Biotechnology in Modern Breeding and Agriculture
Ankica Kondić-Špika, Borislav Kobiljski
Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia
e-mail: [email protected]
Abstract
Improved crops will be needed to feed the world and save land for the conservation of plant
biodiversity in natural habitats. These goals could be achieved through multidisciplinary cooperation among plant breeders, biotechnologists, and other plant scientists. Plant breeding has
a long history of integrating the latest scientific knowledge and innovations to enhance crop
improvement. Methods of modern biotechnology, such as in vitro cultures and marker-assisted
selection (MAS), may assist in the development of high quality crops with improved nutritional
and health characteristics as well as other aspects of added-value. Because of their great potential
and importance, these methods have been applied in plant breeding and seed production at the
Institute of Field and Vegetable Crops, Novi Sad, Serbia for more than three decades. This article
reviews some of the highlights of modern plant biotechnology and the results of its application in
NS breeding programs.
Key words: modern biotechnology, application, breeding
Introduction
In the broadest meaning, biotechnology is the use of biological systems and living organisms to
develop or modify products and processes for specific use. This definition includes the most diverse
processes such as production of wine, beer, bread, dairy products, composting of organic matter,
plant and animal breeding, the production of drugs, etc. Also, biotechnology can be defined as a field
of applied biology that involves the use of living organisms and bioprocesses in industry, technology,
agriculture, medicine, and other fields in which bio-products could be developed. However, all
these examples and definitions are primarily related to the so-called classical biotechnology.
Modern biotechnology covers a wide range of methods and procedures developed in two very
important areas: cell, tissue and organ cultures and molecular biology. The first theory and the
scientific basis for the development of tissue culture have emerged in the 19th and the early 20th
century. However, significant research began in the mid 1930s after the first discovery of natural
growth regulator, auxin indole-3-acetic acid. The development of techniques and methods of
molecular biology has recorded a significant growth in the second half of 1970s. This contributed
to the development of methods for direct manipulation of DNA, known as genetic engineering, and
methods of decoding DNA sequences (Vasil, 1999). These findings very quickly found application in
genetics and plant breeding, and led to the improvement of the old methods with new experimental
tools, and creation of completely new, powerful techniques, such as various types of molecular
markers, DNA fingerprinting, automated DNA sequencing, marker assisted selection (MAS), and
so on.
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Conventional breeding and biotechnology
In traditional plant breeding, new varieties are developed by combining desirable characteristics
from parents and by selecting superior plant phenotypes in the progeny. Conventional plant breeding
has produced a number of highly successful varieties of crops over the centuries. In traditional
breeding, crosses are often made in a relatively uncontrolled manner and consequently the results are
unpredictable. However, traditional breeding programs are time-consuming, and labour-intensive. A
great effort is required to select undesirable from desirable traits, and this is not always economically
effective. Many potential benefits could be lost along the way, as plants that fail to demonstrate the
introduced characteristics are discarded. Conventional plant breeding takes on average 12-15 years to
produce a new crop variety (Moose and Mumm, 2008).
Biotechnology can be used to increase productivity, improve the breeding process, and increase
its efficiency and effectiveness. The genetic base of crop production can be preserved and widen by an
integration of biotechnology tools in conventional breeding (Rajaram, 2005). High quality crops with
improved nutritional and health characteristics as well as other aspects of added-value may be obtained
through multidisciplinary co-operation among plant breeders, biotechnologists, and other plant scientists.
Biotechnology in NS breeding programs
First biotechnology laboratories at the Institute of Field and Vegetable Crops were established in
the early 1980s. At the beginning, biotechnology research involved the development and application
of tissue culture methods and later intensifies the work on molecular methods. Thus, techniques such
as micropropagation, anther and embryo cultures, as well as different marker systems have become
an integral part of the research and breeding programs of wheat, sunflower, rapeseed, sugar beets,
soybeans, maize and other crops in the Institute.
Tissue culture and plant regeneration
Development of micropropagation techniques, known as tissue culture was very important for plant
breeding. Tissue culture techniques enables researchers vegetative propagation of plant material by
excising small amounts of tissue from plants of interest, and then inducing growth and differentiation
of plant tissue on media, to ultimately form a new plant. This new plant carries the entire genetic
information of the donor plant. Exact copies of a desired plant (clones) could thus be produced quickly,
without depending on pollinators, the need for seeds, etc. (Thorpe, 2007).
Specialized plant tissue culture methods have enabled the production of completely homozygous
lines from gametic cells in a shortened time, compared to conventional plant breeding. Plants derived
from gametic cells represent a homozygous array, each having a different genetic contribution from
the parents. Doubled haploid production is widely used not only for plant breeding (De Buyser et al.,
1987; Pauk et al., 1995), but also for basic research (Orshinsky et al., 1990), such as genomic mapping,
haploid transformation and artificial seed production (Tuvesson et al., 2003).
Androgenesis is a common methodology used to develop haploids, and doubled haploids, in major
grain crops. The formation of androgenetic structures and regenerant plants depends on the genotype
of the anther donor plant, its growth environment, culture conditions and their interactions (Lazar et
al., 1985; Kondić and Šesek, 1998; 1999; Tuvesson et al., 2000; Redha and Talaat, 2008; Ljevnaić and
Kondić, 2008). Production of doubled haploid lines (DHL) is commonly used by plant breeders in
order to obtain pure lines in the shortest possible time. In addition to wide hybridizations, the anther
culture method is a usual way to produce DH plants in cereals (Pauk et al., 1995; Ljevnaić et al., 2007).
These techniques were also used in the Institute for the production of wheat DH lines. The produced
DH lines were evaluated for important agronomical traits and the most promising lines were involved
in the wheat breeding programs (Šesek et al., 1994; Kondić-Špika et al., 2007).
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Embryo culture
Distantly related plant species could not be hybridized by traditional crossing methodology,
because the embryos formed following fertilization were often aborted. The development of embryo
rescue technology permitted crop breeders to make crosses among distantly related species by growing
resulting embryos into whole plants through tissue culture (Thorpe, 2007).
Embryo culture could also be used for many other purposes, such as testing of plants for biotic and abiotic
stress tolerance, in vitro selection, induction and use of somaclonal variation, etc. It is often considered
desirable to have a controlled-environment screening system, in which plant reactions to different stresses
could be evaluated uniformly and rapidly. A screening system may become generally acceptable when
it is based on simple selection criteria, provides rapid and accurate screening of large numbers, is nondestructive, reproducible and relates to filed performance (Stoddard et al., 2006). Methods of in vitro culture
meet the desired criteria in many aspects, and because of that they were frequently used as supplement to
classical breeding methods for testing crops tolerance to abiotic stresses (Huang and Graham, 1990; Šesek
et al., 1999; Kondić-Špika and Jevtić, 2002; Stoddard et al., 2006; Kondić-Špika et al., 2006; 2009).
Genetic variability
The presence of wide genetic variability in the starting material is one of the most important
prerequisites for a successful breeding program. There are different methods for assessing genetic
variability of cultivated species, such as morphological, biochemical and molecular markers.
Molecular markers and more recently, high-throughput genome sequencing efforts, have dramatically
increased ability to characterize genetic diversity in the germplasm pool for any crop species. Using maize
as an example, surveys of molecular marker polymorphisms and nucleotide sequence variations have
provided basic information on genetic diversity before and after domestication from its wild ancestor
teosinte, among geographically distributed landraces, and within elite germplasm (Cooper et al., 2004;
Niebur et al., 2004; Buckler et al., 2006). This information enriches investigations of plant evolution and
comparative genomics, contributes to our understanding of population structure, provides empirical
measures of genetic responses to selection, and also serves to identify and maintain reservoirs of genetic
variability for future mining of beneficial alleles (McCouch, 2004; Slade et al., 2005). In addition,
knowledge of genetic relationships among germplasm sources may guide choice of parents for production
of hybrids or improved populations (Dudley et al., 1992; Collard and Mackill, 2008).
A number of authors have successfully used different molecular markers to determine the level of
genetic diversity in collections of various crops. The results of molecular evaluations could be used
for further calculations and construction of dendrograms that allow us to interpret and compare the
results obtained by using different techniques (Prasad et al. 2009). At the Institute of Field and Vegetable
Crops molecular markers were routinely used to analyze the genetic variability of a number of crops,
such as wheat (Kobiljski et al., 2002, Kondić-Špika et al., 2008), sunflower and its wild relatives (Vasić
et al., 2003), rapeseed (Marjanović-Jeromela et al., 2009), alfalfa (Nagl et al., 2010; 2011a), sugar beet
(Nagl et al., 2011b) and other crops.
DNA fingerprinting
Specific DNA profile for a particular organism could be obtained as a result of molecular
characterization by various techniques. These profiles are independent of environment, and consistent
throughout different parts and developmental stages of the organism. Similarity of DNA profiles
depends on the genetic closeness of tested samples. DNA fingerprinting can be used to identify genetic
diversity within breeding populations, to differentiate accessions, cultivars, and species that might be
difficult to characterize due to similar morphological characteristics or indistinct traits, and to identify
plants containing genes of interest such as the confirmation of transformation events (Weising et al.,
2005; Kondić and Kobiljski, 2011).
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Biotechnology in Modern Breeding and Agriculture
DNA profiling of lines and varieties of cultivated species have recently been performed at the
Institute of Field and Vegetable Crops. Thus, DNA profiles of 180 elite wheat varieties, bred in different
breeding centres in Serbia were obtained by using microsatellite molecular markers (Kondić-Špika
et al., 2010). In addition to wheat, DNA profiling of 96 soybean genotypes with 15 SSR markers was
performed, while fingerprinting of 96 maize inbred lines with 40 markers and 96 bean genotypes
with 20 markers are in progress. This work should enable profiling of varieties and lines of cultivated
species, created in Serbia in the past 40-50 years and the opportunity for a reliable and internationally
recognized protection of our results.
Induced mutations
While molecular markers and other tools for genomic analysis have been highly successful in
characterizing existing genetic variation within species, plant biotechnology generates new genetic diversity
that often extends beyond species boundaries (Gepts, 2002; Johnson and McCuddin, 2008). Mutations are
changes in the genetic background of a plant, which occur naturally and sometimes result in the development
of new beneficial traits. In 1940, plant breeders learned that they could make mutations happen faster with a
process called mutagenesis. Radiation or chemicals are used to change the plant’s DNA, the basic molecular
system of all organisms’ genetic material. The goal is to cause changes in the sequence of the base pairs of
DNA, which provide biochemical instructions for the development of plants. Resultant plants may possess
new and desirable characteristics through this modification of their genetic material. During this process,
plant breeders must grow and evaluate each plant from each seed produced.
In the study by Borojević (1978) some evolutionary processes which operate in populations of
wheat after mutagenic treatments, especially those which may be im­portant in plant breeding were
analysed. The material used con­sisted of two populations of wheat after irradiation in the M5, M6
and M7 generations. Irradiation and the processes which ap­peared after treatment increased genetic
variability in popula­tions of wheat. Increased genetic variability allowed an inten­sive operation of
natural selection, which resulted in an increase in the mean values of polygenic characters, especially
those which were the components of adaptive value in wheat. There­fore, artificial selection may be
more effective if were applied in a positive direction, in which natural selection already operated.
More than 3400 plant varieties from 224 different plant species have been developed using
mutagenesis in more than 60 countries throughout the world (FAO/IAEA, 2008). Induced mutation
breeding was widely used in the United States during 1970s, but today few varieties are produced using
this technique. As our understanding of genetics developed, so new technologies for plant variety
development arose. Examples of these that are used today include marker assisted breeding, where
molecular markers associated with specific traits could be used to direct breeding programs.
Marker assisted selection (MAS)
Selection with markers represents the practical application of molecular markers in plant breeding.
Marker assisted selection is the last stage of the process, which involves finding the source of favourable
alleles, detection of polymorphic markers by genome analysis, the correlation between allelic variation
of markers and traits of interest, determination of the effect of candidate genes and quantitative trait
loci (QTLs), checking their stability in multiple environments and different genetic backgrounds
(Treskić et al., 2011). Only after all these phases of testing, it is possible to distinguish markers that can
be potentially useful for implementation of the so-called molecular breeding.
Previous efforts to develop large numbers of molecular markers, high density genetic maps, and
appropriately structured mapping populations have now made routine for many crop species the
ability to simultaneously define gene action and breeding value at hundreds and often thousands of
loci distributed relatively uniformly across entire genomes. The results from such mapping studies
provide greatly improved estimates for the number of loci, allelic effects, and gene action controlling
traits of interest. More importantly, genomic segments can be readily identified that show statistically
204
significant associations with quantitative traits (QTLs). In addition to genetic mapping in families
derived from biparental crosses, new advances in association genetics with candidate genes and
approaches that combine linkage disequilibrium analysis in families and populations further enhance
power for QTL discovery (Holland, 2007; Yu et al., 2008).
For traits exhibiting low to moderate heritability, such as grain yield, QTLs, and their associated
molecular markers often account for a greater proportion of the additive genetic effects than the
phenotype alone. Success in using information about QTLs to increase genetic gain depends greatly
on the magnitude of QTL effects, precise estimation of QTL positions, stability of QTL effects across
multiple environments, and whether QTLs are robust across relevant breeding germplasm. Prediction
of QTL positions is enhanced by further fine mapping, which facilitates testing QTL effects and
breeding values in additional populations (for review see Salvi and Tuberosa, 2005; Yu and Buckler,
2006; Belo´et al., 2008; Harjes et al., 2008).
Numerous studies have been performed at the Institute, both independently and in cooperation
with prestigious institutions in the world, in order to find significant relationships between agronomic
traits and candidate markers for MAS. Thus, using different methodologies and approaches potentially
useful candidate markers were identified: in wheat for the flowering and heading date, yield components
(number and grain weight), quality parameters (protein content and sedimentation), drought tolerance
(Kobiljski et al., 2009; Brbaklić et al., 2010; Neumann et al., 2010; Trkulja et al., 2011; 2012; Dodig et
al., 2011), in sunflower for resistance to broomrape and downy mildew (Panković et al., 2007; Jocić et
al., 2010; Imerovski, 2010; Dimitrijević et al., 2010a), while the intense research on soybean, corn and
sugar beets are in progress.
Biotechnology in seed production
Determination of genetic purity of varieties and hybrids
One of the important components of seed quality is its genetic identity and uniformity, and varietal
purity. Genetic purity of cross-pollinated plant species, especially if the production is based on the use
of hybrids is of particular importance. Causes of varietal impurities can originate from seed production
whether due to uncontrolled cross-pollination, or due to interference of seeds of different varieties/
hybrids (Zlokolica and Taški, 2006). Therefore, in the Laboratory for seed testing of the Institute of
Field and Vegetable Crops genetic purity of varieties and hybrids is routinely controlled. These tests
are based on molecular markers that can be biochemical and DNA markers. Since the techniques
based on DNA molecular markers are more expensive, electrophoretic methods based on storage
proteins and functional proteins/isoenzymes are still widely used (Nikolić, 2010; Taški-Ajduković and
Nagl, 2011). Techniques based on DNA molecular markers are used to solve specific problems that
sometimes occur in seed production, because they allow more precious analyses of the problems and
accurate concussions about the causes.
Thus, phenomena with more atypical plants in seed production of high yielding wheat variety
NS40S has been observed at several locations. Samples from these sites were analyzed by the set
of primers used in our fingerprinting study (Kondić-Špika et al., 2010), and the obtained results
were compared with already identified DNA profile of this variety. The obtained results led to the
conclusion that atypical plants at one of the tested sites do not belong to the NS40S variety, or that
it is a mechanical admixture. In the other two sites the results showed that there was an appearance
of a certain percentage of cross-pollination, probably due to a very open kind of flowering which
the variety has. The emergence of open flowering has been observed in other high-yielding varieties
of wheat, not only in our country but also abroad. For this reason, the future investigations should
be directed towards determining the relationship between this phenomenon and the high yields in
wheat, as well as finding mechanisms which will allow that such problems are prevented in the future.
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Biotechnology in Modern Breeding and Agriculture
Similar problem was observed in the seed production of maize hybrid Radan. At several sites of
the hybrid seed production a significant reduction in corn yield was recorded due to emergency of
the ears with small number of poorly filled grains. DNA analysis with 12 SSR markers showed that
the DNA profile of the samples of corn seed does not match the profile of the standard hybrid Radan.
In this way, molecular marker analysis quickly and unambiguously demonstrated that corn seed from
these sites do not belong to the hybrid Radan and that there was an uncontrolled pollination or seed
mixing.
Determining the degree of homozygosity
and heterozygosity of sugar beet lines
Parental lines of sugar beet hybrids have different abilities of self-fertilization, which results in the
fact that they are very different from each other in the degree of homozygosity. In order to determine
the extent of homozygosity and its possible association with combining ability of lines, the work on the
application of microsatellite markers (SSR) in their analysis was recently started. In the preliminarily
study PCR reactions for 36 primer pairs were optimized. Testing homozygosity of sugar beet lines with
optimized primers will become the routine analysis.
Testing isogenic lines
Use of molecular markers can significantly reduce the number of back crosses required to enter
certain characters in the recipient line (Knapp, 1998). Molecular markers provide a reliable picture of
the variability of the offspring at the molecular level in each generation, thus avoiding the errors that
arise when using phenotypic traits in selection (Moose and Mumm, 2008).
The possibility of using different SSR markers to determine the level of similarity between the
parental lines and progeny obtained by recurrent selection (F6 and F7) was investigated. Three of
the tested markers were selected (ORS 509, ORS 610 and ORS 1209) due to their ability to clearly
differentiate between the recipient and the donor line (Dimitrijevic et al., 2010b). These markers will
further be used in sunflower breeding program to increase the efficiency and reliability of back crosses
and checking uniformity of parental lines.
Conclusions
Many different tools are available for increasing and improving agricultural production. These tools
include methods to develop new varieties such as classical breeding and biotechnology. Traditional
agricultural approaches are experiencing some resurgence today, with renewed interest in organic
agriculture. The role that biotechnology stands to play in sustainable agricultural development is an
interesting topic for the future.
Biotechnology and molecular biology methods have proven to be effective tools in NS breeding of
the most important crop plant species, especially in areas where conventional breeding has reached its
limits. These methods are expected to contribute to increase of yield of NS varieties and hybrids, as well
as improvement of their tolerance to drought and major pathogens. Further work will be focused on
reducing costs and optimizing the conditions for widespread application of biotechnology methods,
in order to improve efficiency of NS breeding programs.
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