IN VITRO SCREENING FOR DROUGHT TOLERANCE IN DIFFERENT
SORGHUM (Sorghum bicolor (L.) Moench) VARIETIES
A Thesis Submitted to the College of Natural and Computational Sciences
Department of Biology, School of Graduate Studies
HARAMAYA UNIVERSITY
In Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE IN GENETICS
By
Yohannes Tsago Chare
April, 2012
Haramaya University
SCHOOL OF GRADUATE STUDIES
HARAMAYA UNIVERSITY
As Thesis Research advisor, we here by certify that we have read and evaluated this thesis
prepared, under our guidance, by Yohannes Tsago Chare, entitled In Vitro Screening for
Drought Tolerance in Different Sorghum (Sorghum Bicolor (L.) Moench) Varieties. We
recommend that it be submitted as fulfilling the Thesis requirement.
Mebeaselassie Andargie (PhD)
Major Advisor
Abuhay Takele (PhD)
Co-advisor
_________________
________________
Signature
__________________
Date
________________
Signature
Date
As member of the Board of Examiners of the Final M. Sc. Thesis Open Defense Examination,
we certify that we have read, evaluated the Thesis prepared by Yohannes Tsago Chare and
examined the candidate. We recommended that the Thesis be accepted as fulfilling the Thesis
requirement for the Degree of Master of Science in Genetics.
______________________
Chairperson
______________________
Internal Examiner
______________________
External Examiner
_________________
_______________
Signature
Date
_________________
Signature
_________________
Signature
ii
_______________
Date
_______________
Date
DEDICATION
I would like to dedicate this thesis to my deceased younger sister Amsal Tsago who always had
something nice to say and to my mother Walolie Wada and my father Tsago Chare who never
gave up on me.
iii
STATEMENT OF AUTHOR
First, I solemnly declare that this thesis is my bonafide work and that all sources of materials
used for this thesis have been duly acknowledged. This thesis has been submitted in partial
fulfillment of the requirements of M. Sc. degree at the Haramaya University and is deposited
at the University Library to be made available to borrowers under rules of the Library. I
solemnly declare that this thesis is not submitted to any other institution anywhere for the
award of any academic degree, diploma, or certificate.
Brief quotations from this thesis are allowable without special permission provided that
accurate acknowledgement of source is made. Requests for permission for extended quotation
from or reproduction of this manuscript in whole or in part may be granted by the head of the
major department or the Dean of the School of Graduate Studies when in his or her judgment
the proposed use of the material is in the interests of scholarship. In all other instances,
however, permission must be obtained from the author.
Signature: …………………
Name: Yohannes Tsago Chare
Place: Haramaya University, Haramaya
Date of Submission: …………………
iv
BIOGRAPHICAL SKETCH
The author was born on May 24, 1988 in Southern Nations Nationalities and People’s Region
(SNNPR), Gamo Gofa Zone, Dita Woreda a small Kebele called Anduro to his father Tsago
Chare Bashe and his mother Walolie Wada. He attended his elementary and junior education
(1-8 grades) at Anduro Elementary School and Chencha Primary and Junior Secondary School
respectively. He also attended his high school education (9-12) at Chencha Senior Secondary
School. Then joined Haramaya University in 2006/07 and graduated with B.Sc. in Plant
Sciences in July 2009.
Soon after graduation, he was employed by the Ministry of Education (MoE) and joined
Haramaya University to pursue graduate studies in 2010 for the M.Sc. degree in Genetics.
v
ACKNOWLEDGMENTS
It would have been difficult to finalize this thesis research without the help of many people and
organizations. First and foremost, my heartfelt appreciation and gratitude goes to my research
advisors Dr. Mebeaselassie Andargie and Dr. Abuhay Takele for their constant instruction,
guidance, intellectual feedback, enthusiasm and invaluable suggestions while designing and
executing the laboratory experiment and during the write-up of the thesis.
I want to express my deepest gratitude and appreciation to the Ministry of Education (MoE) for
permitting me to join the school of graduate studies and financing the study. I also want to
express my deepest gratitude and appreciation to the Ethiopian Institute of Agricultural Research
(EIAR) and Melkassa Agricultural Research Center (MARC) for collaborating and permitting
work place. My sincere gratitude and indebtedness is extended to Abel Debebe and the
biotechnology section staff as well as sorghum improvement section of MARC for their constant
cooperation in sharing their experience during the laboratory work and providing me the
necessary resources on time.
Finally, I would like to thank my uncle Mathewos Bekele and his family for supporting me
through the more difficult times of my life.
vi
ACRONYMS AND ABBREVIATIONS
2, 4-D
2, 4-Dichlorophenoxyacetic Acid
ABA
Abscisic Acid
BA
Benzylaminopurine
CDK
Cyclin-Dependent Kinase
CFW
Callus Fresh Weight
CIE
Callus Induction Efficiency
CL
Coleoptile Length
CRD
Completely Randomized Block Design
CSA
Central Statistical Authority
ECP
Embryogenic Callus Percent
EIAR
Ethiopian Institute of Agricultural Research
FAO
Food and Agricultural Organization
IAA
Indole-3-acetic Acid
IBA
Indole-3-butyric Acid
ICRISAT
International Crop Research Institute for the SemiArid Tropics
IPCC
Intergovernmental Panel on Climate Change
KIN
Kinetin
MARC
Melkassa Agricultural Research Center
MoE
Ministry of Education
MS
Murashige and Skoog
MW
Molecular Weight
NAA
1-Naphthaleneacetic Acid
PEG
Polyethylene glycol
vii
ACRONYMS AND ABBREVIATIONS (Continued)
PRP
Plant Regeneration Percent
QTL
Quantitative Trait Loci
RDW
Root Dry Weight
RL
Root Length
RN
Root Number
RS
Rank Sum
RSDW
Root to Shoot Dry Weight Ratio
SDW
Shoot Dry Weight
viii
TABLE OF CONTENTS
DEDICATION
iii
STATEMENT OF AUTHOR
iv
BIOGRAPHICAL SKETCH
v
ACKNOWLEDGMENTS
vi
ACRONYMS AND ABBREVIATIONS
vii
TABLE OF CONTENTS
ix
LIST OF TABLES
xii
LIST OF FIGURES
xiii
LIST OF TABLES IN THE APPENDIX
xiv
ABSTRACT
xvi
1. NTRODUCTION
1
2. LITERATURE REVIEW
5
2.1 Sorghum
5
2.1.1 Origin, distribution and taxonomy of sorghum
5
2.1.2 World production and economic importance
7
2.2 Plant Water Deficit (Drought) Stress
9
2.3 The Genetics and Mechanisms of Drought Tolerance in Plants
10
2.4 Effects of Drought on Plant Growth and Development
12
2.4.1 Callus growth dynamics
12
2.4.2 Seedling growth and physiology
13
2.5 Sorghum Research for Drought Tolerance in Ethiopia
14
2.6 The Role of Plant Tissue Culture and Application of PEG in Drought Tolerance
Screening
15
ix
TABLE OF CONTENTS (Continued)
2.6.1 Composition of in vitro culture medium
17
2.6.2 Plant hormones and growth regulators
17
2.6.3 PEG as an in vitro drought inducer
19
3. MATERIALS AND METHODS
20
3.1 Description of the Research Site
20
3.2 Experimental Design
20
3.3 Plant Materials
20
3.4 In Vitro Screening
20
3.4.1 Callus induction under PEG stress
20
3.4.2 Plant regeneration from induced callus in PEG stress condition
21
3.5 Data Recorded and Statistical Analysis
22
4. RESULT AND DISCUSSION
23
4.1 Callus Induction, Proliferation and Plant Regeneration
23
4.1.1 The effect of PEG induced moisture deficit on callus induction efficiency (CIE)
23
4.1.2 The effect of PEG induced moisture deficit on callus fresh weight (CFW)
25
4.1.3 The effect of PEG induced moisture deficit on embryogenic callus percent (ECP)
28
4.1.4 The effect of PEG induced moisture deficit on plant regeneration percent (PRP)
30
4.2 Morpho-physiological Study of Regenerated Plants
33
4.2.1 The effect of PEG induced moisture deficit on coleoptile length (CL)
33
4.2.2 The effect of PEG induced moisture deficit on root length (RL)
35
4.2.3 The effect of PEG induced moisture deficit on shoot dry weight (SDW)
37
4.2.4 The effect of PEG induced moisture deficit on root dry weight (RDW)
39
4.2.5 The effect of PEG induced moisture deficit on root shoot dry weight ratio (RSDW) 41
4.2.6 The effect of PEG induced moisture deficit on root number (RN)
43
4.3 Association between In vitro Traits
45
4.4 Mann–Whitney–Wilcoxon Rank Sum Test of the Measured Traits
47
x
TABLE OF CONTENTS (Continued)
5. SUMMARY AND CONCLUSION
51
6. REFERENCES
54
7. APPENDICES
70
Appendix A: Main Effects of Genotypes across PEG levels
81
Appendix B: Main Effect of PEG Levels across Genotypes
74
Appendix C: General ANOVA Table of the Studied Traits
75
Appendix D: Culture Media Composition and Preparation
78
xi
LIST OF TABLES
Tables
Page
1. Mean callus induction efficiency of genotypes for each PEG treatment level…………….…24
2. Mean callus fresh weight of genotypes for each PEG treatment level……………………….27
3. Mean embryogenic callus percent of genotypes for each PEG treatment level……………....29
4. Mean plant regeneration percent of genotypes for each PEG treatment level………………..32
5. Mean coleoptile length of genotypes for each PEG treatment levels…………………………34
6. Mean root length of genotypes for each PEG treatment levels……………………….............36
7. Mean shoot dry weight of genotypes for each PEG treatment level………………….............38
8. Mean root dry weight of genotypes for each PEG treatment level…………………………...40
9. Mean root shoot weight ratio of genotypes for each PEG treatment level…………………....42
10. Mean root number of genotypes for each PEG treatment level………………………….…..44
11. Correlation coefficient of measured traits…………………………………………………...46
12. Rank sum of all traits measured……………………………………………………………...49
xii
LIST OF FIGURES
Figures
Page
1. Callus induction difference due to PEG stress
……………………………………………23
2. Callus growth difference due to PEG stress………………..…………………………………26
3. Plant regeneration difference due to PEG stress…...................................................................31
xiii
LIST OF TABLES IN THE APPENDIX
Appendix Tables
Page
Appendix A: Main Effects of Genotypes across PEG levels………………………………..71
Appendix Table 1: Mean callus induction efficiency, callus fresh weight, embryogenic
callus percent and plant regeneration percent of the tested
genotypes…………………………………………………………………....71
Appendix Table 2: Mean coleoptile length, root length and root number of the tested
Genotypes…………………………………………………………………...72
Appendix Table 3: Mean shoot and root dry weight and root shoot dry weight ratio of the
Tested genotypes……………………...…………………………………….73
Appendix B: Main Effect of PEG Levels across Genotypes…………………………………74
Appendix Table 4: Mean callus induction efficiency, callus fresh weight, embryogenic
callus percent and plant regeneration across the PEG stress levels………...74
Appendix Table 5: Mean coleoptile length, Root length and root number across
the PEG treatment levels……………………………………………………74
Appendix Table 6: Shoot and root dry weight and root shoot dry weight ratio across
the PEG stress levels………………………………………………………..75
Appendix C: General ANOVA Table of the Studied Traits………………………………....75
Appendix Table 7: Analysis of Variance Table for CIE ………………………………………...75
Appendix Table 8: Analysis of Variance Table for CFW ………………………………………75
Appendix Table 9: Analysis of Variance Table for ECP …………………….….………………76
Appendix Table 10: Analysis of Variance Table for PRP ……………….…….………………..76
Appendix Table 11: Analysis of Variance Table for CL ..............................................................76
Appendix Table 12: Analysis of Variance Table for RL ………………………..………………76
xiv
LIST OF TABLES IN THE APPENDIX (Continued)
Appendix Table 13: Analysis of Variance Table for SDW ……………………………….…….77
Appendix Table 14: Analysis of Variance Table for RDW………………...……………………77
Appendix Table 15: Analysis of Variance Table for RSDW…....................................................77
Appendix Table 16: Analysis of Variance Table for RN …………………………………….....78
Appendix D: Culture Media Composition and Preparation…………………………….…..78
Appendix Table 17: Stock solution of macro nutrients…….……………………………………78
Appendix Table 18: Stock solution of micro nutrients….............................................................78
Appendix Table 19: Fe-EDTA (Ethylenediaminetetra acetic acid) solution Preparation…….....79
Appendix Table 20: Vitamins, Amino acids, Carbon and energy sources, growth regulators,
and solidifying agent………………………………………………………..79
Appendix Table 21: Preparation of MS medium (1000 ml)…………………………………......80
Appendix Table 22: Agroecological adaptation of the selected sorghum varieties……………..81
xv
IN VITRO SCREENING FOR DROUGHT TOLERANCE IN DIFFERENT
SORGHUM (Sorghum bicolor (L.) Moench) VARIETIES
ABSTRACT
Drought is one of the complex environmental factors affecting growth and yield of sorghum in
arid and semi-arid areas of the world. Developing crops that have the mechanism to cope with
such drought prone production environments is vital. Callus culture is a novel approach
addressing cultured cells as selection units independent of whole plant. Sixteen elite sorghum
genotypes (Abshir, Chelenko, Raya, Hormat, Gubiye, Gambella-1107, Birmash, Meko, Macia,
Seredo, Misikir, Melkam, 76T1#23, ESH-2, Girana-1, and Teshale) were evaluated using PEG
as osmoticum at cellular level and then plantlet stage for drought tolerance. The factorial
experiment was laid down in a completely randomized design which comprised of a combination
of two factors (genotypes and five PEG stress level; 0, 0.5, 1.0, 1.5, and 2.0% (w/v) treatments).
The data regarding callus induction efficiency, callus fresh weight, embryogenic callus
percentage, plant regeneration percentage, coleoptile length, root length, shoot dry weight, root
dry weight, root shoot dry weight ratio and root number revealed highly significant (P< 0.01)
interaction of genotypes with the PEG treatments. The correlation analysis revealed strong and
significant association between embryogenic callus percent and plant regeneration percent as
well as between shoot dry weight and root dry weight. By taking into consideration all the
measured traits, Mann Whitney rank sum test revealed that 76T1#23 and Teshale followed by
Meko, Gambella-1107 and Melkam showed better drought stress tolerance. Therefore they are
recommended to be used as parents for genetic analysis, gene mapping and improvement of
drought tolerance. Chelenko, Hormat and Raya appear to be sensitive, therefore they are
recommended for crossing and genetic analysis of drought tolerance using diallel mating design
or generation mean analysis and also for the QTL (quantitative trait loci) mapping and marker
assisted selection.
Key words/phrases: Sorghum genotypes, drought tolerance, callus culture, PEG
xvi
1. NTRODUCTION
Sorghum (Sorghum bicolor (L.) Moench), a tropical plant belonging to the family Poaceae, is
the fifth most important crop in many parts of the world and grown for food, feed and industrial
purposes. Being a major crop in many parts of Africa, Asia and Latin America (ICRISAT, 2006),
the crop is the second crop next to maize grown across all agroecologies in Africa (Wortmann et
al., 2006). In Ethiopia, it is the fifth major cereal crop in terms of area and production next teff
(Eragrostis teff), barley (Hordeum vulgare), wheat (Triticum aestivum L.) and maize (Zea mays)
(CSA, 2011). This crop is the major crop in drought stressed lowland areas that cover 66% of the
total arable land in the country (Gebeyehu et al., 2004). The main locations of sorghum
production are the north central, northwestern, western and the eastern mid-altitude areas of the
country (Wortmann et al., 2006).
Compared to other cereals, sorghum is more tolerant to many stresses like heat, drought, salinity
and flooding and tolerates a wide range of soil conditions (Ejeta and Knoll, 2007). However,
sorghum production in Ethiopia has declined due to several causes such as population growth;
land degradation; and use of traditional farm implements (Simon, 2009). In addition, the crop is
usually grown in arid and semi-arid parts of the tropics and sub-tropics where it is affected by
drought during various growth stages (Amjad et al., 2009). This problem is intensified by the
possible global climate change scenarios (IPCC, 2001). For example, Simon (2009) reported that
recurring drought due to deficit of rainfall in semi-arid and dry sub-humid areas of the country is
one of the major causes of underproduction of sorghum. Therefore, drought is one of the most
severe stresses which affect sorghum production in such regions (Bota et al., 2000).
Drought is a worldwide problem and a higher proportion of agricultural land is affected with
varying degrees of drought (Sidari et al., 2008). Under drought conditions, the soil moisture
content throughout the soil profile will be below field capacity, subsequently resulting in poor
seed emergence and stand establishment. It causes water deficit in the plant which is one of the
most severe stresses faced by the sustainable crop productivity all over the world (Bota et al.,
2000). As a result, it restricts expression of full genetic yield potential of a crop. Observations on
sorghum fields suggested that poor seedling establishment and retarded growth early in the
1
seedling stages are a common phenomenon (Abuhay, 2000). The consequence of water deficit
includes its adverse effects on crop yield by obstruction of various essential physiological and
biochemical processes (Farooq et al., 2008). The amount of yield reduction depends not only on
the developmental stage of the crop, but also on genotype, intensity and duration of stress (Sinaki
et al., 2007). Water deficit remains to be the most limiting factor for better plant performance
and higher crop yield (Szilgyi, 2003, as cited in Muhammad et al., 2010). Worldwide, yield
losses each year due to drought are estimated to be around USD 500 million (Sharma and
Lavanya, 2002). Being a complex phenomenon and considered as one of the most important
factors limiting crop yields around the world (Beltrano and Ronco, 2008), drought continues to
be a challenge to agricultural scientists in general and plant geneticists in particular, despite
many decades of research.
In order to thrive under drought stress, plants have developed various drought resistance
mechanisms which include drought avoidance, escape and tolerance (Blum, 1979). Drought
tolerance is the mechanism causing minimum loss of a trait or yield in a water deficit
environment relative to the maximum in a water-constraint free well managed crop (Biswas et
al., 2002).
Variety development for lowland parts of Ethiopia has focused on selection of early maturing
varieties that can escape drought for the last nearly half a century. A number of early sorghum
open-pollinated varieties were developed and released for these areas (Asfaw, 2007). There are,
however, disadvantages to early maturity. Cultivars that mature extremely early tend to be lower
in yield because the plants have a shorter growth period to flower and store nutrients in the grain
(Sleper and Poehlman, 2003).
Previous investigations that had been undertaken to screen cultivars of sorghum for drought
tolerance were mainly focused on post flowering morphological characteristics. For example,
Dagnachew (2008) and Zelalem (2008) evaluated sorghum for post-flowering drought tolerance
using few morphological criteria. Moreover, Addisie (2010) has reported on evaluating sorghum
genotypes for post flowering drought resistance specifically the stay green trait.
2
Even though recent emphasis on field screening under severe drought stress has led to significant
developments in the understanding of drought tolerance, it requires several years and diverse
environmental conditions to get tolerant genotypes(Rosenow et al., 1983). Since screening is
done under field conditions and only in years when rainfall is scarce, such a procedure has
always a drawback of inconvenience and inefficiency (Abuhay, 1997).
On the other hand, in vitro culture studies play tremendous role by providing efficient way of
understanding plant genetic processes in short period of time in a controlled environment. In
vitro culture of plant cells and tissue has attracted considerable interest over recent years because
it provides the means to study plant physiological aspects and genetic processes in addition to
offering the potential to assist in the breeding of improved cultivars by increasing genetic
variability (Wani et al., 2010). In this regard, genetic factors are considered to be a major
contributor to the in vitro responses of cultured tissues. Differences in the production of
embryogenic calli and the regenerated plants have been observed by different workers, and they
were found to be dependent on the genotype (Ezatollah et al., 2012). Therefore, the in vitro
techniques are considered to be an important complement to classical field screening methods for
genotypic variability (Zalc et al., 2004).
For in vitro drought stress induction, one of the most popular approaches is to use high molecular
weight osmotic substances, like polyethylene glycol (PEG) (Muhammad et al., 2010). PEG is a
non penetrable and nontoxic osmotic substance which is used to lower the water potential of the
culture medium and it has been used to simulate drought stress in cultured plant tissues. It is
commonly established that cell lines that adapt PEG show high level of tolerance to drought
stress as compared to cell lines that fall short under the induced stress condition (Abdel-Raheem
et al., 2007). Application of in vitro selection for water deficit stress tolerance for different crops
using PEG has been reported by different authors (Abdel-Raheem et al., 2007; Wani et al.,
2010).
Evaluating local accessions for drought tolerance would play a considerable role in crop
improvement. Local accessions serve as source materials and are still the backbone of
agricultural production in developing world, because they are adapted to various environments
3
and preferred by farmers for various traits under difficult conditions (Addisie, 2010), where
modern cultivars are less reliable. Therefore, it is imperative to screen sorghum local varieties for
their tolerance to drought under in vitro condition through callus culture. With this background,
the present study was aimed to assess the effect of polyethylene glycol induced stress on
callusing, callus growth, embryogenesis, plant regeneration, coleoptile elongation, root
elongation, dry weight accumulation, and root branching of sorghum genotypes and to identify
the superior genotypes from all the accessions for drought tolerance.
Objectives of the Study:
General objective:
 To study the level of tolerance of different sorghum varieties for drought stress at in vitro
condition
Specific Objectives:
 To study callus induction capacity of sorghum genotypes and growth of callus cells in
induced drought stress
 To study embryogenic callus formation and plant regeneration capacity of sorghum
genotypes under induced drought condition
 To study the growth performance of in vitro regenerated sorghum seedlings
4
2. LITERATURE REVIEW
2.1 Sorghum
Sorghum (Sorghum bicolor) (L.) Moench) is a critically important crop in sub-Saharan Africa
because of its drought tolerance. It can tolerate hot and dry conditions and also able to withstand
heavy rainfall along with some water logging. Sorghum can constantly produce yield under
climatic conditions where other cereals fail. Sorghum is indigenous to the semi-arid tropics of
Africa (Amuna et al., 2000; Oria et al., 2000).
Sorghum is a genus with many species and subspecies and there are several types of sorghum,
including grain sorghum, grass sorghum (for pasture and hay), sweet sorghum (for syrups), and
Broomcorn. Grain sorghum and maize (corn) are comparable in production. Grain sorghum
requires less water than corn, so is likely to be grown as a replacement to corn and produce better
yields than corn in hotter and drier areas (Mihiret, 2009)
Sorghum is an important food crop in the semi-arid regions of Ethiopia. Because it is less
dependent on rainfall, and therefore, less affected by fluctuations in environmental conditions.
Still, there is a need to boost its production and adoption both in domestic use and in industrial
technologies for bread and for infant foods (Asfaw et al., 2005).
2.1.1 Origin, Distribution and Taxonomy of Sorghum
Determining the time and place sorghum was domesticated has been a dilemma for historians.
However, it is generally believed that Sorghum domesticated in Africa, more precisely in
Ethiopia, between 5000 and 7000 years ago. From there, it was distributed along the trade and
shipping routes around the African continent, and through the Middle East to India at least 3000
years ago. It then journeyed along the trade routes across Afro-Asian land mass. Sorghum was
first taken to North America in the 1700-1800's through the slave trade from West Africa
(Harlan, 1969). It is believed that African slaves took sorghum seeds with them to the US, and
that is how it was introduced to what is now the first sorghum growing country in the world. It
5
was re-introduced to Africa in the late 19th century for commercial cultivation and spread to
South America and Australia (Vavilov, 1951). Sorghum is now widely cultivated in the dry areas
of Africa, Asia (India and China), the Americas, Europe and Australia (Kimber, 2000; Dicko et
al., 2006). It is also one of the crops for which Ethiopia has been recognized as being a
Vavilovian center of origin and/or diversity (Firew, 2009). Therefore, today sorghum is
cultivated across the world in the warmer and drier climatic areas.
Sorghum is distributed throughout the tropical, semi-tropical, arid and semi arid regions of the
world. More than half of the world’s sorghum is grown in semi-arid tropics of India and Africa,
where it is a staple food for millions of poor people (Mehmood et al., 2008). The crop is also
found in temperate regions and at altitudes of up to 2300 meters in the tropics. It does well even
in low rainfall areas. Sorghum is also termed as “Nature-cared crop” or “the crop camel” because
of its strong resistance to harsh environments such as dry weather and high temperature in
comparison to other crops. This indicates that sorghum has a potential to adapt itself to the given
natural environment. Its ability to produce in areas with marginal rainfall (400 – 600 mm) and
high temperatures (i.e. semi arid tropics and sub tropical regions of the world) where it is
difficult to grow any other cereal makes the crop highly valued (Shewale, 2008).
Sorghum is an important crop in India, Pakistan, Thailand, in central and northern China,
Australia, in the dry areas of Argentina and Brazil, Venezuela, USA, France and Italy where it
has been given various names. For example, sorghum is known dawa in Housa, sorgho in
French, durra Arabic, mashela in Amharic, mtama in Swahili, jowar in Hindi, Kaolian in
Chineese milo in Spanish and sorgo in portuguese (Dicko et al., 2006).
In Africa, a major growing area runs across West Africa, South of the Sahara, through Sudan,
Ethiopia and Somalia. It is also grown in upper Egypt and Uganda, Kenya, Tanzania, Burundi,
and Zambia (Dicko et al., 2006).
.
In Ethiopia, sorghum is an economically, socially and culturally significant crop grown over a
wide range of ecological habitats, in the range of 400-3000 m.a.s.l. (Teshome et al., 2007).
Sorghum is the only most important cereal in the lowland areas of the country because of its
6
drought tolerance. The greater concentration of sorghum production comes from north central,
northwestern, western and the eastern mid-altitude areas of Ethiopia (Wortmann et al., 2006).
The mentioning of the name of sorghum dates back in some writings to 1305 A.D. The current
formal taxonomic concept of the sorghum genus and species is the one established by Moench.
Sorghum was first described by Linnaeus in 1753 under the name of Holcus. Later, the genus
sorghum was separated from the Holcus and the combination of Sorghum bicolor was made by
Moench (Firew, 2009).
The genus Sorghum has 25 recognized species that have been taxonomically classified into five
subgenera or sections: Eusorghum, Chaetosorghum, Heterosorghum, Parasorghum and
Stiposorghum (Price et al., 2005). S. bicolor (L) Moench is a member of the section Eu-sorghum
along with S. propinquum (Kunth) Hitch. And S. halepense (L.) Pers. The remaining 4 sections
contain 19 species native to Africa, Australia, and Asia (Kuhlman et al., 2008). Price et al.
(2005) has reported species of the genus sorghum have chromosome numbers of 2n = 2x = 10,
20, 30 or 40 of which Sorghum Bicolor has 2n = 2x =20.
2.1.2 World Production and Economic Importance
The annual production of sorghum is over 60 million tons on about 45 million hectares, of which
USA and Africa produce 20 and 40%, respectively. Developing countries account for nearly
90% of the world's sorghum area and 77% of the total production (FAO, 2011). It is estimated
that more than 300 million people from developing countries are essentially dependent on
sorghum as a source of energy (Godwin and Gray, 2000). More than 35% of sorghum produced
in Africa is used directly for human consumption and the rest is used primarily for animal feed,
alcohol production and industrial products (Dicko et al., 2006). This makes sorghum one of the
main staple crops for the world's poorest and most food insecure people.
Though the purpose of sorghum production in the developed countries is mainly for animal feed,
in Africa and Asia the grain is mostly used for human consumption. The main foods prepared
from sorghum are: tortillas (Latin America), thin porridge, e.g. bouillie (Africa and Asia), stiff
7
porridge (West Africa), couscous (Africa), injera (Ethiopia), nasha and kisra (Sudan), traditional
beers, e.g. dolo, tchapallo, pito, burukutu, etc. (South Africa), ogi (Nigeria), baked products
(USA, Japan, Africa), etc. Tortillas are a kind of chips prepared from sorghum alone or by
mixing sorghum with maize and cassava where as nasha is a traditional weaning food (infant
porridge) prepared by fermentation of sorghum flour. Ogi is an example of traditionally
fermented sorghum used as weaning food, which has been upgraded to a semi industrial scale
and injera is a locally fermented pancake-like bread prepared from sorghum in Ethiopia
(Yetneberk et al., 2004). Finally, Kisra is a traditional bread prepared from fermented dough of
sorghum (Dicko et al., 2006).
In Ethiopia, sorghum is the fifth among major cereal crop for private peasant holdings in terms
of area and production next to teff (Eragrostis teff), barley (Hordeum vulgare), wheat (Triticum
aestivum L.) and maize (Zea mays) (CSA, 2011). The crop has been grown in different agroecological zones of the country. Based on their adaptation zones within the country, cultivated
sorghums are grouped into highland, intermediate and lowland sorghum (Alemayehu, 2003).
This classification has been made largely based on altitude, length of growing period and amount
and distribution of rainfall (Yilma and Abebe, 1987). These authors indicated that intermediate
zone sorghum grows at an altitude of 1600-1900 m.a.s.l and those of lowlands grow in areas of
altitude less than 1600 m.a.s.l.
Being an indigenous crop to Ethiopia, sorghum exists in tremendous diversity throughout the
growing areas, which contains pockets of geographical isolation, with extremely broad and
valuable genetic base for potential breeding and improvement work in the country and the world
at large. Moreover, in Ethiopia, many efforts have been made to address the drought problem in
sorghum production. Breeding programmes in Ethiopia have released a number of varieties from
lowland areas which give reasonable yield in drought prone areas (Asfaw, 2007).
Sorghum, in a typical production environment, averages between 5 and 15% of its recorded
maximum yield potential (Asfaw et al., 2005). While biotic stresses reduce yield potential in
specific environments, most of the reduction in sorghum yield is attributed to abiotic stress,
primarily drought. In Ethiopia, even if sorghum has tremendous economic importance, its
8
production is affected by different abiotic constraints among which drought is an important stress
(Chala et al., 2007). Yields are still limited by drought because most of the mechanisms of
drought tolerance, their interactive effects, and associated morphological and physiological
modifications and symptoms have not yet been fully identified and understood by plant
geneticists, breeders and physiologists (Abdulai, 2005).
2.2 Plant Water Deficit (Drought) Stress
Plants often encounter variety of environmental factors which can be categorized as biotic or
abiotic in their nature. Biotic environmental factors, resulting from interactions with other
organisms include, infection or mechanical damage by herbivory or trampling, as well as effects
of symbiosis or parasitism. On the other hand, abiotic environmental factors include drought,
salinity, and extreme temperatures, chemical toxicity, and oxidative stress. Abiotic stresses are
serious threats to agriculture and result in the deterioration of the environment that influences
development, growth and productivity of plants (Addisie, 2010). Abiotic stress is the primary
cause of crop loss worldwide, reducing average yields for most major crop plants by more than
50% (Soltani et al., 2006). One of the most important abiotic factors limiting plant growth and
productivity is water stress brought about by drought, which is a widespread problem around the
world. Losses in crop yield due to water stress probably exceed the loss from all other causes
combined (Bartels and Souer, 2004).
Drought, commonly known as water stress, is one of the environmental stresses constraining
global crop production seriously and recent global climate change has made this situation more
serious (Sibel and Birol, 2007; Centritto et al., 2008). Drought is a major environmental factor
that determines plant productivity and plant distribution. It affects more than 10 percent of the
arable land (Bartels and Ramanjulu, 2005). The percentage of drought affected land areas is
more than doubled from 1970s to the early 2000s in the world (Isendahl and Schmidt, 2006).
Agricultural production was also affected by the limitation of water sources because of the
serious drought problem in many countries. For the purpose of crop production, yield
improvement and yield stability under water stress conditions and developing of drought tolerant
varieties is the best option (Siddique et al., 2000). To meet this goal, differential screening based
9
on physical and biochemical processes at both cellular and whole organism levels activated at
early stages of plant development have a great importance.
Larcher (2003) denotes drought as a period without appreciable precipitation, during which the
water content of the soil is reduced to an extent that plants suffer from lack of water. This author
has also stated that drought causes stress in plants if too little water is available in a suitable
thermodynamic state. Consequently, the biological role of water such as serving as solvent,
transport medium, as electron donor in the Hill reaction, and as coolant is often impaired.
Generally, water stress affects plant growth or morphological traits by affecting various essential
physiological and biochemical processes (Farooq et al., 2008). On the other hand, the effect of
water stress is a function of genotype, intensity and duration of stress, weather conditions,
growth and developmental stages of different crop plant species (Sinaki et al., 2007).
2.3 The Genetics and Mechanisms of Drought Tolerance in Plants
Drought tolerance is the capability to survive water-deficit with low tissue water potential. In
most cases, the responses of plants to tissue water-deficit determine their level of drought
tolerance (Blum, 1979). This is achieved by maintaining sufficient cell turgor to allow
metabolism to continue under increasing water deficits. The maintenance of turgor takes place
through osmotic adjustment (accumulation of compatible solutes in cell), increase in elasticity
and decrease in cell size and desiccation tolerance by protoplasmic resistance (Turner and
Kramer, 1980). Furthermore, Gunasekera and Berkowitz (1992) have indicated that osmotic
adjustment enables water uptake to continue under increasing stress in many species and, in
some cases, is associated with maintenance of growth and stable yield under drought. Therefore,
tolerant plants can endure stress by undergoing aforesaid physiological changes in their tissues
thus maintain their cell water potential turgidity at normal level, in spite of soil drought. On the
other hand, plants vary greatly in their capability to tolerate stress conditions; hence some of
them are unable to endure stress so wilt and die (Simmons et al., 1989).
Plants respond to changing drought stimulus with the expression of specific sets of genes that
allow the plants to adapt to the altered environmental conditions. There are two most established
10
approaches to understand the basic genetic responses of plants to drought which involve studying
candidate genes and differential screening. The former is comparing the expression of genes for
the enzymes in drought-induced metabolic pathways under drought versus non-drought
conditions. The expression of these genes is important for drought tolerance and provide useful
information on which genes are expressed as response of drought stimuli. The second approach
uses differential screening to isolate up-regulated genes encoding proteins of known function
associated with desiccation (Sorrells et al., 2007). Most of these genes are induced by the plant
hormone abscisic acid (ABA) (Guerrero et al., 1990; Nordin et al., 1991; Yamaguchi-Shinozaki
et al., 1992). ABA is synthesized through the carotenoid biosynthesis pathway and its
concentration regulated when there are changes in cellular dehydration. Its synthesis becomes
rapid when there is loss of turgor as a result of water deficit. Increased levels of ABA can, in
turn, induce changes in gene expression resulting in stomatal closure in leaves, inhibition of
photosynthesis and the growth of leaves, stems and promote growth of roots (Quarrie and Lister,
1984; Guerrero and Mullet, 1986).
Cell division is the principal determinant of meristem activity and determines the overall plant
growth rate. It has been described that environmental controls of growth rate like water deficit
act by regulating cyclin-dependent kinase (CDK) activity and cell division (Cockcroft et al.,
2000; West et al., 2004). CDKs are a family of protein kinases, each with a positive regulatory
subunit termed a cyclin and the catalytic subunit CDK (den Boer and Murray, 2000). CDKs are
emerging as key players in regulation of cell division and are likely to be regulated at both
transcriptional and post-translational levels in response to stress. For example, maize ZmCdc2 (a
member of the CDK family) was shown to be down regulated by water stress leading to a
decrease in mitotic cell cycling (Setter and Flanningan, 2001).
Cell expansion is a coordinately regulated process at the whole plant level and is influenced by
external stimuli including water availability. The rate of cell expansion is mainly determined by
two parameters, cell wall extensibility and cellular osmotic potential. The enlargement of plant
cells involves control of wall synthesis and expansion, solute and water transport, membrane
synthesis, Golgi secretion, ion transport and other processes (Cosgrove, 1997).
11
2.4 Effects of Drought on Plant Growth and Development
Water stress is one of the stress factors and it has a significant effect on the growth and
development of plants. Tolerance to this abiotic stress is a complex phenomenon, comprising a
number of physio-biochemical processes at both cellular and whole organism levels activated at
different stages of plant development (Mahajan and Tuteja, 2005). Inadequate water supplement
in soil limits the productive potential of several plant species by provoking smaller growth
during the vegetative period as well as the reproductive period (Sibel and Birol, 2007).
Comparative studies on the effect of different plants have showed that water stress reduced the
growth and development while compared to those of unstressed plants. So, a plant or a group of
plants showing better growth with limited soil moisture than other plants in a similar
environments are understood to be drought tolerant (Kumar and Sharma, 2009). Effects of water
stress on growth and development under in vitro condition and early seedling stage have been
documented (Abuhay, 1997; Abuhay, 2000; Ali et al., 2009).
2.4.1 Callus Growth Dynamics
Basically PEG application in culture media callus growth and dramatic decline in callus
formation ability has been reported in various plants which were forced by water deficit
conditions imposed by high molecular weight PEG as in vitro drought inducer (Dragiiska et al.,
1996; Ezatollah et al., 2012; Gopal and Iwama, 2007; Wani et al., 2010). There is an inherent
tolerance in callus of crop plants to PEG- induced drought in culture media (Wani et al., 2010).
This author has also revealed that increased water stress was induced by increased concentration
of PEG which caused a progressive reduction in callus fresh weight. There was normal callus
formation and plant regeneration in the no-stress medium, but increased PEG concentration
decreased callus formation efficiency and plant regeneration in the stress medium. Previously,
Abdel-Raheem et al (2007) stated that callus formation and plant regeneration in PEG- induced
drought condition of the culture medium is higher in relatively tolerant varieties and decreases
with increased level of the stress. Therefore, the inherent capacity for callus formation and
regeneration on growth medium with PEG is an easiest way to identify cultivars which are
genetically tolerant to water deficit.
12
2.4.2 Seedling Growth and Physiology
Arrest of plant growth during stress conditions is a common feature of the physiological
response. Mild osmotic stress leads rapidly to growth inhibition of leaves and stems, whereas
roots may continue to elongate. The inhibition of shoot growth during water deficit is thought to
contribute to solute accumulation and thus eventually to osmotic adjustment. For instance,
hexose accumulation accounts for a large proportion of the osmotic potential in the cell
elongation zone in cells of the maize root tip (Bartels and Ramanjulu, 2005). On the other hand,
continuation of root growth and branching under drought stress is an adaptive mechanism that
facilitates water uptake (Munns et al., 2000).
In the past, different morpho-physiological traits have been potentially utilized for screening
genotypes of different crops under water stress conditions. These include seedling traits like
shoot weight, root weight, root and shoot lengths, root:shoot ratio and coleoptiles length at
seedling stage (Dhanda et al., 2004; Taiz and Zeiger, 2006). Early and rapid elongation of roots
is important indication of drought tolerance. Ability of continued elongation of root under
situation of water stress was remarkable character of tolerant genotypes. Also root weight have
been used as selection index and highest root weight was recorded by drought tolerant cultivars
whereas lowest weight was noted in susceptible ones (Kulkarni and Deshpande, 2007).
Moreover, a survey of literature revealed that morpho-physiological traits such as flag leaf area,
specific leaf weight, leaf dry matter, excised leaf weight loss (Bhutta, 2007), relative dry weight,
relative water content (Colom and Vazzana, 2003), residual transpiration (Sabour et al., 1997 as
cited in Ali et al., 2009) and cell membrane stability (Rahman et al., 2006) had been widely used
as selection parameters contributing towards drought tolerance for various crop plants in addition
to grain yield.
Water availability mostly affects dry matter accumulation in different plant parts. Reduction in
dry matter accumulation and partitioning are typical responses of crop plants when subjected to
moisture deficit (Abuhay, 1997). In previous studies under field condition, dry matter
accumulation in the stem is favored by adequate moisture while the root is favored by moisture
stress (Ramu et al., 2008).
13
Screening sorghum varieties for relative drought tolerance has been attempted by various
workers using different physiological, and biochemical parameters. It has been documented that
root growth, leaf area development, synthesis of epicuticular wax and osmotic adjustment under
stress are some of the guidelines in characterizing the genotypes for stress tolerance in sorghum
(Blum and Sullivan, 1987). Barrs and Weatherly (1962) developed the concept of relative water
content and the reduction in relative water content under stress has been used as a measure of
drought tolerance by several workers. In addition, percent germination, seedling shoot dry
weight, seedling shoot length, and leaf area has been used to investigate performance of different
sorghum genotypes in water deficit conditions (Abuhay, 2000). Pawar (2007) used plant height
at different growth stages, dry weight of different plant parts, and leaf area as physiological
indices for drought tolerance in sorghum.
An understanding of the genetic basis of drought tolerance in crop plants based on various
morpho-physiological traits has been also a pre-requisite for breeders to produce superior
genotype through either conventional breeding methodology or genetic engineering (Mitra,
2001; Chen et al., 2004). Therefore identification and analysis of plant traits with sound and
positive association with drought tolerance and high productivity under drought is necessary
(Rauf and Sadaqat, 2008). Genetic management through cultivation of drought-tolerant crops
and varieties in specific crops is a feasible proposition to exploit such constrained ecosystems.
2.5 Sorghum Research for Drought Tolerance in Ethiopia
The national and regional research institutions in Ethiopia have released significant number of
varieties of sorghum for commercial production. The varieties are broadly divided as highland
varieties such as Alemaya 70, ETS2752, Chiro, ETS1176; mid-altitude varieties such as IS9302,
Birmash, Baji; and lowland varieties such as Gambella 1107, 76T1#23, Seredo, Meko, Teshale,
Gubiye, Abshir (Asfaw et al., 2005). Currently, sorghum breeding in Ethiopia is fully engaged in
different research activities in sorghum drought tolerance.
14
So far, a number of varieties which are sources of drought tolerance genes have been identified
from the Ethiopian gene pools by ICRISAT and other scientists in the region and now they are in
use in different parts of the world to generate drought tolerant/resistant sorghum varieties
(Borrell and Hammer, 2000). In addition to these, the Ethiopian sorghum germplasm is also
noted worldwide as source for useful genes such as cold tolerance, good grain quality, and
disease and insect resistance (Yilma and Abebe, 1987; Doggett, 1988).
Zelalem (2008) evaluated sorghum accessions for post-flowering drought tolerance using some
morphological and agronomic criteria. The author has reported the presence of variation in
tolerance among 165 sorghum accessions evaluated. Dagnachew (2008) has indicated that
estimation of genetic diversity in sorghum is very important in the evaluation of accessions as
possible source of genes for a given trait of interest like trait for drought resistance/ tolerance.
Moreover, Abuhay (2000) investigated genotypic difference among sorghum genotypes starting
from seedling stage in green house under various soil water deficit conditions. This author
emphasized that sorghum genotypes differed in response to variable soil moisture difference.
2.6 The Role of Plant Tissue Culture and Application of PEG in Drought Tolerance
Screening
Plant tissue culture broadly refers to the in vitro cultivation of plants, seeds and parts of the
plants such as leaf, immature and mature embryos, flower buds, anthers, microspores, ovaries,
tissues, single cells, protoplasts and others. Tissue culture technology can potentially regenerate
species of any plant in the laboratory. Improved in vitro tissue culture systems are needed for
manipulation of cereal germplasms for improved performance (Aulinger, 2002; Zhang et al.,
2004).
New complete plants can be obtained from different explants through direct or indirect
morphogenesis and through somatic embryogenesis. Direct morphogenesis is the production of
shoots from explants without passing through callus (unorganized tissue) phase known as organ
culture, which include meristem cultures, shoot cultures, embryo cultures and isolated root
cultures. Whereas indirect morphogenesis refers to induction of shoots through callus phase
15
grouped as unorganized tissue cultures, which include callus cultures, suspension or cell cultures
or anther cultures (Khanna, 2003; Tileye et al., 2005). Plant tissue culture is based on three cell
doctrines (concepts). The first one is plasticity which is the flexibility or adaptability of tissue or
cells to altered chemical and physical factors such as hormones, nutrient elements, fixed carbon
sources, light, temperature, and culture vessels. The other decisive concept is totipotency which
is the capacity of cells or tissues to develop in to any of the structure of the plant; and finally, dedifferentiation in which differentiated cells get de-differentiated in order to pave way for a new
line of development (Hartmann et al., 2002; Khanna, 2003).
Studying the effect of a particular stress using in vitro culture techniques minimize
environmental variations due to defined nutrient media, controlled conditions and homogeneity
of stress application. In addition, the simplicity of such manipulations enables studying large
plant population and stress treatments in a limited space and short period of time. Simulation of
drought stress under in vitro conditions during the regeneration process constitutes a convenient
way to study the effects of drought on the morphogenic responses. Therefore, applying osmotic
stress during the regeneration phase was found to be the most efficient for the selection of
drought tolerant genotypes (Hsissou and Bouharmont, 1994)
Genetic factors are considered to be a major contributor to the in vitro response of cultured
tissues. Differences in the production of embryogenic calli and the regenerated plantlets have
been observed in different times and they were dependent on the genotype (Ganeshan et al.,
2003). In vitro selection for tolerance to abiotic stress depends on the development of efficient
and reliable callus induction and plant regeneration systems.
16
2.6.1 Composition of In Vitro Culture Medium
In most plants, successful regeneration from the callus is carried out after a series of subcultures
in different media. The basic nutritional requirements of in vitro cultured plant cells are very
similar to those utilized by plants in nature. However, the nutritional composition used in vitro
varies depending on the type of protoplasts, cells, tissues, organs, and plant species (Deen and
Mohamoud, 1996). The mineral salts, sugar as carbon source and water are the main components
for most plant tissue culture media. Sugar (most commonly, sucrose) is an important component
in medium and its addition is essential for in vitro growth and development of plants because
photosynthesis is insufficient, due to the growth taking place in conditions unfavorable for
photosynthesis or without photosynthesis (Gamborg and Phillips, 1995).
Cultured cells are normally capable of manufacturing all of the required amino acids. Yet, the
addition of an amino acid or mixtures of amino acids may be used to stimulate cell growth and
facilitate plant regeneration (Mohamed, 1996).
Plant tissues and organs are raised in vitro on artificial media that supply the nutrients necessary
for growth. For healthy and vigorous growth, intact plants need to take up from the soil. The
most commonly used medium is the formulation of Murashige and Skoog (1962). This medium
was formulated for optimal growth of tobacco callus and the development involved a large
number of dose-response curves for the various essential minerals. Mineral nutrients are one of
the most basic constituents of plant tissue culture media. Unlike carbon sources, plant growth
regulators, vitamins, amino acids, gelling agents (agar) and undefined substances that may or
may not be included in any given medium, the macro and micro mineral nutrients are always
present (van Staden et al., 2008).
2.6.2 Plant Hormones and Growth Regulators
The influence of plant growth regulators on the cell culture media differs with the type and
concentration of growth regulator. Auxin (IAA, NAA, 2, 4-D and IBA) is a plant growth
regulator which is known to be distributed universally in higher plants. This compound is
generated by the apical meristems of both shoots and roots and manages the expansion of the
17
tissue cells. Auxin was also proved to be responsible for apical dominance (the inhibition of
lateral bud and lateral root development by the active apical meristem), in the retention or falling
of leaves and flower buds, in flower development and in the initiation and continuance of fruit
development (Davies, 1995).
According to Petrasek et al. (2002), the division and growth of most types of plant cells cultured
in vitro require an external source of auxin. Artificial auxins such as 2, 4-dichlorophenoxyacetic
acid (2, 4-D) are essential constituents of culture media. Generally, the auxin, 2, 4-D, is critical
in the induction of primary calli and embryogenic calli, which was in accordance with the results
of some monocotyledonous plants (Vikrant, 2003; Jogeswar et al., 2007). In cereals, 2, 4-D has
been used for callus induction and maintenance (Pellegrineschi et al., 2004) or for induction of
somatic embryogenesis (Vikrant, 2003).
Cytokinins are other classes of plant hormones used in plant tissue cultures. Considering natural
cytokinins, benzyladenine (BA) or kinetin (6-furfurylaminopurine) are most frequently used in
plant tissue culture systems (Barciszewski et al., 1999). The effect of cytokinins is most
noticeable in tissue cultures where they are used, often in combination with auxins, to stimulate
cell division and control morphogenesis. Applied to shoot culture media, these compounds
overcome apical dominance and release lateral buds from dormancy (van Staden et al., 2008).
It has been reported that the callus production and plant regeneration of sorghum were influenced
by indole-3-acetic acid (IAA), 6-benzyladenine (6-BA) and Kinetin (KT). IAA and
naphthaleneacetic acid (NAA) were supplemented during regeneration of sorghum calli culture
(Hagio, 2002). Indole-3-butyric acid (IBA) was known to induce roots in many cereals such as
rice and sorghum (Jogeswar et al., 2007).
.
18
2.6.3 PEG as an In Vitro Drought Inducer
It appears that in vitro selection for stress tolerance will continue to have its significant place in
the strategy of establishing plant systems with optimal stress reaction and productivity.
Polyethylene-glycol (PEG) of high molecular weight have been long used to simulate drought
stress in plants as non-penetrating and non-toxic osmotic agent which is able to lower the water
potential of the medium in a way similar to soil drying (Abdel-Raheem et al., 2007).
In vitro selection for drought tolerance commonly uses PEG as selection component. This
method has been applied to several plants (Muhammad et al., 2010; Wani et al., 2010; Ezatollah
et al., 2012). A positive correlation between the seed germinating capability at the PEG
containing media and the plant growth under stress condition has been found. PEG could be
applied for stimulating drought because it could inhibit water in such a way that no water is
provided for somatic cell, except for the callus which has particular mechanism for absorbing
water. Only the tolerant callus which bears PEG selection media could increase its tolerance
against drought stress (Lestari, 2006).
Selective media treated with PEG inhibited the growth and development of the soybean explants
and decreased the number of formed somatic embryos. The increasing amount of PEG added to
the selective media brought the deterioration influence of PEG on somatic/callus embryo. In a
study by Lestari (2006), treating tolerant soybean genotypes with 15% solution of PEG most of
the genotypes survived where as in the same study about half of moderately tolerant genotypes
survived.
Selection of the callus of several rice varieties conducted by Prakash et al., (1994) using 5, 10,
15 mg/l of PEG (6000) successfully resulted in drought tolerant plants. In this work, five plants
from three varieties exhibited their tolerance to drought. In another study conducted by Wani et
al. (2010), increased levels of PEG (6000) (0, 0.5, 1.0, 1.5, 2.0 %) were used to create water
stress in callus initiation and plant regeneration in rice. There was reduction in callus induction
ability and plant regeneration efficiency with increasing levels of PEG (6000) stress.
19
3. MATERIALS AND METHODS
3.1 Description of the Research Site
The experiment was conducted in the tissue culture laboratory of Melkassa Agricultural
Research Center which is located 17 km SW of Nazareth town and 117 km away from Addis
Ababa at 8o24’N latitude and 39o21’E longitude and at elevation of 1550 m.a.s.l.
(www.eiar.gov.et, accessed date: 25th April, 2012).
3.2 Experimental Design
Sixteen sorghum varieties from more than 19 released varieties were selected based on their
agro-ecological adaptation (Appendix Table 22). Five level concentrations of PEG were used as
treatments. PEG levels were kept as low as 2% so that it will be possible to get enough
population of regenerated plants for morphological study. The experiment of the study was laid
out in a completely randomized design (CRD) in factorial arrangement with three replications
(Gomez and Gomez, 1976).
3.3 Plant Materials
Seeds of sixteen elite sorghum varieties (Abshir, Chelenko, Raya, Hormat, Gubiye, Gambella1107, Birmash, Meko, ESH-2, Seredo, Misikir, 76T1#23, Melkam, Macia, Girana-1, and
Teshale) were obtained from National sorghum improvement project of Melkassa agricultural
research center.
3.4 In Vitro Screening
3.4.1 Callus Induction under PEG Stress
MS media (Murashige and Skoog, 1962) supplemented with plant hormones and growth
regulators as described by Zhao et al., (2010) for callus induction were prepared and the pH was
adjusted to 5.8 using 1N HCl and 1N NaOH. The media were heated to boiling for proper mixing
with agar before 25ml was dispensed into culture jars and labeled. The media in the jars was
20
closed and sealed with parafilm. The sterilization of media was carried out in an autoclave at 120
o
C under 15 lb/ in2 pressure for 15 minutes. Then the media were kept for three days before
culturing.
The seeds of the germplasm were washed in running tap water for 30 minutes, sterilized in 75%
ethanol for three minutes and in 0.2% HgCl2 for 15-20 minutes, and then washed 3-5 times in
sterilized water. Thirty seeds were cultured per each jar. The jars were sealed with parafilm and
placed in a growth chamber in the dark at 25 ± 2ºC.
After four weeks of incubation, the induced calli were excised and sub-cultured, under the same
growth conditions, and in the same MS medium to which five level concentrations of
polyethylene glycol (PEG) (6000) (0, 0.5, 1.0, 1.5, 2.0 %) has been added (Wani et al., 2010).
The incubation period was four weeks in which the media is replaced with the same media in
two weeks.
3.4.2 Plant Regeneration from Induced Callus in PEG Stress Condition
Resulting calli were excised, transferred, to jars containing 25ml MS basal salts medium
supplemented with plant hormones and growth regulators as described by Zhao et al., (2010) for
shoot initiation in conditions in which PEG (6000) (0, 0.5, 1.0, 1.5, 2.0 %) has been added. The
incubation period was four weeks.
The jars were placed in a growth chamber under fluorescent light and at an ambient temperature
of 25 ± 2ºC. The medium was changed in 15 days and after four weeks, calli with clearly
differentiated shoots were scored as regenerating callus. Rooting was initiated on MS medium
supplemented with 3 mg l-1 indole-3-butyric acid (IBA) under the same conditions. Regenerating
calli, showing shoot and root formations, were transferred to MS basal medium with no
phytohormones to sustain growth of plantlets for four weeks.
21
3.5 Data Recorded and Statistical Analysis
After the end of callus induction period callus induction efficiency (CIE) was recorded as the
number of calli induced divided by total number of seeds cultured × 100. After four weeks of
culturing the excised callus data were also taken for difference in callus fresh weight (CFW). In
the mean time, embryogenic callus percent (ECP) was recorded as the number of embryogenic
callus obtained divided by total number of induced callus X 100. Finally, at the end of plant
regeneration, data for percentage plant regeneration (PRP) was recorded as the number of
plantlets obtained divided by total number of embryogenic calli cultured × 100.
After four weeks of incubation in MS basal medium with no phytohormones, plant parts above
and below callus (shoot and roots) were harvested separately. Roots were washed free of culture
media. Plant height above callus was measured from the surface of the culture media to the tip.
Root length was also measured from the point shoot length was measured down to the tip. Root
branching was measured by visual counting. Each plant part was oven dried at 70 oC for 24 h
before weighing. From the measurements of shoot dry weight and root dry weight, root shoot dry
weight ratio was computed.
Data were analyzed using SAS software 9th Edition. The data analysis was carried out using
analysis of variance for each parameter while the difference between the pair of treatment means
was evaluated at 1% level of significance using Fischer’s LSD.
22
4. RESULT AND DISCUSSION
4.1 Callus Induction, Proliferation and Plant Regeneration
4.1.1 The Effect of PEG Induced Moisture Deficit on Callus Induction Efficiency (CIE)
Sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress levels for
callus induction efficiency (Table 1). There was also highly significant (p<0.01) difference
among the tested genotypes as well as the PEG treatments (Appendix Table 1 and 4). The PEG
resulted in reduction in CIE of all genotypes with increasing level of PEG treatment, indicating
sorghum genotypes are responding differently to PEG stress levels (Fig. 1). The total reduction
in the CIE was quantified as the sum of simple effects (i.e. reduction in each PEG treatment
level) (Table 1).
A) Normal callus induction in the control
B) Reduced callus induction in the 2% PEG
Fig. 1 Callus induction difference due to PEG stress
In variety Meko the PEG stress had lowest effect in mean CIE (1.11), followed by Seredo (2.23)
with increasing level of PEG from 0 to 2% indicating their relative tolerance. The effect of PEG
23
treatment on Teshale and 76 T1#23 was nearly in the same magnitude (4.44), but in the later the
reduction in CIE was more pronounced at 1.5% PEG level. Gubiye exhibited lower reduction in
CIE (5.56.) than 76T1#23. Likewise, in Melkam and Gambella-1107 the reduction in CIE (8.89)
due to the PEG stress was higher than 76T1#23, but the reduction in Gambella-1107 was more
pronounced at 2% PEG level. In Abshir, Girana-1, ESH-2, and Hormat, the effect of the PEG
stress in CIE (18.89) was higher than in Gambella-1107. Also in Macia, Birmash and Misikir,
the reduction in CIE was nearly in the same magnitude (26.67), but higher than Gambella-1107.
The highest effect of PEG on CIE (27.78) was observed on varieties Chelenko and Raya
respectively which indicated the relative susceptibility of these genotypes to PEG stress
treatments.
Table 1: Mean callus induction efficiency (CIE) of genotypes for each PEG treatment level
CIE (%)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD(p<0.01
0.0
53.33
62.22
86.66
48.89
64.44
61.11
56.67
80.00
60.00
35.56
63.33
54.44
48.89
40.00
52.22
61.11
13.39
12.22
0.5
51.11
52.22
82.22
42.22
58.89
57.78
50.00
78.89
41.11
34.44
57.78
53.33
46.67
36.67
51.11
58.89
1.0
47.78
42.22
77.78
37.78
58.89
58.89
37.78
78.89
33.33
28.89
50.00
51.11
48.89
33.33
46.67
57.78
1.5
48.89
36.67
72.22
34.44
56.67
56.67
34.44
77.78
38.89
30.00
38.89
48.89
42.22
25.56
36.67
57.78
2.0
40.00 (13.33)
24.44 (27.78)
58.89 (27.78)
30.00 (18.89)
58.89 (05.56)
52.22 (08.89)
30.00 (26.67)
78.89 (01.11)
33.33 (26.67)
33.33 (02.23)
36.67 (26.67)
45.56 (08.89)
44.44 (04.45)
22.22 (17.78)
36.67 (15.56)
56.67 (04.44)
Numbers in parenthesis are total reductions in mean CIE across the PEG treatment levels
24
In the present experiment, PEG treatment has an effect on callus induction capacity of sorghum
genotypes. The mean CIE decreased drastically in genotypes under higher PEG treatments than
lower PEG treatments. Decrease in CIE is a typical response of explants of crop genotypes when
subjected to PEG stress. In agreement with the result of this study, reduction in CIE has been
reported in many cereal species. According to Matheka et al. (2008) who investigated in maize,
Biswas et al. (2002) in rice, and Abdel-Ghany et al. (2004) in wheat, CIE declined with
increasing PEG stress level. The observed significant reduction in callus initiation and genotypePEG treatment interaction is also in agreement with those observed by Wani et al., 2010.
However, the response of CIE for PEG treatment was genotype dependent. The difference in
decreasing trend in CIE of the genotypes might further explain difference in osmotic regulation
among genotypes, which enables them to maintain osmotic balance to assist initiation of callus
cells under severe stress conditions. According to Begum et al. (2011), in sugarcane, 5 % PEG
treatment resulted in reduction of callus induction from 100 to 86% and the reduction differed in
different genotypes. The same result has been reported by Ezatollah et al. (2012) in bread wheat
and Wani et al. (2010) in rice. Again, such a decrease in CIE in response to PEG stress might be
due to either water shortage which led to profuse mutation in cellular metabolism including
protein functioning and alteration in amount of proteins (Plomion et al., 1999) or altered gene
expression (Visser, 1994) controlling this trait or the genes may express themselves but the
resultant proteins may be denatured due to increased stress.
4.1.2 The Effect of PEG Induced Moisture Deficit on Callus Fresh Weight (CFW)
Sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress levels for
CFW (Table 2). There is also a highly significant (p<0.01) difference among the tested
genotypes as well as the PEG treatments (Appendix Table 1 and 4). In all genotypes, effect of
the PEG treatments resulted in decrease of the mean CFW with increasing level of the PEG from
0 to 2% (Fig. 2). The overall reduction in the CFW was quantified as the sum of simple effects of
each PEG level.
25
A) Normal callus growth in the control B) Reduced callus growth in the 2% PEG
Fig. 2 Callus growth difference due to PEG stress
Variety Meko exhibited the lowest reduction in CFW (34.38) followed by Abshir (72.21), ESH-2
(77.44), 76T1#23 (93.56), Melkam (98.30), Girana-1 (100.39), Gambella-1107 (101.44), Macia
(101.51), Teshale (105.56), Gubiye (116.82), Birmash (118.56) and Seredo (120.13). Misikir
(121.30) and Chelenko (125.90) exhibited higher reduction in CFW. The greatest effect of PEG
treatments on CFW was observed on Raya (128.56) and Hormat (143.83) than the other tested
genotypes, indicating the highest damage to callus growth in these genotypes due to the PEG
stress.
26
Table 2: Mean callus fresh weight (CFW) of genotypes for each PEG treatment level
CFW (mg)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76t1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
380.02
390.92
392.05
385.51
379.48
377.96
375.29
338.23
384.45
379.89
384.12
383.33
376.73
350.62
387.80
384.89
4.80
34.12
0.5
370.05
383.18
376.24
377.48
367.76
381.09
359.15
358.26
376.39
367.61
367.72
369.59
365.12
369.28
378.57
374.27
1.0
348.31
349.83
342.71
338.85
358.48
351.52
307.58
331.88
360.39
342.55
341.10
354.44
339.40
338.08
361.51
353.32
1.5
319.54
281.95
278.30
276.79
322.35
319.61
276.33
295.65
304.14
310.98
274.36
325.19
326.00
286.31
307.34
328.33
2.0
307.81 (072.21)
265.02 (125.90)
263.49 (128.56)
241.68 (143.83)
262.66 (116.82)
276.52 (101.44)
256.71 (118.56)
303.85 (034.38)
282.94 (101.51)
259.76 (120.13)
262.82 (121.30)
285.03 (098.30)
283.17 (093.56)
273.18 (077.44)
287.41 (100.39)
279.37 (105.56)
Numbers in parenthesis are total reductions in mean CFW across the PEG treatment levels
The major effect of PEG stress in callus growth is mainly observed in the form of decrease in
CFW which is a typical response in callus tissue of many crop plants. In the callus cells of
stressed genotypes, a significant reduction in CFW across the five PEG stress levels was found
and the outcome is also in agreement with the results found in soybean (Sakthivelu et al., 2008),
barley (Lührs and Lörz, 1987), wheat (Özgen et al., 1998), durum wheat (Lutts et al., 2004;
Turhan and Baser, 2004) and rice (Khanna and Raina, 1998; Hoque and Mansfield, 2004;
Khalequzzaman et al., 2005; Wani et al., 2010).
Different genotypes respond differently to PEG treatment in terms of callus growth. The same
result also exists in maize (Hamdy and Aref, 2002), and wheat (Abdelsamad et al., 2007).
27
Increasing water stress causes a progressive reduction in growth as expressed in callus fresh
weight. Abdulaziz and Al-Bahrany (2002) studied the callus to varying degree of polyethylene
glycol (PEG)-induced water stress. Their results revealed that increasing water stress induced by
increasing concentration of PEG caused a progressive reduction in callus fresh weight.
According to Sakthivelu et al. (2008), the addition of PEG to the MS medium decreased the
water potential of the media inducing water stress that adversely affected the callus growth.
Several other authors also reported the use of PEG for in vitro drought screening in various crop
plants (Dragiiska et al., 1996; Ezatollah et al., 2012; Gopal and Iwama, 2007; Wani et al., 2010).
Such a decrease in CFW in response to PEG stress might be due to water shortage which affects
development and growth of cells. Cell division and cell growth are the two primary processes
involved in increase of fresh weight. In general, cell division is considered to be less sensitive to
drought when compared with cell enlargement or growth (Sakthivelu et al., 2008). However,
both cell expansion and cell division can be influenced by relatively mild osmotic stress.
Maintenance of cell turgor plays an important role in cell growth (Prasad and Staggenborg,
2008). Addition of PEG-6000 in solid media lowers water potential of the medium that adversely
affect cell division leading to reduced callus growth (Ehsanpour and Razavizadeh, 2005;
Sakthivelu et al., 2008).
Generally, the results of this experiment agrees with previous reports in which genotypic
differences in callus proliferation was reported to be genetically determined in some cereals
including sorghum (Newton et al., 1986), barley (Lührs and Lörz, 1987), rice (Khanna and
Raina, 1998; Hoque and Mansfield, 2004; Khalequzzaman et al., 2005), and wheat (Özgen et al.,
1998; Galovic et al., 2005).
4.1.3 The Effect of PEG Induced Moisture Deficit on Embryogenic Callus Percent (ECP)
The mean ECP of the tested genotypes in each PEG treatment level is presented on Table 3.
There was a highly significant (p<0.01) PEG stress genotype interaction for ECP. The difference
among PEG levels and the genotypes was also highly significant (P<0.01) (Appendix Table 1
and 4). In the entire genotypes mean ECP increased with increasing level of PEG treatment from
28
0 to 1.5% and declined at 2% PEG level. However, in varieties Hormat (1.73), ESH-2 (1.50), and
Raya (1.40), there was an overall reduction in ECP, indicating the highest effect of PEG
treatment on ECP of these genotypes. In Abshir (-0.06) and 76T1#23 (-0.07), slight increase in
ECP was recorded. Melkam (-1.05), Seredo (-1.51), Misikir (-1.91), Gubiye (-2.08), Birmash (2.31), Teshale (-2.52), Macia (-2.60), Gambella-1107 (-2.89), and Chelenko (-3.17) exhibited
higher level of increase in ECP in their respective order. The highest increase in ECP was
attributed to varieties Girana-1(-3.34) and Meko (-3.88), which is an indication of lowest effect
of PEG stress on embryogenic callus formation of these genotypes.
Table 3: Mean embryogenic callus percent (ECP) of genotypes for each PEG treatment level
ECP (%)
PEG
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (p<0.01)
0.0
43.91
10.97
39.73
48.88
38.12
51.10
28.13
21.19
48.89
49.46
51.75
31.94
42.20
41.39
27.85
58.34
6.74
5.93
0.5
45.07
12.97
40.69
48.78
38.52
52.67
29.66
23.49
50.69
51.33
51.39
34.29
40.95
43.86
28.92
59.59
1.0
45.07
12.85
40.33
49.69
40.42
52.99
31.61
27.69
50.72
52.51
53.21
34.67
41.14
44.38
29.08
66.48
1.5
53.19
12.86
41.12
48.35
42.26
55.24
30.03
25.88
53.00
55.84
54.90
41.90
43.44
46.01
31.94
60.65
2.0
43.97 (-0.06)
14.14 (-3.17)
38.33 (1.40)
47.15 (1.73)
40.21 (-2.08)
54.00 (-2.89)
30.44 (-2.31)
25.07 (-3.88)
46.29 (-2.60)
50.96 (-1.51)
53.67 (-1.91)
33.00 (-1.05)
42.27 (-0.07)
39.89 (1.50)
31.19 (-3.34)
60.86 (-2.52)
Numbers in parenthesis are total increase or reduction in mean ECP across the PEG treatment
levels (“-“= Reduction)
29
In the results of the present experiment, PEG induced water deficit has an effect on ECP.
Inconsistent variation in ECP was recorded among genotypes in response to the five PEG stress
levels. The inconsistency could be attributed to the difference in genotypes' strategies and
responses to the induced water deficit conditions.
In contrast to report of Bozhkov and von Arnold (1998) and Luma and Al-Ka’aby (2011), the
mean ECP increased in genotypes under mild PEG stress level. However, the results agree with
those reported by Kim and Moon (2007), Iraqi and Tremblay (2001) and Lipvska et al. (2000) in
which PEG treatment became very effective in inducing somatic embryos. Moon and Park
(2008) also reported significant increase in embryogenesis more than doubling the average
number of somatic embryos with doubling PEG concentration. PEG stress promotes
embryogenesis by provoking osmotic stress (Kim and Moon, 2007, Iraqi and Tremblay, 2001,
Lipvska et al., 2000).
The presence of variation among genotypes also indicates that there is a possibility of improving
the response of different genotypes to PEG induced water stress. These findings agree with those
reported by Gandonou et al. (2005), Ather et al. (2009), Khan et al. (2009), Raza et al. (2010) in
sugarcane, Khanna and Raina, (1998), Hoque and Mansfield, (2004), Khalequzzaman et al.
(2005) in rice; Özgen et al., (1998) in wheat; and Lührs and Lörz, (1987) in barley in which
significant difference in embryogenic callus production was observed.
4.1.4 The Effect of PEG Induced Moisture Deficit on Plant Regeneration Percent (PRP)
The mean PRP of the PEG treatment X genotype interaction is given on Table 4, and the mean
PRP of the PEG treatments and individual genotypes is presented in Appendix Table 1 and 4
respectively. Highly significant (p<0.01) PEG stress level x genotype interaction was recorded
for PRP. The effect of PEG stress on PRP was observed in the form of reduction in mean PRP of
all genotypes with increasing level of PEG from 0 to 2%. The total reduction in the PRP was
quantified as the sum of simple effects of each PEG level.
30
A) Normal plant regeneration in the control
B) Reduced plant regeneration in the 2% PEG
Fig.3 Plant regeneration difference due to PEG stress
The highest effect of the PEG stress in PRP was recorded in variety Chelenko (26.39). In Raya
(23.81) and Girana-1 (23.61) the effect of PEG in PRP was of the same trend and lower than in
Chelenko. Macia (21.11), Hormat (20.73), ESH-2 (20.04), Melkam (19.91), Misikir (19.25),
76T1#23 (19.05), Seredo (18.81), Abshir (17.91), Meko (17.50), and Gubiye (16.47) in their
respective order exhibited decreased plant regeneration due to PEG stress. In Gambella-1107
(11.94), Birmash (11.94) and Teshale (13.33), PEG treatment imposed the lowest reduction in
PRP indicating that these genotypes are the least affected by PEG treatment in terms of plant
regeneration.
31
Table 4: Mean plant regeneration percent (PRP) of genotypes for each PEG treatment level
PRP (%)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Miskir
Melkam
76t1#23
ESH-2
Girana-1
Teshale
CV
LSD(P<0.01)
0.0
31.75
36.31
36.31
33.23
28.97
24.44
24.44
30.00
35.00
31.31
31.75
31.11
31.55
33.14
36.11
30.00
20.58
9.29
0.5
24.60
28.97
28.97
22.22
17.778
24.44
20.00
23.33
28.33
24.83
26.11
27.68
23.81
27.381
30.83
25.00
1.0
18.89
23.41
23.41
20.00
16.667
18.89
13.89
21.67
21.67
20.14
19.76
23.33
18.65
16.67
24.07
20.83
1.5
16.67
16.39
16.39
16.89
13.89
15.28
13.89
17.78
17.50
16.43
14.48
14.48
17.64
14.48
14.29
20.83
2.0
13.83 (17.91)
12.50 (26.39)
12.50 (23.81)
12.50 (20.73)
12.50 (16.47)
12.50 (11.94)
12.50 (11.94)
12.50 (17.50)
13.89 (21.11)
12.50 (18.81)
12.50 (19.25)
11.20 (19.91)
12.50 (19.05)
13.09 (20.04)
12.50 (23.61)
16.67 (13.33)
Numbers in parenthesis are total reductions in mean PRP across the PEG treatment levels
The results of the present study showed that an increment in PEG stress level caused reduction of
PRP in sorghum genotypes which is a typical response of callus cells of many plants. The mean
PRP drastically decreased in some genotypes exposed to PEG stress. The decreasing trend of
PRP with increasing level of PEG stress is found to be in agreement with those reports in many
cereals like in rice (Biswas et al., 2002; Wani et al., 2010), wheat (Barakat and Abdel-Latif,
1995) and sorghum (Smith et al., 1985). According to Wani et al. (2010), supplementing a
month old embryogenic calli with 0, 0.5, 1.0, 1.5, 2.0% PEG (6000) for two cycles each of two
weeks decreased percent plant regeneration while there was normal plant regeneration in the nostress medium. Moreover, Smith et al. (1985) used elevated concentration of PEG during callus
growth and it resulted in no plant regeneration in sorghum.
32
In the current study different sorghum genotypes responded to PEG treatment differently
indicating the genotypic variability. In agreement with the result of this study, genotypic
variability in PRP has been reported in many plant species (Yadav et al., 2000; Yadav and
Chawla, 2001; Schween and Schwenkel, 2003; Galovic et al., 2005; Sakthivelu et al., 2008;
Begum et al., 2011).
The typical decrease in plant regeneration in callus cells of crop plants in response to water stress
is due to water shortage in the cells which leads to a decrease in cell turgor and eventually cell
growth. Addition of PEG-6000 in culture media lowers water potential of the medium that affect
cell division leading to reduced callus growth and consequently influences regeneration
(Ehsanpour and Razavizadeh, 2005; Sakthivelu et al., 2008)
4.2 Morpho-physiological Study of Regenerated Plants
4.2.1 The Effect of PEG Induced Moisture Deficit on Coleoptile Length (CL)
Sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress levels for
CL (Table 5). There was also a highly significant (p<0.01) difference among the tested
genotypes as well as the PEG treatments (Appendix Table 2 and 5). The PEG treatment reduced
mean CL in all genotypes with increasing level from 0 to 2%. The total reduction in the CL was
the sum of simple effects of each PEG level. In varieties Hormat (6.30) and Abshir (6.20) the
effect of the PEG treatment was highest and hence, highest reduction in CL was observed.
Gubiye (5.97), ESH-2 (5.93), Birmash (5.90), Gambella-1107 (5.83), Chelenko (5.73), Misikir
(5.63), Macia (5.60), Seredo (5.57), Raya (5.47), Girana-1 (5.33), Melkam (5.27), and Meko
(5.00) in their respective order exhibited reduction in CL. The least effect of PEG treatment on
CL was attributed to 76T1#23 (4.83) and Teshale (4.13).
33
Table 5: Mean coleoptile length (CL) of genotypes for each PEG treatment levels
CL (cm)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
16.67
15.17
15.10
14.77
14.03
14.20
14.43
14.30
16.63
18.57
14.53
19.30
14.50
14.50
14.73
16.43
10.85
4.32
0.5
15.63
13.87
13.93
13.33
12.87
12.93
12.70
12.87
15.27
17.17
13.23
18.03
13.50
13.23
19.57
16.50
1.0
14.57
12.63
12.47
11.77
11.50
11.50
11.33
11.53
13.93
15.60
11.73
17.70
12.37
11.67
12.50
14.80
1.5
12.90
11.03
10.93
10.00
09.90
09.77
09.97
10.30
12.37
14.37
10.17
15.40
11.20
10.20
10.60
13.40
2.0
10.47 (6.20)
09.43 (5.73)
09.63 (5.47)
08.47 (6.30)
08.07 (5.97)
08.37 (5.83)
08.53 (5.90)
09.30 (5.00)
11.03 (5.60)
13.00 (5.57)
08.90 (5.63)
14.03 (5.27)
09.67 (4.83)
08.57 (5.93)
09.40 (5.33)
12.30 (4.13)
Numbers in parenthesis are total reductions in mean CL across the PEG treatment levels
In the present experiment, PEG induced water deficit has an effect on coleoptile elongation. With
increasing PEG level CL reduced in all tested genotypes due to the PEG stress. According to
Shao et al. (2008) decreases in morphological traits are also common responses of crop plants
when subjected to osmotic stress. The decrease in the traits of the genotypes explains difference
in osmotic regulation, which enables them to maintain cell turgor to assist growth under severe
stress conditions. The variability in the decreasing trend of the genotypes will indicate the
genotypic variability in response to water deficit stress.
The findings of the present study were in line with some earlier studies where water stress
reduced shoot length in wheat (Kamran et al., 2009), maize (Ali et al., 2007), corn (Parmer and
34
Moore, 1966), and pearl millet (Radhouane, 2007) genotypes. Apart from highly significant
water deficit genotype interaction observed in this study, the reduction in coleoptile elongation
was also genotype dependent and this is also found to be in agreement to those reported by
Raziuddin et al. (2010) in wheat, Abuhay, (2000) and Ambika et al. (2011) in sorghum.
Reduced CL was observed in PEG-induced stress since PEG may interfere with water absorption
which leads to reduced cell division and growth. When plants experience drought stress, shoot
growth is arrested in response to changes in internal water status (Simonneau et al., 1993).
4.2.2 The Effect of PEG Induced Moisture Deficit on Root Length (RL)
Sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress levels for
RL (Table 6). There is also a highly significant (p<0.01) difference among the tested genotypes
as well as the PEG treatments (Appendix Table 2 and 5). The mean RL in all genotypes
decreased with increasing level of PEG from 0 to 2%. The total decrement in the RL was
quantified as the sum of simple effects of each PEG level. In varieties Seredo (0.23), 76T1#23
(0.53), Abshir (0.60), Chelenko (0.70), and Meko (0.87) respectively lowest effect of PEG stress
on RL was observed. Likewise, in Raya (0.97) and Birmash (0.97), mean RL was reduced in the
same magnitude due to the PEG treatments, but in the latter the reduction was more pronounced
above 1.5% PEG level. Similarly, in Teshale (1.03) and Melkam (1.03) the reduction in mean RL
is of the same magnitude but in the latter the reduction is more pronounced above 1.5% PEG
level. Gambella-1107 (1.17), Misikir (1.30), Hormat (1.40), and Gubiye (1.47) also exhibited
higher reduction in mean RL. The highest effect of PEG stress and consequently the highest
reduction in mean RL was observed in Macia (1.50), ESH-2 (1.50), and Girana-1 (2.23).
35
Table 6: Mean root length (RL) of genotypes for each PEG treatment levels
RL(cm)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
6.70
5.10
4.13
4.80
7.50
7.60
4.50
6.60
5.30
6.47
5.63
7.33
4.43
5.53
5.70
7.27
6.86
0.78
0.5
6.63
4.80
4.07
4.50
7.37
7.43
4.27
6.23
4.97
7.33
5.30
7.23
4.50
5.23
4.60
7.17
1.0
6.50
4.67
3.70
4.20
7.17
6.96
4.07
6.10
4.73
6.73
4.97
6.73
4.37
4.70
2.27
6.80
1.5
6.20
4.60
3.33
3.57
6.80
6.67
3.80
6.07
4.47
6.53
4.60
6.50
4.10
4.40
3.70
6.40
2.0
6.10 (0.60)
4.40 (0.70)
3.17 (0.97)
3.40 (1.40)
6.03 (1.47)
6.43 (1.17)
3.53 (0.97)
5.73 (0.87)
3.80 (1.50)
6.23 (0.23)
4.33 (1.30)
6.30 (1.03)
3.90 (0.53)
4.03 (1.50)
3.47 (2.23)
6.23 (1.03)
Numbers in parenthesis are total reductions in mean RL across the PEG treatment levels
In the genotypes scrutinized, reduction in RL across the five PEG stress levels was found. The
findings of the present study are similar to some earlier studies where water stress reduced RL in
cereals (Kamran et al., 2009 and Ali et al., 2007). Comparable results have been reported by
various authors like Parmer and Moore (1966) in corn and Radhouane (2007) in pearl millet.
The decreasing trend in RL in crop plants under moisture deficit is due to many factors. Root
growth decreases when moisture deficit stress occurs during seedling stage, mainly because of
decreased carbon partitioning to roots (Batts et al., 1998). In contrast, the response of root
growth to drought can be variable; under moderate moisture stress, root growth can be greater
because of increased partitioning of carbohydrates to roots, whereas, severe drought often limits
root growth (Prasad et al., 2008). The extent and pattern of root development are closely related
36
to the ability of the plant to absorb water and the tolerant genotypes have higher capacity of this
character. Price et al. (1997) reported that root growth is an important component of the
adaptation of rice genotypes to water deficit. Correspondingly, Nagarajan and Rane (2000)
recorded decreased RL in response to water stress.
The stressed sorghum genotypes exhibited significant difference in RL. The result was however
in contrast with Addisie (2010) in which water-stressed plants of sorghum genotypes tended to
produce less RL, but the accessions did not differ significantly in overall.
4.2.3 The Effect of PEG Induced Moisture Deficit on Shoot Dry Weight (SDW)
Sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress levels for
SDW (Table 7). There was also highly significant (p<0.01) difference among the tested
genotypes as well as the PEG treatments (Appendix Table 3 and 6). The mean SDW in all
genotypes decreased with increasing level of PEG from 0 to 2%. The total reductions in the
SDW are the sum of simple effects of each PEG level. The lowest effect of PEG stress was
recorded in variety 76T1#23 (0.02), followed by Girana-1 (0.06), Misikir (0.06), and Gambella1107 (0.08) for SDW. Teshale (0.08), Birmash (0.09), Macia (0.10), Meko (0.11), Gubiye (0.11),
ESH-2 (0.12), Melkam (0.12), Abshir (0.14), and Hormat (0.16) exhibited higher reduction in
SDW due to the PEG stress. The highest effect of the PEG stress was recorded in Raya (0.18),
Chelenko (0.36) and Seredo (0.44) respectively, indicating the relative susceptibility of the
genotypes to the induced moisture stress.
37
Table 7: Mean shoot dry weight (SDW) of genotypes for each PEG treatment level
SDW(mg)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Miskir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
1.87
1.98
1.83
1.63
1.65
1.65
1.64
1.67
1.69
2.38
1.63
2.26
1.64
1.64
1.64
1.97
3.39
0.12
0.5
1.84
1.73
1.82
1.61
1.63
1.63
1.61
1.62
1.66
2.05
1.61
2.23
1.57
1.58
1.62
1.95
1.0
1.82
1.64
1.75
1.52
1.59
1.59
1.56
1.59
1.63
2.01
1.55
2.17
1.55
1.55
1.59
1.89
1.5
1.75
1.61
1.68
1.49
1.54
1.57
1.53
1.57
1.59
1.99
1.53
2.15
1.55
1.55
1.54
1.87
2.0
1.66 (0.14)
1.59 (0.36)
1.57 (0.18)
1.44 (0.16)
1.49 (0.11)
1.54 (0.08)
1.51 (0.09)
1.54 (0.11)
1.56 (0.10)
1.93 (0.44)
1.55 (0.06)
2.11 (0.12)
1.51 (0.02)
1.52 (0.12)
1.53 (0.06)
1.86 (0.08)
Numbers in parenthesis are total reductions in mean SDW across the PEG treatment levels
The results of the present study showed that an increment in PEG stress level caused a significant
decrease of SDW. Decrease in SDW is a common response of crop plants when subjected to
moisture deficit stress. The variability in the decreasing trend of the trait explains difference in
osmotic regulation of the genotypes, which enables them to maintain cell turgor to assist growth
under severe moisture deficit conditions (Shao et al., 2008). SDW decreases manly due to
increased partitioning of solutes to the root (Prasad et al., 2008).
In the genotypes tested, reduction in SDW across the PEG stress levels was found and the
outcome is also in agreement with Ramu et al. (2008) and Addisie (2010). The variation in SDW
under different drought stress conditions would help to screen tolerant accessions from sensitive
38
ones because tolerant genotypes have less reduction in SDW than susceptible ones under water
stress conditions (Salem, 2003). The present study showed significant PEG treatment X genotype
interaction in terms of SDW which is in contradiction with Abuhay (2000). The possible reason
for the contradicting result might be the experimental set up used in this study might be the
variation in experimental set up and environment employed in this study. In this study, osmotic
stress of PEG rather than soil moisture deficit was used for inducing water deficit stress.
Moreover, the study was conducted in controlled environment.
4.2.4 The Effect of PEG Induced Moisture Deficit on Root Dry Weight (RDW)
In terms of RDW, sorghum genotypes also exhibited highly significant (p<0.01) interaction with
PEG stress levels (Table 8). Highly significant (p<0.01) difference among the tested genotypes
as well as the PEG treatments (Appendix Table 3 and 6) was also observed. RDW decreased
across each PEG treatment level and the total reduction was quantified as the sum of simple
effects of each PEG treatment level. Variety 76T1#23 (0.027) revealed lowest reduction in mean
RDW with increasing level of PEG stress. In Gambella-1107 and Girana-1, RDW reduced in the
same magnitude (0.033), but in the earlier the reduction was more intense after 1 % PEG level.
Also in Macia (0.034), Teshale (0.036), Hormat (0.036), Meko (0.043), Raya (0.047), Gubiye
(0.047), Seredo (0.051), ESH-2 (0.057), and Melkam (0.058) PEG stress caused higher reduction
in RDW in their respective order. The highest reduction was recorded in varieties Birmash
(0.060), Misikir (0.060), Chelenko (0.063) and Abshir (0.091) respectively which indicates the
highest damage to RDW in the genotypes due to the PEG stress.
39
Table 8: Mean root dry weight (RDW) of genotypes for each PEG treatment level
RDW(mg)
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76t1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
0.52
0.48
0.47
0.46
0.47
0.46
0.46
0.47
0.48
0.59
0.43
0.64
0.47
0.45
0.45
0.56
3.27
0.03
0.5
0.49
0.46
0.46
0.45
0.46
0.46
0.45
0.44
0.47
0.55
0.41
0.62
0.46
0.43
0.44
0.56
1.0
0.47
0.43
0.43
0.44
0.44
0.43
0.42
0.43
0.46
0.54
0.38
0.59
0.45
0.41
0.43
0.53
1.5
0.44
0.41
0.40
0.41
0.41
0.39
0.41
0.43
0.44
0.52
0.37
0.55
0.45
0.40
0.42
0.52
2.0
0.39 (0.091)
0.39 (0.063)
0.39 (0.047)
0.39 (0.036)
0.39 (0.047)
0.38 (0.033)
0.39 (0.06)
0.42 (0.043)
0.42 (0.034)
0.53 (0.051)
0.36 (0.06)
0.54 (0.058)
0.44 (0.027)
0.39 (0.057)
0.41 (0.033)
0.51 (0.036)
Numbers in parenthesis are total reduction in mean RDW across the PEG treatment levels
According to Rhodes and Samara (1994), a means of increasing drought tolerance is by
decreasing osmotic potential by accumulation of solutes. Generally plants accumulate some
kinds of organic and inorganic solutes in the cytosol to raise osmotic pressure and thereby
maintain both turgor and the driving gradient for water uptake. Under mild drought stress,
pattern of resource allocation generally root growth is favored rather than shoot growth. Severe
stress conditions often decrease root growth (Batts et al., 1998). Prasad et al. (2008) found that
timing of drought stress has great influence on partitioning of carbohydrates. Drought Stress
occurring during early growth stage shifts partitioning to the roots.
In addition, Seghatoleslami et al. (2008) observed that the major effect of water stress is mainly
expressed in the form of increase in RDW. However, in the result of current study decreasing
40
trend in RDW was observed. The possible reason for the decreasing trend of RDW observed in
this study might be the growth environment employed in this study. For the genotypes to store
significant carbohydrate to the roots, they have to accumulate solutes through photosynthesis. In
this study, the required nutrients were supplied through culture media; the plants were not
exposed to natural sun light and the experiment was quitted in 30 days.
In some previous investigations, decrease of RDW across water deficit stress levels was
reported. Therefore, the decrease in RDW of the studied genotypes across water deficit levels is
in line with Ramu et al. (2008) and Addisie (2010). The dissimilarity in RDW under different
drought stress conditions would help to screen tolerant genotypes from sensitive ones (Salem,
2003).
4.2.5 The Effect of PEG Induced Moisture Deficit on Root to Shoot Dry Weight Ratio
(RSDW)
For RSDW sorghum genotypes exhibited highly significant (p<0.01) interaction with PEG stress
levels (Table 9). There was also highly significant (p<0.01) difference among the tested
genotypes as well as the PEG treatments (Appendix Table 3 and 6). The mean RSDW in all
genotypes decreased, except in varieties Chelenko and Seredo with increasing level of PEG from
0 to 2%. Variety Chelenko (0.003) and Seredo (0.019) exhibited a slight overall increase in
RSDW, whereas slight reduction was recorded in Raya (0.003), Meko (0.008), and 76T1#23
(0.011). Correspondingly, in Teshale (0.011), Girana-1 (0.012), Hormat (0.013), Macia (0.013),
ESH-2 (0.019), Gubiye (0.021), Birmash (0.021), and Melkam (0.027) exhibited higher
reduction in RSDW. The highest reduction in RSDW was attributed to Gambella-1107 (0.029),
Misikir (0.031), and Abshir (0.041), which indicates that the genotypes are highly sensitive to
PEG-induced moisture deficit.
41
Table 9: Mean root shoot weight ratio (RSDW) of genotypes for each PEG treatment level
RSDW (mg)
PEG
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
0.275
0.24
0.255
0.282
0.283
0.280
0.279
0.281
0.281
0.253
0.261
0.284
0.286
0.274
0.277
0.285
3.86
0.02
0.5
0.266
0.264
0.255
0.278
0.281
0.279
0.281
0.273
0.285
0.267
0.252
0.278
0.291
0.272
0.271
0.284
1.0
0.258
0.264
0.243
0.292
0.279
0.271
0.268
0.272
0.284
0.266
0.247
0.271
0.284
0.264
0.270
0.282
1.5
0.248
0.254
0.240
0.275
0.269
0.249
0.270
0.272
0.274
0.262
0.244
0.255
0.287
0.260
0.270
0.276
2.0
0.234 (-0.041)
0.245 (+0.003)
0.252 (-0.003)
0.270 (-0.013)
0.262 (-0.021)
0.251 (-0.029)
0.258 (-0.021)
0.273 (-0.008)
0.268 (-0.013)
0.273 (+0.019)
0.231 (-0.031)
0.257 (-0.027)
0.275 (-0.011)
0.255 (-0.019)
0.265 (-0.012)
0.274 (-0.011)
Numbers in parenthesis are total reductions or increase in mean RSDW across the PEG treatment
levels (“+“=increase)
Prasad et al. (2008) found that timing of drought stress has great influence on partitioning of
carbohydrates. If drought stress occurs during early vegetative growth stages, the partitioning
shifts toward roots rather than shoots, which ends up in increased RSDW. Therefore, the
outcome is mainly due to decreased shoot weight rather than increased root weight. In addition,
Seghatoleslami et al. (2008) observed that the major effect of water stress is mainly expressed in
the form of increase in RDW.
42
However, in the result of current study decreasing trend in RSDW was observed. The possible
reason for the decreasing trend of RSDW observed in this study might be the result of decrease
in RDW. RSDW decreased because it is a function of RDW.
In the genotypes investigated, reduction in RSDW across the PEG stress levels was found and
the outcome is also in agreement with Ramu et al. (2008) and Addisie (2010). The variability in
decreasing trend of RSDW under different drought stress conditions would practically assist in
screening tolerant accessions from sensitive ones (Salem, 2003).The significant PEG treatment X
genotype interaction in terms of RSDW seen in this study contradicts the results obtained by
Abuhay (2000). In this study, the tested sorghum genotypes showed significant difference in
terms of RSDW, and this is also in contrast to Abuhay (2000). The possible reason for the
contradicting results might be the experimental set up used in this study might be the variation in
experimental set up and environment employed in this study.
4.2.6 The Effect of PEG Induced Moisture Deficit on Root Number (RN)
The mean RN in each PEG treatment level, genotypes and main PEG treatments is given in
Table 10 and Appendix Table 2 and 5 respectively. Highly significant (p<0.01) PEG X
interaction with genotypes for RN was observed. The average RN increased with increasing level
of PEG treatment in each variety. Variety Abshir exhibited the highest increase in RN (12.00)
with increasing level of PEG treatment followed by Melkam (11.00). In Gubiye, Gambella-1107,
Birmash, and Macia, increase in RN was 7.33 for each. ESH-2 (6.67), Misikir (6.00), 76T1#23
(6.00), Teshale (5.33), Seredo (5.00), Raya (4.33), Chelenko (3.33), and Hormat (3.33)
respectively exhibited lower RN than variety Birmash. The lowest increase in RN was attributed
to Meko (3.00) and Girana-1(2.67) respectively indicating that elevated PEG level triggered
lowest root branching in these genotypes.
43
Table 10: Mean root number (RN) of genotypes for each PEG treatment level
RN
PEG (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV
LSD (P<0.01)
0.0
22.67
25.33
17.00
21.00
19.33
20.33
17.33
18.00
14.00
26.33
19.66
27.00
13.00
17.33
19.33
21.33
11.23
5.52
0.5
24.00
26.67
17.67
19.67
20.67
22.00
20.33
18.66
16.66
26.00
20.00
27.00
16.00
21.33
20.33
22.67
1.0
28.00
29.33
18.67
21.00
23.67
24.33
22.00
22.00
16.67
27.00
20.00
34.00
16.00
20.33
20.67
24.33
1.5
31.00
25.67
22.00
21.67
24.67
26.00
22.00
24.33
19.33
29.00
23.67
35.33
17.00
21.33
24.00
26.00
2.0
34.64 (12.00)
28.66 (03.33)
21.33 (04.33)
24.33 (03.33)
26.66 (07.33)
27.67 (07.33)
24.66 (07.33)
26.00 (03.00)
21.33 (07.33)
31.33 (05.00)
25.66 (06.00)
38.00 (11.00)
19.00 (06.00)
24.00 (06.67)
25.33 (02.67)
28.33 (05.33)
Numbers in parenthesis are total increase in mean RN across PEG treatment levels
Genotypes showed increase in RN across increasing PEG stress levels which is in agreement
with Forster et al. (2004) in barley. Abd Allah et al. (2011) also reported that water deficit favors
the growth of seminal and lateral roots in seedlings of rice. There was increase in RN with
increasing water deficit treatment.
The results obtained in this study are also in agreement with Begum et al. (2011) who reported
that root initiation on culture media supplemented with different level of PEG increased RN in
sugarcane after one week. The RN was highest in tolerant genotypes and lowest in susceptible
ones.
44
Such an increase in RN in response to PEG induced water stress might be due to limited water up
take by the amount of roots in a particular volume of growth media (Passioura, 2002) and
enhanced root branching can reduce drought stress
The ability to maintain a variable root number among sorghum accessions may contribute to the
crop drought tolerance; since, RN is among root characteristics that are responsible for
adaptability to water stress (Abdallah, 2009).
4.3 Association between In vitro Traits
Callus induction efficiency (CIE) had positive significant correlation with traits such as CFW,
PRP, CL, and RL (Table 11). In contrast, CIE showed a significant negative correlation with
ECP and RN. It also exhibited non-significant relation with SDW, RDW, and RSDW. CFW
exhibited strong positive correlation with PRP and also positively associated with CL, RL, SDW,
RDW, and RSDW. However, it was negatively correlated with RN. No significant correlation
was observed between CFW and ECP. ECP had strong positive correlation with PRP and also
positively correlated with RL, SDW, and RDW. In contrast, no significant correlation was
observed between ECP and CL, RSDW, and RN. PRP showed positive correlation with CL, RL,
SDW, RDW, and RSWR, but negatively correlated with RN. CL also revealed positive
correlation with RL, SDW, RDW, and RSDW, but no correlation with RN. RL exhibited positive
correlation with SDW, RDW, RSDW, and RN. Likewise, SDW showed positive relation RDW
and RN, but no relation with RSDW. RDW revealed positive correlation with RSDW and RN.
Finally RSDW exhibited negative correlation with RN.
45
Table 11: Correlation coefficient (r) of measured traits (P<0.05)
CIE
CFW
ECP
PRP
CL
RL
SDW
RDR
RSDW
CIE
1
CFW
0.349*
1
ECP
-0.125*
0.001
1
PRP
0.332*
0.731*
0.783*
1
CL
0.162*
0.626*
0.041
0.549*
1
RL
0.257*
0.341*
0.259*
0.200*
0.357*
1
SDW
0.069
0.328*
0.110*
0.322*
0.596*
0.480*
1
RDW
0.099
0.451*
0.140*
0.387*
0.653*
0.546*
0.873*
1
RSDW
0.064
0.336*
0.070
0.203*
0.225*
0.188*
-0.075
0.413*
1
RN
-0.188*
-0.326*
-0.045
-0.385*
-0.045
0.326*
0.398*
0.231*
-0.312*
CIE=Callus induction efficiency
CFW=Callus fresh weight
ECP=Embryogenic callus percent
PRP=Plant regeneration percent
CL=Coleoptile length
RL=Root length
SDW=Shoot dry weight
RDW=Root dry weight
RSDW=Root shoot dry weight ratio
RN=Root number
46
RN
1
The present investigation revealed that CIE is positively correlated with PRP which is in
agreement to Malahat (2001) in which the number of regenerated plants was observed to be
determined by callus induction. This suggests that genotypes with high callus induction also
caused an increase in the number of plants obtained from regeneration medium. In the present
study, CIE was negatively correlated with ECP which is in contrast to Gandonou et al. (2005)
who found no correlation between CIE and ECP. The possible reason might be the incorporation
of PEG stress in this study the effect of which was observed in the form of decrease in CIE, but a
slight increase in ECP. The presence of significant correlation between callus induction
frequency and regeneration capacity of callus might indicate that callus induction and
regeneration capacity may be controlled by the same mechanisms. The result also exhibited
positive relation between ECP and PRP which is also in agreement with Gandonou et al. (2005).
The highly significant and strong correlation observed between the ability of cultivars to produce
embryogenic callus and their capacity for regeneration indicate that embryogenic callus
percentage constitute a good index for callus ability to regenerate later on plantlets. The cultivars
that presented high embryogenic callus percentages at the first weeks of culture have high chance
to regenerate plants after several weeks of culture.
Positive correlation between SDW and RDW is also found to be in agreement with those
reported by Ali et al. (2009) in sorghum. In those reports, RL and RSDW were also positively
associated. Dhanda et al. (2004) revealed positive association of RL with CL which is in
agreement with the results of this study, negative correlation with RSDW in wheat which is in
contrast to the result of this study. Moreover, Matsuura et al. (1996) reported a positive
correlation between drought tolerance and root length in sorghum and millet. This suggested that
these characters could be selected simultaneously with their positive effects on drought tolerance
in different stages of crop growth in sorghum.
4.4 Mann–Whitney–Wilcoxon Rank Sum Test of the Measured Traits
To determine the most desirable drought tolerant genotypes based on all traits measured, mean
rank sum method was used according to Ezatollah et al., (2012). In this method, all the indices
mean rank and standard deviation of ranks of all in vitro drought tolerance criteria were
47
calculated and summed (RS) (Table 12). Based on this criterion the most desirable drought
tolerant genotypes were identified.
48
Table 12: Rank sum of all traits measured
Variety
CIE
CFW
ECP
PRP
CL
RL
SDW
RDW
RSDW RN
Rank
Mean
Rank
SD
RS
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76t1#23
ESH-2
Girana-1
Teshale
08
15
16
11
05
07
13
01
12
02
14
06
04
10
09
03
02
14
15
16
10
07
11
01
08
12
13
05
04
03
06
09
12
03
13
15
07
04
06
01
16
09
08
10
11
14
02
05
06
16
15
12
04
02
03
05
13
07
09
10
08
11
14
01
15
10
06
16
14
11
12
03
08
07
09
04
02
13
05
01
03
04
06
12
13
10
07
05
14
01
11
09
02
15
16
08
12
15
14
13
09
04
06
08
07
16
03
11
01
10
02
05
16
15
08
06
09
02
13
07
04
10
14
12
01
11
03
05
16
01
03
06
11
14
12
04
09
02
15
13
05
10
07
06
9.1
10.6
10.8
12.1
08.5
06.5
08.8
05.0
09.7
07.7
10.4
08.2
04.7
10.4
08.0
05.3
5.88
5.76
4.64
3.63
3.72
4.06
3.77
4.29
3.86
4.90
3.72
3.71
3.53
3.47
5.54
3.09
14.98
16.36
15.44
15.74
12.22
10.56
12.57
09.29
13.56
12.60
14.12
11.91
08.23
13.88
13.54
08.39
01
13
12
14
03
04
05
15
06
11
08
02
09
07
16
10
CIE=Callus induction efficiency
CFW=Callus fresh weight
ECP=Embryogenic callus percent
PRP=Plant regeneration percent
CL=Coleoptile length
RL=Root length
SDW=Shoot dry weight
RDW=Root dry weight
RSDW=Root shoot dry weight ratio
RN=Root number
SD=Standard deviation
RS=Rank Sum
49
By taking in to consideration all indices, genotypes: 76T1#23(RS= 8.23) and Teshale (RS= 8.39)
followed by genotypes: Meko (RS= 9.29), Gambella-1107(RS= 10.56) and Melkam (RS=11.91)
were the most drought tolerant genotypes, respectively. While genotypes: Chelenko (RS =16.36),
Hormat (RS = 15.74) and Raya (RS= 15.44) were the most sensitive to drought, therefore they
are recommended for crossing and genetic analysis of drought tolerance using diallel mating
design or generation mean analysis and also for the QTL (quantitative trait loci) mapping and
marker assisted selection. The same procedures have been used for screening quantitative
indicators of drought tolerance in wheat, maize, and in rye by Mohammadi et al. (2011) and
Ezatollah et al. (2012).
.
The detection of superior genotypes 76T1#23, Teshale, Meko, Gambella-1107 and Melkam for
drought tolerance at cellular level together with their high potential for callus induction leads to
the conclusion that a hybridization breeding procedure using these superior plant materials
supplemented with in vitro selection for drought tolerance might be beneficial for improving
these traits in sorghum. Hence, callus culture can be useful to speed up sorghum improvement.
50
5. SUMMARY AND CONCLUSION
The present investigation was aimed to evaluate the relative tolerance of different sorghum
genotypes for drought tolerance with respect to callusing, callus growth, embryogenic callus
formation, plant regeneration and various morpho-physiological traits. To achieve the objectives,
16 elite sorghum genotypes were taken and laboratory experiment was conducted in tissue
culture laboratory of Melkassa Agricultural Research Center. PEG 6000 was used as in vitro
drought inducer. The experiment was laid down in factorial experiment with CRD design with
three replications. MS media (Murashige and Skoog, 1962) supplemented with plant hormones
and growth regulators were used for callusing, embryogenic callus formation, and plant
regeneration. The growth of the regenerated plants was sustained on MS basal medium with no
phytohormones.
Measurements on callusing, callus proliferation, embryogenic callus formation, plant
regeneration and growth performance of the regenerated plants were made in PEG stress
condition. There was also highly significant (p<0.01) PEG stress genotype interaction for CIE,
CFW, ECP, PRP, CL, RL, SDW, RDW, RSDW, and RN. In all genotypes, all traits decreased
with increasing level of PEG from 0 to 2% except ECP and RN. ECP exhibited slight
inconsistency in response to PEG treatments, whereas mean RN increased with increasing level
of PEG treatment. In terms of CIE, varieties Meko, Seredo, and Teshale found to be tolerant to
PEG induced moisture stress. Varieties Meko, Abshir, and ESH-2 found to be tolerant to PEG
induced moisture stress in terms of CFW. Slight decrease in ECP in varieties Hormat and ESH-2,
observed while there was general increment in mean CIE in all other genotypes. Varieties Meko,
Girana-1, and Chelenko exhibited higher tolerance in terms of ECP. Sorghum varieties Teshale,
Gambella-1107, Birmash revealed highest capacity of plant regeneration under PEG stress.
Under PEG stress, varieties Teshale, 76T1#23, and Meko exhibited higher coleoptiles elongation
indicating the relative tolerance of the genotypes. In terms of RL, Seredo, 76T1#23, Abshir
exhibited higher tolerance than the tested genotypes. Varieties 76T1#23, Girana-1, and Misikir
had relatively higher SDW, whereas 76T1#23, Gambella-1107, and Girana-1 revealed higher
RDW under the PEG stress. On the other hand, varieties Chelenko, Seredo and Raya had higher
RSDW, while Abshir, Melkam, and Gubiye exhibited higher mean RN under PEG stress. The
51
correlation analysis revealed strong positive association between ECP and PRP. SDW and RDW
were also revealed strong positive correlation.
By taking in to consideration all indices measured in this study, Mann–Whitney–Wilcoxon Rank
Sum test revealed varieties 76T1#23 and Teshale followed by genotypes Meko, Gambella-1107
and Melkam were the most drought tolerant genotypes, respectively. On the contrary, genotypes:
Chelenko, Hormat and Raya were the most sensitive to drought.
In conclusion, the results of the present study pointed out the need for using in vitro screening
when evaluating drought tolerance of sorghum accessions. Under in vitro water deficit condition
the accessions demonstrated variation in response to all of the measured parameters under
osmotic water stress at cellular level. The genotypes demonstrated variation in response to most
of measured parameters at plant level. Moreover, genotypes which are recommended for drought
prone areas appeared to be tolerant at cellular and early plant growth stage. Thus, the variations
elucidated the existing genotypic difference among sorghum genotypes in response PEG induced
water deficit stress. In consideration to all indices, sorghum genotypes: 76T1#23, Teshale, Meko,
Gambella-1107 and Melkam appeared to be more tolerant for PEG induced drought stress than
all genotypes tested. The results also showed that sorghum accessions present a remarkable array
of similar behaviors in response to PEG induced water stress conditions. Therefore, this indicates
that the traits are recommendable as potential screening tools for drought stress in sorghum under
in vitro condition.
On the basis of the various traits measured the present study identified 76T1#23, Teshale, Meko,
Gambella-1107 and Melkam as tolerant which showed higher performance under in vitro
condition. It is recommended that further work on identification of desirable traits of the selected
set of genotypes through marker assisted breeding should follow, so that utilization of the
measured traits for selection of drought tolerance could be further integrated with the
inheritance/genetics and molecular background of the genotypes selected. In addition to these,
Meko, Raya, Macia, Abshir, Girana-1, Teshale, Gambella-1107, Seredo, Misikir, Chelenko were
selected for their suitability for callus culture and plant regeneration. In the current study,
Genotypes like Chelenko, Hormat and Raya were the most sensitive to drought, therefore they
52
are recommended for crossing and genetic analysis of drought tolerance using diallel mating
design or generation mean analysis and also for the QTL (quantitative trait loci) mapping and
marker assisted selection. Since the study was carried out under controlled culture media
condition, extrapolating the results would require careful consideration of environmental
conditions in the field. Therefore, testing the same set of genotypes under real condition to find
the relative tolerance should follow.
The number of sorghum genotypes utilized in this study was limited only to 16. Similar approach
is recommended for other accessions. Moreover, similar study can be used as complementary to
field study for varieties being developed through diffferent research programs or for those which
are online to be released for commercial production. The concentrations of PEG used in this
study were kept as low as below 2% to obtain enough population of regenerated plants for
morphological study. Further study using elevated level of PEG concentration is recommended.
In the current study, only PEG was used as in vitro drought inducer. Further study using other
osmotica like mannitol or in combination with PEG is recommended.
The results also showed that sorghum genotypes have a incredible array of similar behaviors in
response to PEG induced water stress conditions. Similar study can be extrapolated for other
drought sensitive crops.
53
6. REFERENCES
Abd Allah, A. A., A. Shimaa, B. Badawy, A. Zayed and A. A. El.Gohary. 2011. The role of root
system traits in the drought tolerance of rice (oryza sativa L.). World Academy of Science,
Engineering and Technology 80
Abdallah, A. A. 2009. Genetic studies on leaf rolling and some root traits under drought
conditions in rice (Oryza sativa L.). African Journal of Biotechnology 8: 6241-6248
Abdel-Ghany, H. M, A. A. Nawar, M. E. Ibrahim, A. El-Shamarka, M. M. Selim and A. I.
Fahmi. 2004. Using tissue culture to select for drought tolerance in bread wheat. Proceedings of
the 4th International Crop Science Congress Brisbane, Australia, 26 Sep – 1 Oct
Abdel-Raheem, A. T., A. R. Ragab, Z. A. Kasem, F. D. Omar and A. M. Samera. 2007. In vitro
selection for tomato plants for drought tolerance via callus culture under polyethylene glycol
(PEG) and mannitol treatments. African Crop Science Conference Proceedings 8: 2027-2032
Abdelsamad, A., O. E. El – Sayed and F. Ibrahim. 2007. Development of drought tolerance
haploid wheat using biochemical genetic markers on in vitro culture. Journal of Applied Sciences
Research, 3(11):1589 -1599
Abdulai, L. A. 2005. Morphological and physiological response of Sorghum (Sorghum bicolor
L. Moench) to different patterns of drought. M.Sc. thesis, Ghana
Abdulaziz, M. and A. M. Al- Bahrany. 2002. Callus growth and praline accumulation in
response to polyethyleneglycol-induced osmotic stress in rice (Oryza sativa L.). Journal of
Biological Sciences, 5(12): 1294-1296
Abuhay Takele. 1997. Genotypic variability in dry matter production, partitioning and grain
yield of teff (Eragrostis tef (Zucc.) Trotter) under moisture deficit. SINET. Ethiopian Journal of
Science, 20: 177-188
Abuhay Takele. 2000. Seedling emergence and of growth of sorghum genotypes under variable
soil moisture deficit. Acta Agronomica Hungarica, 48(1): 95-102
Addisie Yalew. 2010. Evaluation of sorghum (Sorghum bicolor) genotypes for post-flowering
drought resistance (stay-green trait). M.Sc. Thesis. Addis Ababa University
Alemayehu Bekelle. 2003. Variability and interrelationship among yield and yield related traits
of lowland sorghum. M.Sc. Thesis Haramaya University, Haramaya, Ethiopia
54
Ali, G., A. C. Rather, A. Ishfaq, S. A. Dar, S. A. Wani and M. N. Khan. 2007. Gene action for
grain yield and its attributes in maize (Zea mays L.). International Journal of Agricultural
Science, 3(2): 767-788.
Ali, M. A., A. Abbas, S. Niaz, M. Zulkiffal and S. Ali. 2009. Morpho-physiological criteria for
drought tolerance in sorghum (Sorghum Bicolor) at seedling and post-anthesis stages.
International Journal of Agriculture and Biology, 11: 674–680
Ambika, R., A. Rajendran, R. Muthiah, A. Manickam, P. Shanmugasundaram and A. J. Joel.
2011. Indices of Drought Tolerance in Sorghum (Sorghum bicolor L. Moench) Genotypes at
Early Stages of Plant Growth. Research Journal of Agriculture and Biological Sciences, 7(1):
42-46
Amjad, M. A., S. Niaz, A. Abbas, W. Sabir and K. Jabran. 2009. Genetic diversity and
assessment of drought tolerant sorghum landraces based on morph physiological traits at
different growth stages. Plant Omics Journal, 2(5):214-227
Amuna, P., F. Zotor, S. Sumar, and Y. T. Chinyanga. 2000. The role of traditional
cereal/legume/fruit-based multimixes in weaning in developing countries. Journal of Nutrition
and Food Sciences, 30(3): 116-122
Asfaw Adugna, Tesfaye Teklu and Abera Deresa. 2005. Sorghum production and research
experiences. Production guideline. Ethiopian Agricultural Research Organization,
Asfaw Adugna. 2007. Assessment of yield stability in sorghum. African Crop Science Journal
15(2): 83 – 92
Ather, A., S. Khan, A. Rehman, and M. Nazir. 2009. Optimization of the protocol for callus
induction, regeneration and acclimatization of sugarcane. Pakistan Journal of Botany, 41(2):
815-820
Aulinger, E. 2002. Combination of in vitro androgenesis and biolistic transformation: an
approach of breeding transgenic maize (Zea mays L.). A Doctoral Thesis, Swiss Federal Institute
of Technology, Zurich, pp115
Barakat, M. N. and T. H. Abdel-Latif .1995. Somatic embryogenesis in callus from mature and
immature embryo culture of wheat. Alexandrian Journal of Agricultural Research, 40: 77-95.
Barciszewski, J. S. I., S. G., Rattan, G. Siboska, and B. F. C. Clark. 1999. Kinetin. Plant Science
148:37–45
55
Barrs, H. D. and Weatherly, P. E. 1962. Re-examination of relative turgidity for estimating water
deficits in leaves. Australian Journal of Biological Science, 15:413- 428.
Bartels, D. and S. Ramanjulu. 2005. Drought and salt tolerance in plants. Critical Reviews in
plant sciences, 24:23-58
Bartels, D., and E. Souer. 2004. Molecular responses of higher plants to dehydration. In: Plant
Responses to Abiotic Stress (eds H. Hirt and K. Shinozaki), pp. 9–38. Springer-Verlag, Berlin
and Heidelberg, Germany
Batts, G. R., R. H. Ellis, J. I. L. Morison, P. N. Nkemka, P. J. Gregory, and P. Hadley. 1998.
Yield and partitioning of crops of contrasting cultivars of winter wheat in response to CO2 and
temperature in field studies using temperature gradient tunnels. Journal of Agricultural Sciences,
130:17–27
Begum, M. K., M. O. Islam, M. A. S. Miah, M. A. Hossain and N. Islam. 2011. Production of
somaclone in vitro for Drought Stress Tolerant Plantlet Selection in Sugarcane (Saccharum
officinarum L.). The Agriculturalist, 9(1 and 2): 18-28
Beltrano, J., and M. G. Ronco. 2008. Improved tolerance of wheat plants (Triticum aestivum L.)
to drought stress and rewatering by the arbuscular mycorrhizal fungus Glomus claroideum:
Effect on growth and cell membrane stability. Brazil Journal of Plant Physiology, 20:29-37
Bhutta, W. M. 2007. The effect of cultivar on the variation of spring wheat grain quality under
drought conditions. Cereal Research Community, 35:1609–1619
Biswas, J., B. Chowdhury, A. Bhattacharya, and A. B. Mandal. 2002. In vitro screening for
increased drought tolerance in rice. In Vitro Cell Developmental Biolology, 38:525–530
Blum, A. 1979. Genetic improvement of drought resistance in crop plants: A case for sorghum.
In: Stress Physiology in Crop Plants, pp 429-455, (Mussel, H. and Staples, R. C., eds). John
Willey and Sons, New York.
Blum, A. and C. Y. Sullivan. 1987. The comparative drought resistance of land races of sorghum
and millet from dryland humid regions. Annals of Botany, 57: 835-846.
Borrell, A. K. and G. L. Hammer. 2000. Nitrogen dynamics and the physiological basis of stay
green in sorghum. Crop Sciences 40:1295-1307
56
Bota, A. J., F. O. Nachtergaele and A. Young. 2000. Land resource potential and constraints at
regional and country levels, World’s Soil Resources Report 90, FAO Land and Water
Development Division, Rome
Bozhkov, P. V., and S. von Arnold. 1998. Polyethylene glycol promotes maturation but inhibits
further development of Picea abies somatic embryos. Physiologia Plantarum 104: 211-224
Centritto, M., M. Lauteri, M. C. Monteverdi and R. Serraj. 2008. Leaf gas exchange, carbon
isotope discrimination, and grain yield in contrasting rice genotypes subjected to water deficits
during the reproductive stage. African Journal of Biotechnology 7: 2341-2352
Chala, A., M. B. Brurberg, and A. M. Tronsmo. 2007. Prevalence and intensity of sorghum
anthracnose in Ethiopia. Journal of SAT Agricultural Research 5(1): 145- 298
Chen, G., M. Sagi, S. Weining, T. Krugman, T. Fahima, A. B Korol, and E. Nevo. 2004. Wild
barley eibi1 mutation identifies a gene essential for leaf water conservation. Planta 219: 684-693
Cockcroft, C. E., B. G. W. den Boer, J. M. S. Healyy, and J. A. H. Murray. 2000. Cyclin D
control of plant growth rate in plants. Nature 405: 575–579.
Colom, M. R. and C. Vazzana. 2003. Photosynthesis and PSII functionality of drought-resistant
and drought sensitive weeping lovegrass plants. Environmental Experimental Botany, 49: 135–
144
Cosgrove, D. J. 1997. Relaxation in a high stress environment: The molecular basis of extensible
cell walls and cell enlargement. Plant Cell, 9: 1031–1041
CSA (Central Statistical Authority of Ethiopia): Area, Production and Yield of Crops for Private
Peasant Holdings for Meher Season 2010/11 (2003 E.C.). Addis Ababa, Ethiopia; 2011.
Dagnachew Bekelle. 2008. Genetic diversity study in Sorghum bicolor germplasm accessions
collected from the major drought prone areas of Ethiopia based on quantitative and qualitative
traits. M. Sc. Thesis, Addis Ababa University, Addis Ababa
Davies, P. J. 1995. Introduction to Plant Hormones, P. J. Davies, ed. (Dordrecht, the
Netherlands: Kluwer Academic Publishers), pp. 1–38
Deen, S. C. and M. Mohamoud. 1996. Comparative study between saponin and natural auxin on
root growth of Rosemary (Rosmarinus Officinal is L.) cutting. Acta Horticulture 426: 635-642
57
den Boer, B. G.W. and J. A. H. Murray. 2000. Triggering the cell cycle in plants. Trends in Cell
Biology 10: 245–250
Dhanda, S. S., G. S. Sethi, and R. K. Behl. 2004. Indices of drought tolerance in wheat genotypes
at early stages of plant growth. Journal of Agronomy Crop Science 190: 6–12
Dicko, M., H. Harry, Gruppen, Alfred, S. Traoré, Alphons, G. J. Voragen, Willem, and J. H. van
Berkel. 2006. Sorghum grain as human food in Africa: relevance of content of starch and
amylase activities. African Journal of Biotechnology 5 (5): 384-395
Dogget, H. 1988. Sorghum 2nd edition. Longman. Green Co. Ltd. London pp512
Dragiiska, R., D. Djilianov, P. Denchev, and A. Atanassov. 1996. In vitro selection for osmotic
tolerance in alfalfa (Medicago sativa). Bulgarian Journal of Plant physiology 22: 30–39
Ehsanpour, A. A., and A. Razavizadeh. 2005. Effect of UV-C on drought tolerance of alfalfa
(Medicago sativa) callus. American Journal of Biochemistry and Biotechnology 1(2): 107-110
Ejeta Gebissa and J. E. Knoll. 2007. Marker-assisted selection in sorghum In: Varshney R. K.
and Tuberosa R (ed.) Genomic-assisted crop improvement: Vol. 2: Genomics applications in
crops, 187-205
Ezatollah, F., B. Jamshidi, K. Cheghamirza and J. A. Teixeira da Silva. 2012. Evaluation of
drought tolerance in bread wheat (Triticum aestivum L.) using in vivo and in vitro techniques.
Annals of Biological Research, 3 (1):465-476
FAO: Food and Agricultural Organization
www.faostat.fao.org (Accessed: 23/03/12)
statistical
database
(FAOSTAT).
2011.
Farooq, M., S. M. A. Basra, A. Wahidi, Z. A. Cheema, M. A. Cheema and A. Khaliq. 2008.
Physiological role of exogenously applied glycinebetaine in improving drought tolerance of fine
grain aromatic rice (Oryza sativa L.). Journal of Agronomy and Crop Sciences 194: 325–333
Firew Mekbib. 2009. Folksong based appraisal of bioecocultural heritage of sorghum (Sorghum
bicolor (L.) Moench): A new approach in ethnobiology. Journal of Ethnobiology and
Ethnomedicine 5:19
Forster, B. P, R. P. Ellis, J. Moir, V. Talame, M. C. Sanguineti, R. Tuberosa, D. This, B. TeulatMerah, I. Ahmed, S. A. E. E. Mariy, H. Bahri, M. Ouahabi, N. El Zoumarou-Wallis, M. El-
58
Fellah, and M. Ben Salem. 2004. Genotype and phenotype associations with drought tolerance in
barley tested in North Africa, Annals of Applied Biology 144, 157–168.
Galović, V., Z. Kotaranin, and S. Denčić. 2005. In vitro assessment of wheat tolerance to
drought. Genetika 37(2): 165-171
Gamborg, O. L. and G. C. Phillips. 1995. Plant cell, tissue and organ culture. Spring-Verlag
Berlin Heidelberg, pp 21- 22
Gandonou, C. H., T. Errabii, J. Abrinii, M. Idaomar, F. Chibi, and N. S. Senhaji. 2005. Effect of
genotype on callus induction and plant regeneration from leaf explants of sugarcane. African
Journal of Biotechnology 4(11): 1250-1255
Ganeshan, S., M. Båga, B. L. Harwey, B. G. Rossnagel, G. J. Scoles and R. N. Chibbar. 2003.
Production of multipleshoots from thiadiazuron-treated mature embryos and leaf-base/apical
meristems of barley (Hordeum vulgare). Plant Cell, Tissue and Organ Culture 73: 57–64
Gebeyehu Adugna, Tadesse Tesso, K. Balate, H. Michael. 2004. Development of sorghum
varieties and hybrids for dryland areas of Ethiopia. Uganda Journal of Agricultural Sciences
9:594-605
Godwin, I. D, and S. J. Gray. 2000. Overcoming productivity and quality constraints in sorghum:
the role for genetic engineering. In Transgenic cereals; O'Brien L., Henry R. J. Eds. Minnesota
USA; pp.153-177
Gomez, K.A. and Gomez, A. A. 1976. Statistical Procedures for agricultural Research with
Special Emphasis on rice. Philippines, International Rice Research
Gopal, J. and K. Iwama. 2007. In vitro screening of potato against water stress mediated through
sorbitol and polyethylene glycol, Plant Cell Report, 26: 693-700
Guerrero, F. D. and J. E. Mullet. 1986. Increased abscisic acid biosynthesis during plant
dehydration requires transcription. Plant Physiology, 80: 588-591
Guerrero, F. D., J. T. Jones and J. E. Mullet. 1990. Turgor-responsive gene transcription and
RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three
inducible genes. Plant Molecular Biology 15: 11-26
Gunasekera, D. and G. A. Berkowitz. 1992. Evaluation of contrasting cellular-level acclimitation
responses to leaf water deficits in three wheat genotypes. Plant Sciences, 86: 1-12
59
Hagio, T. 2002. Adventitious shoot regeneration from immature embryos of sorghum. Plant
Cell, Tissue and Organ Culture 68: 65-72
Hamdy, M. and El. Aref. 2002. Employment of maize immature embryo culture for
improvement drought tolerance. Proceeding of the 3rd Scientific Conference of Agriculture
Sciences, Faculty of Agriculture, Assiut University, Assiut, Egypt. pp. 463 -477
Hartmann, H. T., D. E. Kester and F. T. Davis. 2002. Plant Propagation: Principles and Practices
6th ed. Prentice- Hall International, Inc., Englewood Cliffs, New Jersey, pp 647
Harlan, J. R. 1969. Ethiopia: A centre of diversity. Economic botany, 23:309- 314
Hoque, E. H. and J. W. Mansfield. 2004. Effect of genotype and explant age on callus induction
and subsequent plant regeneration from root-derived callus of Indica rice genotypes. Plant Cell
Tissue and Organ Culture 78: 217–223
Hsissou, D. and J. Bouharmont. 1994. In vitro selection and characterization of drought tolerant
plants of durum wheat (Tritium durum. Desf). Agronomie 2: 65-70
ICRISAT (International Crops Research Institute for the Semi-Arid Tropics). 2006. Sorghum
Genetic Enhancement: Research Process, Dissemination and Impacts. Andhra Pradesh, India
IPCC (Intercontinental Panel on Climate Change). 2001. Climate Change in 2001. Available at
http:// www. ipcc.ch; accessed on sep.1, 2011
Iraqi, D. and F. M. Tremblay. 2001. The role of sucrose during maturation of black spruce (Picea
mariana (Mill.) BSP) and white spruce (Picea glauca (Moench) Voss) somatic embryos,
Physiologia Plantarum 111: 381-388
Isendahl, N., and G. Schmidt. 2006. Drought in the Mediterranean-WWF policy proposal, A.
WWF Report, Madrid
Jogeswar, G., D. Ranadheer, V. Anjaiah, and P. B. K. Kishor .2007. High frequency somatic
embryogenesis and regeneration in different genotypes of Sorghum bicolor (L.) Moench from
immature inflorescence explants. In Vitro Cell, and Developmental Biology of plants 43: 159166
60
Kamran, M., M. Shahbaz, M. Ashraf and N.A. Akram. 2009. Alleviation of drought-induced
adverse affects in spring wheat (Triticum aestivum L.) using proline as a pre-sowing seed
treatment. Pakistan Journal of Botany 41(2): 621-632.
Khalequzzaman, M., N. Haq, M. E. Hoque and T. L. Aditya. 2005. Regeneration efficiency and
genotypic effect of 15 Indica type Bangladeshi rice (Oryza sativa L.) landraces. Plant Tissue
Culture 15: 33–42
Khan, I. A., M. U. Dahot, N. Seema, S. Yasmeen, S. Bibi, G. Raza, A. Kharti and M. H Naqvi.
2009. Direct regeneration of sugarcane plantlets: a tool to unravel genetic heterogeneity.
Pakistan Journal of Botany, 41(2): 797-814
Khanna, H. K and S. K. Raina. 1998. Genotype × culture media interaction effects on
regeneration response of three Indica rice cultivars. Plant Cell, Tissue and Organ Culture 52:
145–153
Khanna, V. K. Plant tissue culture practice. Kalyani Publishers. Printed in India. Xpress Grafics,
Delhi-28. 2003
Kim, Y. W. and H. K. Moon. 2007. Enhancement of somatic embryogenesis and plant
regeneration in Japanese larch (Larix leptolepis). Plant Cell, Tissue and Organ Culture 88: 241245
Kimber, C. T. 2000. Origins of domesticated sorghum and its early diffusion into India and
China. In: Sorghum: Origin, History, Technology, and Production, (C. Wayne Smith and R.A.
Frederiksen, eds), John Wiley & Sons, New York, pp 3-98
Kuhlman, L. C., B. L. Burson, P. E. Klein, R. R. Klein, D. M. Stelly , H. J. Price and W. L.
Rooney. 2008. Genetic recombination in Sorghum bicolor x S. macrospermum interspecific
hybrids. Genome 51
Kulkarni, M. and U. Deshpande. 2007. In Vitro screening of tomato genotypes for drought
resistance using polyethylene glycol. African Journal of Biotechnology, 6 (6): 691-696
Kumar, A. and K. D. Sharma. 2009. Physiological responses and dry matter partitioning of
summer Mungbean (Vigna radiata L.) genotypes subjected to drought conditions, Journal of
Agronomy and Crop Sciences 195: 270–277
Larcher, W. 2003. Physiological Plant Ecology: Ecophysiology and Stress Physiology of
Functional groups. Springer, New York.
61
Lestari, E. G. 2006. In Vitro selection and somaclonal variation for biotic and abiotic stress
tolerance. Indonesian Center for Agricultural Biotechnology and Genetic Resources Research
and Development (ICABIOGRAD), Bogor 16111
Lipvska, H., H. Svobodova, J. Albrechtova, L. Kumstyrova, M. Vagner and Z. Vondrakova.
2000. Carbohydrate status during somatic embryo maturation in Norway spruce. In Vitro
Cellular and Developmental Biology-Plant 36: 260-267
Lührs, R. and H. Lörz. 1987. Plant regeneration in vitro from embryogenic cultures of springand winter-type barley (Hordeum vulgare L.) varieties. Theoretical and Applied Genetics 75:
16–25
Luma, H. A. and H. K. Al-Ka'aby. 2011. The effect of water stress on callus and somatic
embryos formation of rice (Oryza sativa L.) cv. Jasmine cultured in vitro. Journal of Basrah
Researches (Sciences), 37(3)
Lutts, S., M. Almansouri, J. M. Kinet. 2004. Salinity and water stress have contrasting effects on
the relationship between growth and cell viability during and after stress exposure in durum
wheat callus. Plant Sciences, 167: 9-18
Mahajan, S. and N. Tuteja. 2005. Cold, salinity and drought stress: An overview. Archives of
Biochemistry and Biophysics 444:139-158
Melahat, A. B. 2001. Callus Induction and Plant Regeneration from Mature Embryos of Oat
(Avena sativa L.). Turkey Journal of Biology 25 (2001) 427-434
Matheka, J. M., E. Magiri, A. O. Rasha, and J. Machuka. 2008. In vitro selection and
characterization of drought tolerant somaclones of tropical maize (Zea mays L.). Biotechnology
7(4): 641-650.
Matsuura, A., S. Inanaga and Y. Sugimoto. 1996. Mechanism of interspecific differences among
four graminaceous crops in growth response to soil drying. Japanese Journal of Crop Science
65: 352-360
Mehmood, S., A. Bashir, A. Amad, and Akram. 2008. Molecular characterization of regional
Sorghum bicolor varieties from Pakistan. Pakistan Journal of Botany 40: 2015-2021
62
Mihiret Kassa Alemu. 2009. The Effect of natural fermentation on some anti-nutritional factors,
minerals, proximate composition and sensory characteristics in sorghum based weaning food.
M.Sc. thesis Addis Ababa University
Mitra, J .2001. Genetics and genetic improvement of drought resistance in crop plants. Current
Science 80: 758-763
Mohamed, M. A. H, P. J. C. Harris, and J. Henderson. 2000. In vitro selection and
characterization of a drought tolerant clone of Tagetes minuta. Plant Sciences, 159: 213-222
Mohamed, M. S. 1996. Biochemical studies on fenugreek by using tissue culture techniques. M.
Sc. Thesis, Faculty of Agriculture, Cairo University, Egypt
Mohammadi, M., R. Karimizadeh, M. Abdipour. 2011. Evaluation of drought tolerance in bread
wheat genotypes under dryland and supplemental irrigation conditions, Australian Journal of
Crop Sciences, 5(4):487– 493.
Moon, H. K. and S. Y. Park .2008. Somatic embryogenesis and plantlet production using
rejuvenated tissues from serial grafting of a mature Kalopanax septemlobus tree. In Vitro
Cellular and Developmental Biology 44:119-127
Muhammad, H., S. A. Khan, Z. K. Shinwari, A. L. Khan, N. Ahmad and In-Jung Lee. 2010.
Effect of polyethylene glycol induced drought stress on physio-hormonal attributes of soybean.
Pakistan Journal Botany, 42(2): 977-986
Munns, R., J. B. Passioura, J. Guo, O. Ehazen and G. R. Cramer. 2000. Water relations and leaf
expansion: Importance of timing. Journal of Experimental Botany 51: 1495–1504
Murashige, T., A. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco
tissues culture, Physiology of Plants 15: 473-497
Nagarajan, S. and J. Rane. 2000. Relationship of seedling traits with drought tolerance in spring
wheat cultivars. Indian Journal of Plant Physiology 5: 264-270.
Newton, R., J. S. Bhaskaran, J. D. Puryear, and H. R. Smith. 1986. Physiological changes in
cultured sorghum cells in response to induced water stress. Plant Physiology 81; 626-629
Nordin, K., P. Heino, and E. T. Palva. 1991. Separate signal pathways regulate the expression of
a low temperature induced gene in Arabidopsis thaliana (L.) Heynh. Plant Molecular Biology,
16: 1061-1071
63
Oria, M. P., B. R. Hamaker, J. D. Axtell and C. P. Huang. 2000. A highly digestible sorghum
mutant cultivar exhibits a unique folded structure of endosperm protein bodies. PNAS 2000;
97(10): 5065–5070
Özgen, M., M. Türet, S. Altinok and C. Sancak. 1998. Efficient callus induction and plant
regeneration from mature embryo culture of winter wheat (Triticum asetivum L.) genotypes.
Plant Cell Reports 18: 331–335
Ozgen, M., M. Turet, S. Ozcan and C. Saneak. 1996. Callus induction and plant regeneration
from immature and mature embryos of winter durum wheat cultivars. Plant Breeding 115: 455–
458
Parmer, M. J. and R. P. Moore. 1966. Effect of stimulated drought by PEG solution on corn (Zea
mays L.) germination and development. Agronomy Journal 58: 391 392
Passioura, J. B. 2002. Soil conditions and plant growth. Plant Cell and Environment 25:311–318
Pawar, K. N. 2007. Physiological indices for drought tolerance in rabi sorghum. PhD Thesis,
University of Agriculture Sciences, Dharwad. India
Pellegrineschi, A., R. M. Brito, S. Mclean, and D. Hoisington. 2004. Effect of 2, 4
dichlorophenoxyacetic acid and NaCl on the establishment of callus and plant regeneration in
durum and bread wheat. Plant Cell, Tissue and Organ Culture 77: 245–250
Petrasek, J., M. E. Kner, D. Morris, and E. Z. Malova. 2002. Auxin efflux carrier activity and
auxin accumulation Studies on rape explants cultured in vitro. Plant Cell, Tissue and Organ
Culture 77:225-248
Plomion, C., P. Costa, C. Dubos, J. M. Frigerio, J. M. Guehl, and A. Queyrens. 1999. Genetical,
physiological and molecular response of Pinus pinaster to a progressive drought stress. Journal
of Plant Physiology 155: 120-129
Prakash, A. H., S. N. Vajranabhaiah and R. Chandrashekhara. 1994. Changing patterns in water
relation and solute contents during callus development under stress in sunflower (Helianthus
annum L.) Advanced in Plant Science 7: 223-225
Prasad, P. V. V. and S. A. Staggenborg. 2008. Impacts of Drought and/or Heat Stress on
Physiological, Developmental, Growth, and Yield Processes of Crop Plants. Department of
Agronomy, Kansas State University, Manhattan
64
Prasad, P. V. V., S. R. Pisipati, R. N. Mutava and M. R. Tuinstra, 2008. Sensitivity of grain
sorghum to high temperature stress during reproductive development. Crop Science 48: 1911–
1917
Price, A. H., D. S. Virk, A. D. Tomos. 1997. Genetic dissection of root growth in rice (Oryza
sativa L.). Theoretical and Applied Genetics 95:132–142
Price, H. James, S. L. Dillon, G. Hodnett, W. L. Rooney, L. Ross and J. S. Johnston. 2005.
Genome evolution in the genus Poaceae. Annals of Botany 95:219-227
Quarrie, S. A. and P. G. Lister. 1984. Effects of inhibition of protein synthesis on abscisic acid
accumulation in wheat. Plant physiology, 114: 309-314
Radhouane, L. 2007. Response of Tunisian autochthonous pearl millet (Pennisetum glaucum (L.)
R. Br.) to drought stress induced by polyethylene glycol (PEG) 6000. African Journal of
Biotechnology, 6:1102-1105
Rahman, M., I. Ullah, M. Ashraf, J. M. Stewart and Y. Zafar. 2006. Genotypic variation for
drought tolerance in cotton (Gossypium hirsutum L.): Productivity, osmotic adjustment and
cellular membrane stability. International Symposium on Strategies for Crop Improvement
against Abiotic Stresses, pp 14
Ramu, S. V., S. P. Palaniappan and R. Panchanathan. 2008. Growth and Dry matter Partitioning
of Sorghum under Moisture Stress Condition. Journal of agronomy and crop science, 166(4):273277
Rauf, S., and H. A. Sadaqat. 2008. Identification of physiological traits and genotypes combined
to high achene yield in sunflower (Helianthus annuus L.) under contrasting water regimes.
Australian Journal of Crop Science 1:23-30
Raza, G., K. Ali, Z. Mukhtar, S. Mansoor, M. Arshad and S. Asad. 2010. The response of
sugarcane (Saccharum officinarum L.) genotypes to callus induction, regeneration and different
concentrations of the selective agent (geneticin-418). African Journal of Biotechnology 9(51):
8739-8747
Raziuddin, Z., J. Swati, B. Bakht, M. Ullah, M. Shafi, M. Akmal and G. Hassan.2010. In situ
assessment of morpho-physiological response of wheat (Triticum aestivum L.) genotypes to
drought. Pakistan Journal of Botany 42(5): 3183-3195
65
Rhodes, D. and Y. Samara.1994. The effect of water stress on callus and somatic embryos
formation of rice (Oryza sativa L.) cv. Jasmine cultured in vitro.CRC press. 347- 361
Rosenow, D. T., J. E. Quisenberry, C. W. Wendt, and L. E. Clark. 1983. Drought tolerant
sorghum and cotton germplasm. Agricultural water management 7:207-222
Sabour, I., O. Merah, S. El Jaafari, R. Paul and P. Monneveux. 1997. Leaf osmotic potential,
relative water content and leaf excised water loss variations in oasis wheat landraces in response
to water deficit. International Archives of Physiology, Biochemistry and Biophysics 105: 14
Sakthivelu, G., M. K. A. Devi, P. Giridhar, T. Rajasekaran, G. A. Ravishankar, T. Nedev and G.
Kosturkova. 2008. Drought-induced alterations in growth, osmotic potential and in vitro
regeneration of soybean cultivars. General and Applied Plant Physiology Special Issue, 34 (1-2):
103-112
Salem, M. 2003. Response of durum and bread wheat genotypes to drought biomass and yield
components. Asian Journal of Plant Sciences, 2:290-93
Schween, G. and H. G. Schwnkel. 2003. Effect of genotype on callus induction, shoot
regeneration and phenotypic stability of regenerated plants in the greenhouse of Primula spp.
Plant Cell, Tissue and Organ Culture 72: 53 – 61
Seghatoleslami, M., K. and E. Majid. 2008. Effect of drought stress at different growth stages on
yield and water use efficiency of five Proso millet (Pannicum millaceum L.) genotypes. Pakistan
Journal of Botany 40: 1427-1432
Shao, H. B., L. Y. Chu, M. A. Shao, A. C. Jaleel and M. Hong-Mei. 2008. Higher plant
antioxidants and redox signaling under environmental stresses. Comptes Rendus Biologies 331:
433–441.
Setter, T. L. and B. A. Flannigan. 2001. Water deficit inhibits cell division and expression of
transcripts involved in cell proliferation and endoreduplication in maize endosperm. Journal of
Experimental Botany 52: 1401–1408
Sharma, K., and M. Lavanya. 2002. Recent developments in transgenics for abiotic stress in
legumes of the semi arid tropics In: Ivanaga M (ed) Genetic engineering for crop plants for
abiotic stress, JIRCAS Working Report No. 23: 61-73, Tsukuba, Japan
Shewale, D. S. 2008. Studies in the enzymatic depolymerization of natural polysaccharides. PhD
Thesis, Institute of Chemical Technology, University of Mumbai
66
Sibel, T. and T. Birol. 2007. Some physiological responses of drought stress in wheat genotypes
with different ploidity in Turkey. World Journal of Agricultural Sciences 3: 178-183
Sidari, M., C. Mallamaci and A. Muscolo. 2008. Drought, salinity and heat differently affect
seed germination of Pinus pinea. Journals of Forest Research 13: 326-330
Siddique, M. R. B., A. Hamid, and M.S. Islam. 2000. Drought stress effects on water relations of
wheat. Botanical Bulletin of Academia Sinica 41(1):35-39
Simmons, M. W., D. A. Kudrna and R. L. Warner. 1989. Reduced accumulation of ABA during
water stress in a molybdenum cofactor mutant of barely. Plant Physiology, 90: 728-733
Simon, K. 2009. Crop monitoring in Ethiopia. European Commission, Joint Research Center, 4:
1-19
Simonneau, T., R. Habib, J. P. Goutouly, and J. G. Buguet. 1993. Diurnal changes in stem
diameter depend upon variation in water content: Direct evidence from peach trees. Journal of
Experimental Botany, 44:615–621
Sinaki, J. M., E. M. Haravan, A. H. S. Rad, G. Noormohammadi, and G. Zarei. 2007. The effects
of water deficit during growth stages of canola (Brassica napus L.). American-Eurasian Journal
of Agricultural and Environmental Sciences 2: 417-422
Sleper, D. A. and J. M. Poehlman. 2003. Breeding Field Crops. 5th Edition, Blackwell
Publishing, pp 230
Smith, R. H. S. Bhaskaran and F. R. Miller. 1985. Screening for drought tolerance in sorghum
using cell culture. In Vitro Cellular and Developmental Biology 21: 10
Soltani, A., M. Gholipoor, and E. Zeinali. 2006. Seed reserve utilization and seedling growth of
wheat as affected by drought and salinity. Environmental and Experimental Botany 55:195-200
Sorrells, M. E., A. Diab and M. Nachit. 2007. Comparative genetics of drought tolerance.
International center for advanced Mediterranean studies Agronomic studies (www.ciheam.org
accessed date 30/03/2012)
Szilgyi, L. 2003. Influence of drought on seed yield components in common bean. Bulgarian
Journal of Plant Physiology, Special Issue, 320-330
67
Taiz, L. and E. Zeiger. 2006. Stress physiology. In: L. Taiz, and E. Zeiger, eds. Plant Physiology,
4th ed., pp. 671–681. Sinauer Associates, Inc., Sunderland, MA
Teshome Alemayehu, D. Patterson, Asfaw Demissie, J. K. Torrance, and J. T. Arnason. 2007.
Changes of Sorghum bicolor landrace diversity and farmers’ selection criteria over space and
time, Ethiopia. Genetic Resource and Crop Evolution, 54:1219–1233
Tileye Fayissa, M. Wellander and Legesse Negash. 2005. Micropropagation of Hagenia
abyssinica. Plant Cell, Tissue and Organ Culture 80: 119-127
Turhan, H. and I. Baser. 2004. Callus induction from mature embryo of winter wheat (Triticum
aestivum L.). Asian Journal of Plant Sciences, 3(1):17-19
Turner, M. and V. Kramer. Adaption of Plant to Water and High Temperatures stress. Willey
Inters Science, New York. 1980
Van Staden, J., E. Zazimalova and E. F. George. 2008. Plant growth regulators II: Cytokinins,
their analogues and antagonists. In: George EF, Hall MA, De Klerk GJ (eds) Plant propagation
by tissue culture, 3rd eds. Springer, Dordrecht, pp 205–226
Vavilov, N. 1951. The origin, variation, immunity and breeding of cultivated plants. Chronicles
of Botany, 13:37-39
Vikrant, A. R. 2003. Somatic embryogenesis or shoot formation following high 2, 4-D treatment
of mature embryos of Paspalum scrobiculatum (L). Biologia Plantarum 46: 297–300
Visser, B. 1994. Technical aspects of drought tolerance. Biotechnology and Development
Monitor 18: 5
Walter, H. 1963. The water supply of desert plants. In the water relation of plants. pp: 190- 205.
Butter, A. J. and P. W. Whitchead (eds) Wiley and Sons. New York
Wani, S. H., P .A. Sofi, S. S. Gosal and N. B. Singh. 2010. In vitro screening of rice (Oryza
sativa L) callus for drought tolerance. Communications in Biometry and Crop Science 5 (2),
108–115
West, G., D. Inze, and G. T. S. Beemster. 2004. Cell cycle modulation in the response of the
primary root of Arabidopsis to salt stress. Plant Physiology 135: 1050–1058
68
Wortmann, C. S., M. Mamo C. Mburu E. Letayo, Girma Abebe, K. C. Kayuki, M. Chisi, M.
Mativavarira, S. Xerinda and T. Ndacyayisenga. 2006. Atlas of Sorghum (Sorghum bicolor (L.)
Moench): Production in Eastern and Southern Africa. University of Nebraska, Lincoln
Yadav, M. K., N. K. Singh and G. K. Garg, 2000. Development of lines of Indian wheat
genotypes for efficient regeneration using mature embryos. In: Symposium on Biotechnology for
sunstainable agriculture, 27 – 29 April 2000, G. B. Plant University of Agriculture and
Technology, Pantnagar, India
Yadava, R., and H. S. Chawla. 2001. Interaction of genotype, growth regulators and amino acids
on plant regeneration from different developmental states of influorescence in wheat. Journal of
Genetics and Breeding, 55: 261 – 297
Yamaguchi-Shinozaki, K., M. Koizumi, S. Urao, and K. Shinozaki. 1992. Molecular cloning and
characterization of cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana:
Sequence analysis of one cDNA clone that encodes a putative transmembrane channel protein.
Plant Cell Physiology, 33(3): 217-224
Yetneberk Senait, H. L. De Kock, L. W. Rooney and J. R. N. Taylor. 2004. Effects of sorghum
cultivar on injera quality. Cereal Chemistry 81:314–321
Yilma Kebede and Abebe Menkir. 1987. Current activities, research recommendations and future
strategies of sorghum improvement program in Ethiopia Paper presented at the 19th National
Crop Improvement Program in Ethiopia. April 22-26, 1987, Addis Ababa, Ethiopia
Zalc, J. M., H. B. Wicr, K. K. Kidwell and C. M. Steber. 2004. Callus induction and plant
regeneration from mature embryos of a diverse set of wheat genotypes. Plant Cell Tissue and
Organ Culture 76, 277–281
Zelalem Mengiste. 2008. Evaluation of post-flowering drought resistance property of Ethiopian
sorghum accessions collected from the drought prone northern part. M. Sc. Thesis Addis Ababa
University, Addis Ababa
Zhang, L., F. Chen, C. Elliott and A. Slater. 2004. Efficient procedures for callus induction and
adventitious shoot organogenesis in sugar beet (Beta vulgaris L.) breeding lines. In Vitro
Cellular and Developmental Biology 40:475-481
Zhao, L., S. Liu, and S. Song. 2010. Optimization of callus induction and plant regeneration
from germinating seeds of sweet sorghum (Sorghum bicolor Moench). African Journal of
Biotechnology 9(16), 2367-2374
69
7. APPENDICES
70
Appendix A: Main Effects of Genotypes across PEG levels
Appendix Table 1: Mean callus induction efficiency, callus fresh weight, embryogenic callus
percent and plant regeneration percent of the tested genotypes
Variety
CIE (%)
CFW (mg)
ECP (%)
PRP (%)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV (%)
LSD(p<0.01)
48.22
43.56
75.56
38.67
59.56
57.33
41.78
78.89
41.33
32.44
49.33
50.67
46.22
31.56
44.67
58.44
13.39
6.36
345.14
334.18
330.56
324.06
338.15
341.34
315.01
325.57
341.66
332.16
326.02
343.51
338.08
323.49
344.53
344.04
4.80
15.26
46.24
12.76
40.04
48.57
39.90
53.20
29.97
24.66
49.92
52.02
52.98
35.16
41.99
43.11
29.80
61.19
6.74
2.65
21.16
24.00
23.51
20.97
17.96
19.23
17.44
21.33
23.00
21.04
20.92
22.19
20.20
20.91
23.20
22.67
20.58
3.17
CIE=Callus Induction efficiency
CFW=Callus fresh weight
ECP=Embryogenic callus percent
PRP=Plant regeneration percent
71
Appendix Table 2: Mean coleoptile length, root length and root number of the tested genotypes
Variety
CL(cm)
RL (cm)
RN( cm)
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76t1#23
ESH-2
Girana-1
Teshale
CV (%)
LSD(p<0.01)
14.05
12.43
12.41
11.67
11.27
11.35
11.39
11.66
13.85
15.74
11.71
16.89
12.25
11.63
14.03
14.69
10.85
1.93
6.43
4.71
3.68
4.65
6.97
7.02
4.03
6.15
4.65
6.66
4.97
6.82
4.65
4.71
3.95
6.77
6.86
0.35
28.07
27.13
19.33
21.27
23.00
24.07
21.27
21.80
17.60
27.93
21.80
32.27
16.20
20.87
21.93
24.53
6.86
2.47
CL=Coleoptile length
RL=Root length
RN=Root number
72
Appendix Table 3: Mean shoot and root dry weight and root shoot dry weight ratio of the tested
genotypes
Variety
SDW(mg)
RDW(mg)
RSDW
Abshir
Chelenko
Raya
Hormat
Gubiye
Gambella-1107
Birmash
Meko
Macia
Seredo
Misikir
Melkam
76T1#23
ESH-2
Girana-1
Teshale
CV (%)
LSD(p<0.01)
1.57
1.70
1.73
1.54
1.58
1.59
1.57
1.60
1.63
2.07
1.57
2.18
1.58
1.56
1.59
1.91
3.39
0.05
0.46
0.43
0.43
0.43
0.43
0.42
0.42
0.44
0.45
0.55
0.39
0.59
0.45
0.42
0.43
0.54
3.27
0.014
0.26
0.25
0.25
0.28
0.28
0.27
0.27
0.27
0.28
0.26
0.25
0.27
0.29
0.27
0.27
0.28
3.86
0.0098
SDW=Shoot dry weight
RDW=Root dry weight
73
RSDW=Root shoot dry weight ratio
Appendix B: Main Effect of PEG Levels across Genotypes
Appendix Table 4: Mean callus induction efficiency, callus fresh weight, embryogenic callus
percent and plant regeneration across the PEG stress levels
PEG (%)
CIE (%)
CFW(mg)
ECP (%)
PRP (%)
0.0
0.5
1.0
1.5
2.0
CV (%)
LSD(p<0.01)
58.06 a
53.33 b
49.38 c
46.04 d
42.64 e
13.39
3.55
378.20 a
371.36 a
345.00 b
297.91 c
274.46 d
4.80
8.53
41.05a
40.36b
39.96b
39.39b
37.86c
6.74
1.12
31.84a
25.26b
20.17c
16.03d
12.93e
20.58
2.31
CIE=Callus induction efficiency
ECP=Embryogenic callus percent
CFW=Callus fresh weight
PRP=Plant regeneration percent
Appendix Table 5: Mean coleoptile length, root length and root number across the PEG
treatment levels
PEG Level (%)
CL(cm)
RL(cm)
RN
0
0.5
1.0
1.5
2
CV (%)
LSD(p<0.01)
15.49 a
14.87 b
12.98 c
11.41 c
11.41 d
10.85
1.08
5.91 a
5.73 b
5.29 c
5.11 d
4.82 e
6.86
0.20
19.94
21.23
23.00
24.56
26.69
11.25
1.38
CL=Coleoptile length
RL=Root length
RN=Root number
74
e
d
c
b
a
Appendix Table 6: Shoot and root dry weight and root shoot dry weight ratio across the PEG
stress levels
PEG Level (%)
SDW (mg)
RDW (mg)
RSDW
0.0
0.5
1.0
1.5
2.0
CV (%)
LSD(p<0.01)
1.79 a
1.74 b
1.69 c
1.66 d
1.62 e
3.39
0.03
0.49 a
0.48 b
0.45 c
0.44 d
0.42 e
3.27
0.0079
0.27 a
0.27 a
0.27 a
0.26 b
0.26 b
3.86
0.0055
SDW=Shoot dry weight
RDW=Root dry weight
RSDW=Root shoot dry weight ratio
Appendix C: General ANOVA Table of the Studied Traits
Appendix Table 7: Analysis of Variance Table for CIE
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
3660.80
631.52
392.74
642.67
5327.73
MS
244.053
157.881
6.546
4.017
F
60.76
39.31
1.63
MS
1291.6
96411.8
539.1
257.0
F
5.02
375.08
2.10
Appendix Table 8: Analysis of Variance Table for CFW
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
19373
385647
32343
41127
478491
75
Appendix Table 9: Analysis of Variance Table for ECP
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
35055.5
431.3
535.5
1244.0
37266.2
MS
2337.03
107.82
8.93
7.78
F
300.58
13.87
1.15
MS
57.34
2731.92
17.21
19.03
F
3.01
143.54
0.90
MS
44.928
258.640
3.446
4.127
F
10.89
62.67
0.83
MS
23.7329
9.6038
0.3159
0.1356
F
175.00
70.82
2.33
Appendix Table 10: Analysis of Variance Table for PRP
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
860.1
10927.7
1032.8
3045.2
15865.8
Appendix Table 11: Analysis of Variance Table for CL
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
673.92
1034.56
206.73
660.35
2575.57
Appendix Table 12: Analysis of Variance Table for RL
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
355.993
38.415
18.954
21.699
435.061
76
Appendix Table 13: Analysis of Variance Table for SDW
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
8.6231
0.8840
0.4284
0.5332
10.4687
MS
0.57488
0.22100
0.00714
0.00333
F
172.51
66.32
2.14
MS
0.04314
0.03822
0.00040
0.00022
F
194.30
172.11
1.81
MS
0.00194
0.00216
0.00015
0.00011
F
18.20
20.19
1.36
Appendix Table 14: Analysis of Variance Table for RDW
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
0.64716
0.15287
0.02405
0.03553
0.85961
Appendix Table 15: Analysis of Variance Table for SRDW
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
0.02917
0.00863
0.00870
0.01709
0.06359
77
Appendix Table 16: Analysis of Variance Table for RN
Source
Variety
PEG
Variety*PEG
Error
Total
DF
15
4
60
160
239
SS
3883.67
1368.92
343.75
1076.00
6672.33
MS
258.911
342.229
5.729
6.725
F
38.50
50.89
0.85
Appendix D: Culture Media Composition and Preparation
Appendix Table 17: Stock solution of macro nutrients
SN
Chemical
Concentration (mg/L) stock (20X)
1
Ammonium nitrate (NH4NO3)
1650.0
2
Potassium nitrate (KNO3)
1900.0
3
Magnesium sulphate (MgSO4.7H2O)
370.0
4
Potassium dibasic phosphate (KH2PO4)
170.0
5
Calcium Chloride (CaCl2)*
440.0
Appendix Table 18: Stock solution of micro nutrients
SN
Chemical
Concentration (g/L) stock (200X)
1
Boric acid (H2Bo3)
6.2
2
Manganese Sulphate (MnSO4.4H2O)
22.3
3
Zinc Sulphate (ZnSO4.7H2O)
8.6
4
Sodium moligdate (Na2MoO4)
0.25
5
Copper Sulphate (CuSO4.5H2O)
0.025
6
Cobalt Chloride (CoCl2.6H2O)
0.025
7
Potassium Iodide (KI)*
0.83
78
Appendix Table 19: Fe-EDTA (Ethylenediaminetetra acetic acid) solution Preparation
SN
Procedures (200X)
1
Add 1L double distilled water in a beaker
2
Heat until boiling point
3
Separate in to three parts
4
Dissolve separately 3.73 g of Na2EDTA and 2.78 g of FeSO4.7H2O
5
Add FeSO4.7H2O solution over Na2EDTA solution, not vice versa
6
Fill up to 1L in accurate vessel
7
Store in a glass dark vessel inside refrigerator
Appendix Table 20: Vitamines, Amino acids, Carbon and energy sources, growth regulators, and
solidifying agent
SN
Chemical
Concentration (mg/L) (200X)
1
Casain hydrolysate
500
2
Proline
600
3
Thiamine
0.1
4
Myoinositol
100
3
Polyvinyl pyruvate
10
4
Ascorbic acid (Vitamine c)
10
5
Sucrose
30g
6
Agar
1.2g
7
2,4-Dichlorophenoxyacetic acid (2,4-D)
4
8
Kinetin
0.2
9
Indole-3-acetic acid (IAA)
1
10
6-Benzylaminopurine (6-BA)
3
79
Appendix Table 21: Preparation of MS medium (1000 ml)
SN
Stock Solution
Amount/1000mL
1
MS Macro nutrient solution (20X)
50 ml
2
MS Micro nutrient solution (200X)
5 ml
3
MS Vitamin (200X)
5 ml
4
Iron (200X)
5 ml
5
MS Amino Acids (200X)
5ml
6
Casain hydrolysate
500mg
7
Myoinositol
100 mg
8
→ Add BAP, 2, 4, D, Kinetin, and IAA as needed at this stage
→ Make final volume to 1000 ml by double distilled water
→ Set pH at 5.8
→ Add agar 1.2 gm/L , melt the agar in microwave oven
→ Sterilize the media for 15 minutes
→ After autoclaving, gently swirl the medium to mix the agar. When the agar is
completely dissolved and mixed, the medium should be dispensed.
80
Appendix Table 22: Agroecological adaptation of the selected sorghum varieties
S. No
Variety
Year of
Release
Breeder/Maintainer
1
Abshir
2000
MARC/EIAR
2
Chelenko
2005
MARC/EIAR
3
4
5
6
7
Hormat
Raya
Misikir
Girana-1
Gubiye
2007
2005
2007
2007
2000
SRARC/ARARI
8
Gambella1107
1976
MARC/EIAR
9
10
11
Birmash
Meko
Macia
1989
1997
2007
MARC/EIAR
12
Seredo
1986
MARC/EIAR
13
Melkam
2009
MARC/EIAR
14
76T1#23
1979
MARC/EIAR
MARC/EIAR
ESH-2
2009
MARC/EIAR
16
Teshale
2002
MARC/EIAR






























MARC/EIAR
15
Agroecology







81
Altitude: <1600m
Rainfall: 600 mm
north Shewa, Kobbo and Meiso
Altitude:<1850
Rainfall: 900-1200mm
Altitude: <1600m
Rainfall: 500-800 mm
early maturing
Wollo and Sirinka
Altitude: <1600m
early maturing and drought resistant
Rainfall: 600 mm
north Shewa, Kobbo and Meiso
Altitude: (<1600m)
Rainfall: 600 mm
Gambella,Yabello, Jijga Kobo,
Shewa robit
Altitude:<1850
Rainfall: 900-1200mm
Altitude: <1600m
Rainfall: 500-800 mm
Has stay green trait
Altitude: <1600m
Rainfall: 500-800 mm
Drought tolerant
Altitude :<1600 m
Rainfall: 500-800 mm
Melkassa, Mieso, Kobo
Altitude:<1600m
Rainfall: 600 mm
North Wello, Cheffa , north Shewa
and Meiso
very early maturing
Altitude :<1600 m
Rainfall: 500-800 mm
Melkassa, Mieso, Kobo
Altitude: <1600m
Rainfall: 500-800 mm
north Wollo and north Shewa
82