, English, French, Russian and
Spanish texts are equally authentic, shall be deposited with the Secretary-General of the
United Nations.
IN WITNESS WHEREOF the undersigned, being duly authorized to that effect, have signed
this Protocol.
DONE at Montreal on this twenty-ninth day of January, two thousand.
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Annex I
INFORMATION REQUIRED IN NOTIFICATIONS
UNDER ARTICLES 8, 10 AND 13
(a) Name, address and contact details of the exporter.
(b) Name, address and contact details of the importer.
(c) Name and identity of the living modified organism, as well as the domestic
classification, if any, of the biosafety level of the living modified organism in the State of
export.
(d) Intended date or dates of the transboundary movement, if known.
(e) Taxonomic status, common name, point of collection or acquisition, and characteristics
of recipient organism or parental organisms related to biosafety.
(f) Centres of origin and centres of genetic diversity, if known, of the recipient organism
and/or the parental organisms and a description of the habitats where the organisms may
persist or proliferate.
(g) Taxonomic status, common name, point of collection or acquisition, and characteristics
of the donor organism or organisms related to biosafety.
(h) Description of the nucleic acid or the modification introduced, the technique used, and
the resulting characteristics of the living modified organism.
(i) Intended use of the living modified organism or products thereof, namely, processed
materials that are of living modified organism origin, containing detectable novel
combinations of replicable genetic material obtained through the use of modern
biotechnology.
(j) Quantity or volume of the living modified organism to be transferred.
(k) A previous and existing risk assessment report consistent with Annex III.
(1) Suggested methods for the safe handling, storage, transport and use, including
packaging, labelling, documentation, disposal and contingency procedures, where
appropriate.
(m) Regulatory status of the living modified organism within the State of export (for
example, whether it is prohibited in the State of export, whether there are other restrictions,
or whether it has been approved for general release) and, if the living modified organism is
banned in the State of export, the reason or reasons for the ban.
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(n) Result and purpose of any notification by the exporter to other States regarding the
living modified organism to be transferred.
(o) A declaration that the above-mentioned information is factually correct.
Annex II
INFORMATION REQUIRED CONCERNING LIVING MODIFIED
ORGANISMS INTENDED FOR DIRECT USE AS FOOD OR FEED, OR
FOR PROCESSING UNDER ARTICLE 11
(a) The name and contact details of the applicant for a decision for domestic use.
(b) The name and contact details of the authority responsible for the decision.
(c) Name and identity of the living modified organism.
(d) Description of the gene modification, the technique used, and the resulting
characteristics oi the living modified organism.
(e) Any unique identification of the living modified organism.
(f) Taxonomic status, common name, point of collection or acquisition, and characteristics
of recipient organism or parental organisms related to biosafety.
(g) Centres of origin and centres of genetic diversity, if known, of the recipient organism
and/or the parental organisms and a description of the habitats where the organisms may
persist or proliferate.
(h) Taxonomic status, common name, point of collection or acquisition, and characteristics
of the donor organism or organisms related to biosafety.
(i) Approved uses of the living modified organism, (j) A risk assessment report consistent
with Annex III.
(k) Suggested methods for the safe handling, storage, transport and use, including
packaging, labelling, documentation, disposal and contingency procedures, where
appropriate.
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Annex III
RISK ASSESSMENT
Objective
1. The objective of risk assessment, under this Protocol, is to identify and evaluate the
potential adverse effects of living modified organisms on the conservation and sustainable
use of biological diversity in the likely potential receiving environment, taking also into
account risks to human health.
Use of risk assessment
2. Risk assessment is, inter alia, used by competent authorities to make informed
decisions regarding living modified organisms.
General principles
3. Risk assessment should be carried out in a scientifically sound and transparent manner,
and can take into account expert advice of, and guidelines developed by. relevant
international organizations.
4.
Lack of scientific knowledge or scientific consensus should not necessarily be
interpreted as indicating a particular level of risk, an absence of risk, or an acceptable risk.
5. Risks associated with living modified organisms or products thereof, namely, processed
materials that are of living modified organism origin, containing detectable novel
combinations of replicable genetic material obtained through the use of modern
biotechnology, should be considered in the context of the risks posed by the non-modified
recipients or parental organisms in the likely potential receiving environment.
6. Risk assessment should be carried out on a case-by-case basis. The required information
may vary in nature and level of detail from case to case, depending on the living modified
organism concerned, its intended use and the likely potential receiving environment.
Methodology
1. The process of risk assessment may on the one hand give rise to a need for further
information about specific subjects, which may be identified and requested during the
assessment process, while on the other hand information on other subjects may not be
relevant in some instances.
8. To fulfil its objective, risk assessment entails, as appropriate, the following steps:
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(a) An identification of any novel genotypic and phenotypic characteristics associated with
the living modified organism that may have adverse effects on biological diversity in the
likely potential receiving environment, taking also into account risks to human health;
(b) An evaluation of the likelihood of these adverse effects being realized, taking into
account the level and kind of exposure of the likely potential receiving environment to the
living modified organism;
(c) An evaluation of the consequences should these adverse effects be realised;
(d) An estimation of the overall risk posed by the living modified organism based on the
evaluation of the likelihood and consequences of the identified adverse effects being
realized;
(e) A recommendation as to whether or not the risks are acceptable or manageable,
including, where neeessary, identification of strategies to manage these risks; and
(f) Where there is uncertainty regarding the level of risk, it may be addressed by requesting
further information on the specific issues of concern or by implementing appropriate risk
management strategies and/or monitoring the living modified organism in the receiving
environment.
Points to consider
9. Depending on the case, risk assessment takes into account the relevant technical and
scientific details regarding the characteristics of the following subjects:
(a) Recipient organism or parental organisms. The biological characteristics of the recipient
organism or parental organisms, including information on taxonomic status, common name,
origin, centres of origin and centres of genetic diversity, if known, and a description of the
habitat where the organisms may persist or proliferate;
(b) Donor organism or organisms. Taxonomic status and common name, source, and the
relevant biological characteristics of the donor organisms;
(c) Vector. Characteristics of the vector, including its identity, if any, and its source or
origin, and its host range;
(d) Insert or inserts and/or characteristics of modification. Genetic characteristics of the
inserted nucleic acid and the function it specifies, and/or characteristics of the modification
introduced;
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(e) Living modified organism. Identity of the living modified organism, and the differences
between the biological characteristics of the living modified organism and those of the
recipient organism or parental organisms;
(f) Detection and identification of the living modified organism. Suggested detection and
identification methods and their specificity, sensitivity and reliability;
(g) Information relating to the intended use. Information relating to the intended use of the
living modified organism, including new or changed use compared to the recipient
organism or parental organisms; and
(h) Receiving environment. Information on the location, geographical, climatic and
ecological characteristics, including relevant information on biological diversity and centres
of origin of the likely potential receiving environment.
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GLOSSARY
Agrobacterium
A naturally occurring soil microorganism (bacterium) that produces crown gall disease in
the wild; it does so by introducing part of its genetic material into the plant to direct it to
make compounds it needs to live. A small piece of genetic material was isolated from this
bacterium and is used to insert genes into plants in the process of genetic modification.
Allergy
Adverse overreaction of the body's self-defence system, caused by the production of
antibodies against specific substances. Asthma, hayfever and intolerance to milk or egg
are familiar examples of allergies.
Amino acids
Building blocks of proteins. About 20 different amino acids are commonly used by cells
to make proteins.
Antibiotic
Chemical substance produced by some bacteria and fungi, or produced synthetically, that
inhibits the growth of, or kills, other microorganisms.
Antibiotic resistance
Resistance mechanisms to antibiotics exist that render cells 'immune' to the antibiotic; the
genes for these characteristics are found in certain organisms. The genes are used in some
genetic engineering experiments as tools to identify cells that have received new DNA.
Antibodies
A class of proteins (known as immunoglobulins) formed in the body in response to the
presence of antigens (foreign proteins and other compounds), which bind to the antigen
and inactivate it.
Bacillus thuringiensis (Bt)
A naturally occurring microorganism that produces a toxin that only kills organisms with
alkaline stomachs, namely insect larvae. The toxin (in the form of a whole killed
organism) has been used for biological control for decades. The genetic information that
encodes the toxin has now been indentified and moved into plants to make them insect
tolerant.
Bacterium
Simplest form of life that exists as a single cell without a nucleaus (distinct structure that
contains the genetic information of the cell). Also known as a prokaryote.
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Bases
Building blocks of DNA made up of nitrogen and carbon atoms. There are two types of
bases: purines (adenine and guanine, known as A and G) and pyrimidines (cytosine and
thymine, known as C and T). The bases pair up in the DNA double helix, and the order of
bases determines the genetic code.
Chromosome
A thread-like structure, consisting mostly of DNA and supporting proteins, that contains
the genetic information (in the form of genes) that instruct the cell on its function. Genes
are arranged in a particular order on chromosomes. Chromosomes are found in the
nucleus of the cell and organisms contain differing but characteristic numbers of
chromosomes, which are arranged in pairs in 'higher' plants and animals.
Cloning
Means of isolating particular parts of the genome as small fragments of DNA, and
making copies of and studying the sequence in another organism. Can also mean the
process of producing, by non-sexual means, an identical copy of an organism.
DNA
Deoxyribonucleic acid. The chemical building block of the genetic information in the cell
from which genes are composed; it specifies the characteristics of most living organisms.
DNA consists of a long molecule of repeating units (each unit containing deoxyribose (a
sugar), a phosphoric acid and a base) joined together in a particular order. DNA is usually
found as two complementary strands twisted into the shape of a double helix.
DNA code
The sequence (order) of DNA bases in a gene, which make up the instructions for a
particular characteristic of the organism.
Gene
The segment of DNA on a chromosome that contains the information necessary to make
a protein. Genes are the unit by which biological traits are inherited.
Gene flow
The incorporation of genes from one organism into the array of genes in another
population of organisms.
Gene mapping
Determination of the relative locations of genetic information (genes) on chromosomes.
Gene modification
The manipulation of a living organism's genetic make-up by eliminating, modifying or
adding copies of specific genes (often from other oganisms) through modern molecular
biology techniques. Also called 'gene splicing', 'recombinant DNA (rDNA) technology' or
'genetic engineering'.
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Genetically modified organism (GMO)
Term used to refer to an organism modified by the methods of genetic modification.
Genetics Study of the pattern of inheritance of genetic information in organisms.
Genome Entire genetic material in an organism, comprising all chromosomes.
Genomics Molecular characterisation of all the genes and gene products of a species.
Herbicide
Any chemical substance that is toxic to plants; usually used to kill specific unwanted
plants, especially weeds.
Insecticide
A chemical used to kill or control certain populations of insect pests. In agriculture,
insecticides are used to control insect pests that feed on crops or carry plant disease.
Marker
Identifiable physical location on a chromosome, the inheritance of which can be
monitored. Antibiotic resistance marker genes (usually of bacterial origin) render cells
"immune" to the antibiotic; these genes can be used as tools to identify plant cells that
have received new DNA through genetic modification.
Mutant
Organism that differs from its parent because of a mutation in its genetic code. Also
called a variant.
Mutation
Genetic change caused by natural phenomena or an agent that causes mutations (such as
radiation). 'Stable' mutations in genes are passed on to an organism's offspring; 'unstable'
mutations are not.
Nucleotide
Strings of thousands of nucleotides form a DNA or RNA molecule. Nucleotides are
composed of phosphate, sugar and one of four bases (adenine, guanine, cytosine, and
uracil or thymine). Three bases form a codon, which specifies a particular amino acid;
amino acids are strung together to form proteins.
Nucleus
Central compartment in cells of 'higher' organisms (eukaryotes); it houses most of the
heritable genetic information in the cell.
Pathogen Any organism capable of producing disease.
Pesticide
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A chemical substance (such as an insecticide or fungicide) that kills harmful organisms
and is used to control pests, such as insects, weeds, or microorganisms.
Plasmid
Independent, free-floating circular piece of DNA in a bacterium, capable of making
copies of itself in a host cell. Plasmids can be used in genetic modification experiments to
clone genes from other organisms and make large quantities of their DNA. [UCB
Promoter
A control region of a gene that determines in which tissue, and at when, a gene product is
produced.
Ribonucleic acid (RNA)
Chemical chains made up of the sugar ribose attached to a phosphoric acid and a base.
Different types of RNA exist in cells, some of which serve as the immediate code for
proteins, others of which are involved in the physical process of protein synthesis. RNA
can also serve instead of DNA as the only genetic information in certain viruses.
Tissue culture
Process of introducing living tissue into culture in the laboratory, where tissues or cells
can be grown for extended periods of time.
Transformation
Process of introducing into an organism new genetic information that can be stably
maintained.
Transgenic plant
Genetically modified plant, or offspring of genetically modified plants. The transgenic
plant usually contains genetic material that has been rearranged, or a foreign gene (a
transgene) from an unrelated organism such as a virus, animal or other plant.
Vaccine
Use of a killed or debilitated organism, or a part of its contents, that is capable of
inducing protection against the disease caused by that organism. Scientists are working to
develop oral vaccines which can be produced in the edible plants of transgenic plants
(e.g. fruits).
Value-added
Trait introduced into an organism or plant that gives that organism added value, such as
the capability to produce a pharmaceutical substance.
Virus
Small genetic element, composed of either DNA or RNA, that is protected by a protein
coat. Viruses cannot make copies of themselves without invading another (host) cell, and
using some of its cellular machinery. A virus is capable of existing either inside or
outside a host cell.
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Dr. Kauser A. Malik
Patron in Chief
Dr. Ahmad Mukhtar Khalid
Chairman, Organizing Committee
Dr. Yusuf Zafar
Course Organizer
ORGANIZING COMMITTEES
Reception & Registration Committee
Mr. W. Sarwar,Photographer
Coordination Committee.
Dr. Yusuf Zafar, CSO.
Dr. Javed A. Qureshi,PSO
Dr. Shahid Mansoor, PSO.
Mr. Nasir Ahmad, S.O.
Mr. Muhammad Asif, S.O.
Member
Dr. Fauzia Yusuf , CSO.
Dr. Farooq Latif,PSO.
Miss. Ayesha N. Fatima
Miss. Asma Aslam, S.O.
Miss. Farwa Nargis, T.F.
Convenor
Member
Member
Member
Member
Transport Committee
Dr. Z.M.Khalid,CSO.
Mr. Munir A. Anwar,SSO.
Mr. Anwar-ul-Haq, S.O.
Mr. Atiq-ur-Rehman,A.O
Convenor
Member
Member
Member
Promotion Shield & Certificate
Committee.
Mrs. Rubina Tabassum,PSO. Convenor
Mr. Mazhar Hussain, S.S.O. Member
Mr. Zafar Iqbal, S.O.
Member
Mr. Atiq-ur-Rehman,A. O Member
Entertainment Committee.
Dr. M. Shahid Baig,PSO
Dr. M.Hamid Rashid,SSO.
Mr. A. Jamal Hashmat, S.O.
Mr. Munir A. Anwar, SSO.
Mr. M. Jamil, Supdt.
Convenor
Member
Member
Member
Member
Publication Committee.
Dr. M. Afzal Ghauri, PSO
Mr. Arsalan H. Zaidi, S.O
Mr. Muhammad Asif, S.O.
Convenor
Member
Member
Finance Committee
Dr. M.I. Rajoka, CSO.
Dr. Shahid Mansoor, PSO.
Mr. Bakhshish Jilani, Sr.A.O
Malik M. Aslam, P.O.
Convenor
Member
Member
Member
Accommodation Committee
Mr. Sohail Hameed, PSO
Convenor
Mr. Munir A. Anwar, SSO Member
Miss. Razia Tahseen, S.O. Member
Mr. Atiq-ur-Rehman, A.O Member
Mr. Tariq Mahmood, SSA. Member
Audio-Video & Photographic
Committee
Dr. Aftab Bashir,PSO
Convenor
Mr. Mazhar Hussain, SSO Member
Ch. M. Muddassar,S.O
Member
Mr. Javed. Iqbal, Microscopist Member
Convenor
Member
Member
Member
Member
Press & Media/Liason Committee
Dr. M. Sarwar Khan, SSO. Convenor
Mr. Hassan Ali Amin, A.E. Member
Mr. Atiq-ur-Rehman,A.O
Member
First Aid Committee.
Dr. Ayesha Tariq, M.O
Convenor
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NIBGE-FAO Workshop on GMO Detection
LIST OF PARTICIPANTS
Mr. Hamayun Ghulam Ahmed
Seed Production Manager
SAWA-AG Net work,
2Km, Multan Road, Sahiwal
Ph: 0441-221172, 221272
Fax: 0441-221372
Email: [email protected]
Mr. Nadeem Austin
Monsanto Pakistan Agri Tech (Pvt) Ltd.
ABN AMRO Building
2nd Floor, 310-Upper Mall,
P.O.Box No. 10148, Lahore-5400
Tel: 042-111-106-106/ Fax: 0425877152
Mr. Naveed Ahmed
Quality Manager
Khawaja Food Ltd. Main G.T. Road
Sadhoke,
Distt Gujranwala
Ph:0431-265411-2/ Fax:0431-265410
Email: [email protected]
Mr Muhammad Aqeel Bhutto
Assistant Research Associate
Institute of Biotechnology and Genetic
Engineering,
University of Sindh, Jamshoro
P: 0221-882550
Email: [email protected]
Dr. Shafiq Ahmad
Senior Research Officer
ENGRO Chemical Pakistan Ltd
39, Street-1, Zikarya Town
Boson Road, Multan
Tel: 061-575601/ Fax: 061-575603
Email: [email protected]
Mr. M. Ikram Chowdhry
Resham Seed Corporation
Grain Market, Rahim Yar Khan
Ph:0731-70381-2/ Fax:0731-84382
Email: [email protected]
Mr Iftikhar Ali
Principal Scientific Officer
Nuclear Institute for Food and
Agriculture (NIFA), P O Box 446,
Peshawar
Ph: 091-2964060-62/ Fax: 091-2964059
Email: [email protected]
Mr Safdar Ali
Entomologist
Department of Plant Protection,
10-A Infantry Road, Lahore
Ph: 021-6818403/ Fax: 021-6308742
Mr. Muhammad Azeem Asad
Associate Research Officer
Rice Research Institute
Kala Shah Kaku Dist. Lahore
Ph:042-7980368/ Fax:042-7980361
Email: [email protected]
Mr Muhammad Faheem
Organic Inspector
Bhombal and Co. Room No. 39-40, Old
Rally Building, Talpur Road, Karachi
Ph: 021-2436394-8/ Fax: 021-2417004
Email: [email protected]
Mr. Mahmood-ul-Hassan
Lecturer
Department of PBG and Genetic
University of Arid, Rawalpindi
Ph:051-9290151 Email:
[email protected]
Mr. Imtiaz Hussain
Assistant Entomology (Quarantine)
Federal Dept. Plant Protection,
Jhang road, Faisalabad
Ph: 041-652753/ Fax: 041-673299
Email: [email protected]
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NIBGE-FAO Workshop on GMO Detection
Mr. Syed Jawad Hussain
Director
Office, EPB, Gulistan Colony No 2,
Sheikhupura Road , Faisalabad
Ph: 041-9210202/ Fax: 041-9210204
Email: [email protected]
Mr. Azhar Iqbal
Business Development Manager
ICI House, 63 Mozang Road, Lahore
Ph: 042-6307101/ Fax: 042-6307104
Email: [email protected]
Dr Nayyer Iqbal
Senior Scientific Officer
NIAB, P O Box 128,
Jhang Road, Faisalabad
Ph:041- 654221-30/ Fax:041-654213
Mr. Tariq Parvez Islam
Manager Seed
Kisan Supplies Services, 645-C Faisal
Town, Lahore
Ph: 5168255-56/ Fax: 5167649
Email: [email protected]
Mr Abdus Sami Kazi
Bhombal and Co. Room No. 39-40, Old
Rally Building, Talpur Road, Karachi
Ph: 021-2436394-8/ Fax: 021-2417004
Email: [email protected]
Mr. A Tahir Khan
Manager Seed
ICI House, 63 Mozang Road, Lahore
Ph: 111100200/ Fax: 042-6307104
Email: [email protected]
Dr. Muhammad Qayyum Khan
Associate Professor
Faculty of Agriculture,
Dept Plant Br & Mol Gen,
Rawalakot, Azad Kashmir
P: 058710-42688,43193/ Fax: 05871042826
Email: [email protected]
Ms. Asia Khatoon
Scientific Officer
CCRI,P O Box 572, Multan
Ph: 061-9200340-1/ Fax: 061-9200342
Email: [email protected]
Mr. Hamid Malik
Manger Export
Roberts Rice, 35KM Main GT Road,
Muridke
Distt. Lahore
Ph:042-7990877/ Fax:042-7992077
Email: [email protected]
Dr. Shaukat Iqbal Malik
Associate Professor
Center for Biotechnology and
Informations,
BUITMS, Quetta
Ph: 081-92010-51 ext 257/ Fax: 0819201064
Email: [email protected]
Ms. Tanveer Fatima Miano
Lecturer
Horticulture Dept. Sindh Agriculture
University
Tandojam
Ph: 0303-6153538
Mr. Aamir Mahmood Mirza
Manager PA/GA/RA
Monsanto Pakistan Agri Tech (Pvt) Ltd.
ABN AMRO Building
2nd Floor, 310-Upper Mall,
P.O.Box No. 10148, Lahore
Tel: 042-111-106-106;Fax: 042-5877152
Email:[email protected]
nto.com
Mr. Nadeem Zafar Mirza
Syngenta Pakistan LTD. 90-Industrial
Area Kotlakhpat
Lahore
Ph:042-5153059/ Fax:042-5153069
Email: [email protected]
170
NIBGE-FAO Workshop on GMO Detection
Mr. Hamid Mukhtar
Lecturer
Biotechnology Research Centre,
G.C University, Lahore
Fax: 042-9211634
Mr Muhammad Naeem
Seed Analyst
Federal Seed Certification & Research
Department, G-9/4, Mauve area,
Islamabad
Ph:051- 9260126/ Fax: 051- 9260234
Email: [email protected]
Mr Ijaz Ahmad Rao
Journalist
Bhawalpur
Tel: 0621-874861
Email: [email protected]
Mr. Syed Hassan Raza
Director
Neelum Seed Company
10 Gardezi Colony, Mall Road
Multan Cantt.
Mobile: 0303-7972747
Mr M. Hammad Nadeem Tahir
Lecturer
Department Plant Breeding and Genetic
University of Agriculture
Faisalabad
Mr. Syed Hassan Raza
Director
Neelum Seed Company
10 Gardezi Colony, Mall Road
Multan Cantt. Mobile: 0303-7972747
Dr Bushra Sadia
Assistant Botanist
Agriculture Biotech. Research Institute
(AARI), Jhang Road, Faisalabad
Ph: 041-551142, Email:
[email protected]
Ms. Kiran Shafiq
Research Associate
Biotechnology Research Centre, Botany
Dept., G C University, Lahore
Ph: 042-9211634/ Fax: 042-7242820
Email: [email protected]
Mr Mehboob Ali Sial
Senior Scientific Officer
Nuclear Institute of Agriculture (NIA),
Tandojam, Sindh
Ph: 0221-765750, Fax: 0221-765284
Mr M. Hammad Nadeem Tahir
Lecturer
Department Plant Breeding and Genetic
University of Agriculture
Faisalabad
Ph: 041-9200161-70 Ext 2921
Dr. Tahira Yasmin
Scientific Officer
Institute for Plant & Environmental
Protection,
NARC, Islamabad
Ph: 051-9255177/ Fax: 051-9255036
Email: [email protected]
171
NIBGE-FAO Workshop on GMO Detection
SUPPLIERS
Deal in
ABI Applied Biosystems
Equipments & Chemicals
Local Distributor:
(Real time PCR)
AMS Analytical Measuring Systems (Pvt.) Ltd.
9-Malik Complex, 3rd Floor
80 West Blue Area, Islamabad
Tel: 92-51-2277212, 2277391, Fax: 92-51-2277132
Email: [email protected]
Web site: www.anaams.com
BIO-RAD
Local Distributor:
Chemical House
G P O Box 1138, 2-Albert St.
Behind Commercial Building
The Mall, Lahore-54000, Pakistan
Tel: 042-7223098, 7351690 & 7241019-20,
Fax: 92-42-7225983
Email: [email protected]
Equipments & Chemicals
(Real time PCR)
Scientific Supplies (Pvt.) Ltd
121-A, Block 2,
PECHS,
Karachi-75400
PO Box No. 8956, Pakistan
Tel.: (92-21) 4555617
Fax: (92-21) 4557446
E-mail: [email protected]
Equipments & Chemicals
The World Wide Scientific
(Science and technology services)
45, 50-B, Syed Plaza
30-Ferozepure Road, Lahore
Tel.: (92-42) 7552355, Fax: (92-42) 7553255
Email: [email protected]
Enzymes, Chemicals and Kits
KHB Traders
18-B, Kareem Block,
Allama Iqbal Town, Lahore
Tel: 042-7845012
Email: [email protected]
GMO testing Kits & Chemicals
Lucky Lab International
F-6, Usmania Centre,
Muhammad Ali Society,
Commercial Market, Karachi, Pakistan
Tel.: (92-21) 4383054, 4383055, 4383057
Fax: (92-21) 438356
E-mail: [email protected]
Equipments, Thermal cyclers
NIBGE-FAO Workshop on GMO Detection
AUTHORS INDEX
Name
Page No.
Asif, M.
37, 70, 98, 109
Bashir, A.
60
Kabaki, N.
11
Khalid, A. M.
21, 90
Khan, M. S.
64
Malik, K. A.
06
Mansoor, S.
57
Nasim, A.
21
Rahman, M.
79
Shinwari, Z. K.
21
Zafar, Y.
16, 30, 90
172