STUDIES ON GENETIC TRANSFORMATION OF BLACK GRAM (VIGNA MUNGO L.) WITH COLD INDUCED TRANSCRIPTOME GENE (ICE- 1) FOR ABIOTIC STRESS TOLERANCE A THESIS SUBMITTED TO THE ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY, BHUBANESWAR, ODISHA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE (BIOTECHNOLOGY) By KUMARA SWAMY R.V DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY COLLEGE OF AGRICULTURE ORISSA UNIVERSITY OF AGRICULTURE & TECHNOLOGY BHUBANESWAR, ODISHA 2013 THESIS ADVISOR: DR. K. C.SAMAL
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STUDIES ON GENETIC TRANSFORMATION OF BLACK GRAM (VIGNA MUNGO L.) WITH COLD
INDUCED TRANSCRIPTOME GENE (ICE- 1) FOR ABIOTIC STRESS TOLERANCE
A
THESIS SUBMITTED TO THE ORISSA UNIVERSITY OF AGRICULTURE AND TECHNOLOGY,
BHUBANESWAR, ODISHA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN AGRICULTURE (BIOTECHNOLOGY)
By
KUMARA SWAMY R.V
DEPARTMENT OF AGRICULTURAL BIOTECHNOLOGY COLLEGE OF AGRICULTURE
ORISSA UNIVERSITY OF AGRICULTURE & TECHNOLOGY BHUBANESWAR, ODISHA
2013
THESIS ADVISOR: DR. K. C.SAMAL
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CERTIFICATE - I
This is to certify that the thesis entitled �STUDIES ON
GENETIC TRANSFORMATION OF BLACK GRAM (VIGNA
MUNGO L.) WITH COLD INDUCED TRANSCRIPTOME GENE
(ICE- 1) FOR ABIOTIC STRESS TOLERANCE� submitted in partial
fulfillment of the requirement for the award of the Degree of �MASTER
OF SCIENCE IN AGRICULTURAL BIOTECHNOLOGY� to the
Orissa University of Agriculture & Technology, Bhubaneswar is a record
of bona fide research work carried out by KUMARA SWAMY R.V.
(Adm.No.08ABT/11) under my guidance and supervision. No part of the
thesis has been submitted elsewhere for any degree or diploma or
published in any other form. All sorts of help and sources of information
availed during this investigation have been duly acknowledged.
This is to certifying that the thesis entitled �Studies on genetic
transformation of black gram (Vigna mungo L.) With cold induced
transcriptome gene (ICE- 1) for abiotic stress tolerance� submitted by
KUMARA SWAMY R.V (Adm.No.08ABT/11) to the Orissa University
of Agriculture & Technology, Bhubaneswar�751003 in partial fulfillment
of the degree of �MASTER OF SCIENCE IN AGRICULTURAL
BIOTECHNOLOGY� has been approved by the student�s advisory
committee after oral examination in collaboration with external examiner.
ADVISORY COMMITTEE:
CHAIRMAN: Dr. K.C. Samal
Associate Professor Department of Agricultural Biotechnology College of Agriculture OUAT, Bhubaneswar _________________
MEMBERS:
Prof. G.R. Rout Professor and Head Department of Agricultural Biotechnology College of Agriculture OUAT, Bhubaneswar __________________
Prof. M. Kar Vice- chancellor OUAT, Bhubaneswar.
EXTERNAL EXAMINER: ____________________
ACKNOWLEDGEMENT
As a pretty long and eventful journey of my life is nearing an end, it is the right moment to extol my profound gratitude to the virtues of those who have directly or indirectly helped me during the tenure of this work. At this moment of accomplishment, my heart is overwhelmed with gratitude and I wish if these words could convey the subtle feelings.
I express my sincere gratitude to Prof. M. Kar, Vice-Chancellor and my advisor for their valuable advice and necessary suggestions during my work.
I am ineffable in expressing my heartfelt gratitude and indebtedness to the chairman of my advisory committee, Dr. K.C Samal, Associate Professor, Department of Agricultural Biotechnology, College of Agriculture, OUAT, Bhubaneswar, for his sweet gentility pulsated with undiminished enthusiastic support, inspiring guidance, constant supervision and encouragement throughout the course of my research period and teaching me how to be cool and patient in adverse situations.
I thank with great honour and reverential gratitude to Prof. G. R. Rout, Professor and Head, Department of Agricultural Biotechnology, College of Agriculture, OUAT, Bhubaneswar for his valuable advice, moral support, constructive criticism, relevant suggestion and facilities provided during the course of this investigation.
I also owe my deep sense of gratitude and sincere thanks to Dr. A.B. Das, Associate Professor and I.C Mohanty Assistant Professor, Department of Agricultural Biotechnology for their timely advice, valuable suggestions and help at the time of need.
I thank with heartfelt gratitude and indebtedness to my great teachers who make my strengthen Dr. R.L Ravi Kumar, Professor, UAS(B), Dr. P.H. H. Ramanjini Gowda ,Professor, UAS(B), Dr.Mallikarjuna Gowda, Associate Professor UHS(B) and Dr.M.A. Shankar, Dean Director of Research, University of Agricultural Sciences, Bengaluru.
I would especially like to express my heartful gratitude to Prof. Lingaraj Sahaoo Dept. Biotechnology, Indian Institute of Technology, Guwahati, for providing gene construct as well as constant inspiration. I would also like to express my sincere thanks and gratitude to Department Seed Sciences for providing quality seed materials for my research work.
I also avail this opportunity to express my sincere gratefulness to my beloved seniors Mrs. Subhadra, Ms. Netravati ,Mr. Sairam, Mr. Anupam, Ms. Dipti, Mr. Pardip, Mr. Ravimdra, Ms. Divya, Mr.Yogesh, Ms. Seema, Ms. Bhanupriy, Mr. Pravin, Ms.
Sunanda, Ms. priyadharshini, Ms. Amrita, and Mr. Rahul for their prompt help and cooperation for the entire period of study.
Words fail to express my indebtedness with veneration and devotion to my friends Kundansingh, Ratanpal, Shyam, Shantosh, Shital, Ranjeev, Krishna and Ashuthosh who helped me in several ways for the completion of this venture. Due to their kind co-operation and friendly nature, I couldn�t recognise how the time passed away.
I also like to thank my juniors Kirath singh, Sachin, Navanath, Nihar, Dhamadri, and, Pallavi and Rinny for their help.
I am in dearth of words to thank my best friends Akash D., Raghu R , Rajani H.G., Rashmi H.P., Pramod, Prasanna, Prabhu, Sukruth and Vinyaraj who stood by me during all the hard times.
I wish to extend my sincere thanks Amiya, Preedip.M, Preedip B., Naik babu and saria for the help and assistance.
The financial assistance in form of DBT fellowship from the Department of Biotechnology, Ministry of Science and Technology, Government of India is greatly acknowledged.
I wish to extend my sincere gratitude to Mr. Atratran Kar, for his kind co-operation neat & clean editing and formatting of manuscript.
Above all, I am forever beholden to my loving parents, Vishwanath and Pramila B.S, Brother Chandan R.V and family members for their constant prayers, affection, moral support, personal sacrifice and sincere encouragement throughout the period of my studies.
Finally I bow to the lotus feet of Kalikamba Devi whose grace had endowed me the inner strength, patience, will power and health to complete this endeavour successfully.
A word of apology to those I have not mentioned in person and a note of thanks to one and all who worked for the successful completion of this endeavour.
Date: Place: Bhubaneswar (Kumara Swamy R.V.)
Name of student : KUMARA SWAMY R.V Admission No. : 08ABT/11 Title of the thesis : Studies on genetic transformation of black gram
(Vigna mungo L.) with cold induced transcriptome gene (ICE- 1) for abiotic stress tolerance
Degree for which thesis : M.Sc. (Agri.) Biotechnology is submitted Name of department : Department of Agri. Biotechnology, College of
Agriculture Orissa University of Agriculture & Technology Bhubaneswar, Odisha.
Year of submission : 2013 Name of the advisor : Dr. KAILASH CHANDRA SAMAL Associate professor, Dep. of Agri. Biotechnology, CA, OUAT, Bhubaneswar
ABSTRACT
Black gram (Vigna mungo L. Hepper, syn. Phaseolus mungo L.) is one of the
most important pulse crops grown in India. It belongs to the family Fabaceae. It is a
day neutral, warm season, annual legume crop commonly grown in semi-arid to sub-
humid low land tropics and sub-tropics. It is originated in India and has been
cultivated from ancient times. India is the largest producer and consumer of black
gram in the world. The production and productivity of black gram is severely
affected by number of biotic and abiotic stresses. Low temperature seriously
affects the productivity and production of black gram in the Northern and
Eastern parts of India. Classical breeding for tolerance to low temperature have
achieved limited success due to the absence of adequate and satisfactory level of
genetic variability within the available germplasm and genes conferring resistance to
biotic and abiotic stress are mostly available in many wild relatives. But these genes
can be successfully transferred to cultivated crop like black gram, employing
biotechnological tools. ICE1 gene, one of the potential genes conferring resistance to
abiotic stress, has been identified and isolated from Arabidopsis. It is an upstream
transcription factor and a positive regulator of CBF-3 which plays a critical
role in cold tolerance in Arabidopsis. The present study was under taken to
introduce cold tolerance in the popular cultivar of Blackgram �T-9�. For this
purpose, an efficient in-vitro regeneration and transformation protocol was first
standardized. Callus was induced from leaf as well as shoot tip explants on MS
medium supplemented with 3 mg/l 2.4.D, 1mg/l Kinetin and 0.2% cocoanut water.
In spite of several combinations of Phytohormones, vitamins and other organic
compounds in MS medium, organogenesis from callus was not achieved. Direct
regeneration through multiple shoot induction was achieved using cotyledonary
node and shoot tip as explants. Multiple shoots were induced on MS medium
supplemented with 3.0 mg/l BAP and 0.05mg/l IBA. MS medium supplemented
with TDZ (0.1mg/l) and IBA (0.05mg/l) also induced better multiple shoot
induction. Multiple shoots were subcultured on the shoot elongation MS medium
fortified with 1.0 mg/l GA3. The best rooting from multiple shoots were achieved
on MS medium fortified with 0.5 mg/l NAA as well as 0.25 mg/l NAA. The genetic
transformation through co-cultivation has been established by using cotyledonary
node explants inoculated with EHA-105 Agrobacterium strain harbouring a binary
vector pCAMBIA2301 containing Neomycin phosphotransferase (nptII) gene as
selectable marker, ß-glucuronidase (GUS) as a reporter gene and ICE-1gene.
Important parameters like optical density, pre-culture period and co-cultivation time
were standardized to maximize the transformation frequency. Optical density of 0.6
at 600nm, co-cultivation period of 3 days (72 hours) and Pre-culture period of 4
days (96 hours) were found suitable for optimum transformation and better survival
frequency. Lethal dose of Kanamycin was found to be 80 mg/l which inhibits the
growth and proliferation of untransformed/ control plants. Transformation
efficiency on the basis of Kanamycin selection was found to be 9.23%. Transient
GUS expression percentage was observed about 95% in transformed shoots after
screening on selection medium containing antibiotics. Transformed plantlets were
hardened in the greenhouse in pots containing soil: sand: vermicompost (1:1:1).
Based on PCR analysis with nptII primer transformation efficiency was found to be
about 1.53%.
CONTENTS
CHAPTER PARTICULARS PAGE
I INTRODUCTION 1-9
II REVIEW OF LITERATURE 10-62
III MATERIALS AND METHODS 63-79
IV EXPERIMANTL RESULT 80-103
V DISCUSSION 104-111
VI SUMMARY AND CONCLUSION 112-114
BIBLIOGRAPHY i-xliii
APPENDIX xliv-liii
LIST OF TABLES
TABLE TITLE PAGE
1 Taxonomic classification of Black gram 1
2 Nutrition composition of black gram 5
3 Area, Production and Productivity of Blackgram in Major States (2011-12)
9 Effect of surface sterilants on the aseptic culture and survival of explant
81
10 Effect of plant growth regulators (PGR) on callus induction of black gram cultivar �T-9�
82
11 Effect of plant growth regulators on shoot multiplication from shoot tip and cotyledon node
85
12 Effect of plant growth regulators on shoot elongation of multiple shoots
89
13 Effect of plant growth regulators on rooting of multiple shoots
91
14 Detection of lethal concentration of Kanamycin for selection medium
94
15 Effect of pre-culture period on co-cultivation 95
16 Effect of Duration on co-cultivation 96
17 Determine the sensitivity of Agrobacterium to various level of cefotaxime
98
18 Transient GUS expression percentage 101
19 Transformation efficiency based on kanamycin selection and PCR analysis
101
LIST OF PLATES
PLATE TITLE PAGE
1 Botanical description of black gram 3
2 Explant source for regeneration and transformation 65
3 Effect of plant growth regulator on callus induction from leaf explant
84
4 Effect of plant growth regulator on callus induction from shoot tip explant
84
5 Effect of plant growth regulators on shoot multiplication from shoot tip and cotyledon node
87
6 Effect of plant growth regulators shoot elongation of multiple shoots
90
7 Effect of plant growth regulators on rooting of multiple shoots
90
8 Hardening of plantlets 93
9 Determination of Kanamycin sensitivity 100
10 Kanamycin based selection transformants 100
11 GUS histochemical assay 102
12 PCR analysis of Putative transformants using nptII as Primer
103
LIST OF FIGURS
FIGURE TITLE PAGE
1 Scheme illustrating the molecular response of plants to low-temperature stress
20
2 The CBF (C-repeat-binding factor) pathway in plants. 22
3 Schematic map of vector pCAMBIA2301 66
4 Linear map of T-DNA region of vector pCAMBIA2301
66
5 Effect of plant growth regulator on callus induction of the black gram cultivar T-9
88
6. Effect plant growth regulator on shoot multiplication from shoot tip and cotyledon node
88
7. Effect of plant growth regulator on shoot elongation of multiple shoot
92
8. Effect of plant growth regulator on rooting of multiple shoot
92
9 Effect of pre-culture period on co-cultivation 97
10. Effect of duration on co-cultivation 97
ABBREVIATION
BAP : Benzylamino purine CaMV : Cauliflower Mosaic Virus cDNA : Complementary deoxyribonucleic acid cm : centimetre cv. : Cultivar DMSO : Dimethylsulphoxide DREB : Drought responsive binding element EDTA : Ethylene diamine tetra acetic acid Fig. : Figure g : Gram GFP : Green Fluorescent Protein GUS : â-glucuronidase ha : Hectare HCl : Hydrochloric acid h : Hours hpt : Hygromycin phosphotransferase IAA : Indole-3-acetic acid IBA : Indole-3- butyric acid Kn : Kinetin l : Litre LB : Luria Bertani Luc : Luciferase M : Molar mg : Milligram min. : Minute ml : Millilitre MS : Murashige and Skoog medium M.W. : Molecular weight mg/l : Milligram per Litre N : Normal NAA : á-Naphthalene acetic acid NaOH : Sodium hydroxide Npt : Neomycin phosphotransferase nm : Nanometre OD : Optical density PCR : Polymerase chain reaction rpm : Revolution per minute TE : Tris-EDTA TDZ : Thidiazuron T : Treatment pH : Hydrogen ion concentration µM : Micro molar µg : Microgram 2,4-D : 2,4-Dichlorophenoxy acetic acid
1
INTRODUCTION
Black gram (Vigna mungo L. Hepper) (2n-22) is popularly known as
Urad in Hindi, Biri in Odia and Uddu in Kannada. It belongs to the Family
Fabaceae. It is the third most important pulse crop in India after chickpea and
pigeon pea and grown mostly as a fallow crop in rotation with cereals. Its
reference that has also been found in Vedic texts such as Kautilya�s �Arthasasthra�
and in �Charak Samhita� lends support to the presumption of its origin in India.
India is the largest producer and consumer of black gram in the world.
1.1 TAXONOMICAL CLASSIFICATION OF BLACK GRAM
Black gram belongs to the sub-genus Ceratotropis of the genus Vigna.
The genus �Vigna�, together with the closely related genus Phaseolus, forms a
complex taxonomic group, called Phaseolus-Vigna complex. Verdcourt (1970).
Two botanical varieties have been recognized in V.mungo. V.mungo var.
mungo is the cultivated form of black gram and V.mungo var.silvestris is the
wild ancestral form of black gram.They are diploid in nature with 2n=2x=22.
Blackgram have small genome sizes estimated to574 Million Base pairs (Mbp).
Table.1 Taxonomic classification of Black gram
Kingdom Plantae Subkingdom Tracheobionta Super division Spermatophyta Division Magnoliophyta Class Magnoliopsida Subclass Rosidae Order Fabales Family Fabaceae Genus Vigna Species Mungo
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2
1.2 BOTANICAL DESCRIPTION
Black gram is an erect or sub-erect herb, 0.5-1.3m tall with stem lightly
ridged, covered with brown hairs and much branches from the base. The leaves
are large, trifoliate and are also hairy, generally with a purplish tinge. Flower is
pale yellow. The seed colour exhibits a wide range of variations from yellow,
and green mottled with black. Pod colour is either black or brown or pale gray
when mature and 100 seeds weight is 3-7g.
1.3 GEOGRAPHICAL DISTRIBUTION
It is widely cultivated in Indian subcontinent and to lesser extent in
Thailand, Australia and other South Pacific countries. It is popular pulse crop
of India, Pakistan, Burma, Bangladesh, Ceylon and most of the African
countries. It is an important pulse crop grown throughout the country.
In India black gram is very popularly grown in Andhra Pradesh,
Orissa, Maharashtra Madhya Pradesh, Uttar Pradesh, West Bengal, Punjab,
Haryana, Karnataka, Tamil Nadu and Bihar.
1.4 CULTIVATION CONDITIONS
Black gram is a warm weather crop and grows in areas receiving an
annual rainfall ranging from 600 to 1000 mm. It is mainly cultivated in a
cereal-pulse cropping system primarily to conserve soil nutrients and utilize the
left over soil moisture, particularly, after rice cultivation. Hence, although it is
grown in all the seasons, majority of black gram cultivation falls in either Rabi
or late Rabi seasons, particularly in peninsular India. The optimum temperature
range for growth is 27-30ºC. A dry harvest period is desirable as this forces the
crop to mature and reduces the risk of weather damage, although black gram is
3
Plate-1 Botanical Description of black gram A. Black gram plant. B. Inflorescence of black gram C. Dissected flower D. Immature pod E. mature seeds of Black gram
4
more susceptible than green gram. Black gram grows on most soils, with a
preference to loamy soil condition with a pH of 5.5-7.5. It comes up well on water
retentive soils but cannot stand saline and alkaline conditions. Root growth can be
restricted on heavy clays, with a consequent limitation to growth. Black gram is
more tolerant to water logging as compared to green gram.
1.5 AGRICULTURAL IMPORTANCE
This crop is itself a mini-fertilizer factory, as it has a unique
characteristic of maintaining and restoring soil fertility through fixing
atmospheric nitrogen in symbiotic association with Rhizobium bacteria, present
in the root nodules. The crop is suitable for inter cropping with various crops
such as cotton, sorghum, pearl millet, green gram, maize, soybean, groundnut,
for increasing production and maintaining soil fertility.
1.6 FOOD AND NUTRITION VALUE OF BLACK GRAM
Among the grain legumes, black gram is the second most important food
grain in the world for his protein content (first is soybean). Grain legumes contain
2 to 3 times more protein than cereals, ranging approximately between 20 to 40
per cent. This protein is rich in essential amino acids such as arginine, leucine,
lysine, isoleucine, valine and phenylalanine etc. In addition to being an important
source of human food and animal feed. The presence of fats with low degree of
saturation has contributed significantly to the increasing popularity of blackgram.
In the South Asia, black gram is used to make �dal�, which is the most
common dish made from various kinds of split legumes with spices. In the
Southeast and East Asian countries, it is used to make various kinds of sweet,
bean jam, sweetened bean soup, vermicelli, and bean sprout. Due to various
health benefits of black gram, they are extensively used in the preparation of
5
dishes in China, Taiwan, Thailand, Pakistan, Korea, Southeast Asia and India.
They are eaten whole and as sprouts. The starch extracted from black gram is used
in making noodles. They are also used in making desserts and soups. Black gram
is a wonderful food that helps in reducing weight. Urad beans are rich in protein,
vitamin A, B, C, E and are a perfect source of minerals such as potassium, iron
and calcium. According to Chinese medicine, the sprouts are considered as a
cooling food with anti-cancerous properties. They are used to treat inflammations
that can arise as a result of infections, hypertension and heat strokes.
Black grams have high fiber content and complex carbohydrates that
are helpful in digestion. The complex carbohydrates balance the blood sugar
levels in the body and halt the rise of sugar levels just after meal consumption.
These properties of black gram are beneficial for people suffering from high
cholesterol or diabetes. The high nutritional value of Urad bean gave this pulse
a special importance in Ayurveda medicine.
Blackgram is one of the rich sources of vegetable protein and some
essential minerals and vitamins for the human body.
proteins and enzymes required for Osmoprotectants biosynthesis (Yamaguchi-
Shinozaki & Shinozaki 2006). An outline of these processes is given in Fig. 1
20
Fig. 1 Scheme illustrating the molecular response of plants to low-temperature stress, realised as changes in transcriptome, proteome, metabolome and phenotype.
2.5 THE MOLECULAR BASIS OF COLD TOLERANCE (CBF / DREB RESPONSIVE PATHWAY)
The CBF / DREB responsive pathway provides one of the most
important routes for the production of cold responsive proteins. The major cis-
acting element involved in CBF / DREB is DRE (dehydration-responsive
element) / CRT. Two major groups of transcription factors bind to DRE/ CRT
in low-temperature signalling, and DREB-2 during osmotic stress (Nakashima
& Yamaguchi-Shinozaki, 2006). CBF1, 2 and 3 are all responsive to low
temperature, and their encoding genes are present in tandem on Arabidopsis
21
thaliana chromosome 4. The genes carry the conserved AP2 / ERF domain
DNA-binding motif. CBF2 / DREB1C is a negative regulator of both CBF1 /
DREB1B and CBF3 / DREB1A. CBF3 is thought to regulate the expression
level of CBF2 (Chinnusamy, 2006). Thus, the functions of CBF1 and CBF3
differ from those of CBF2, and act additively to induce the set of CBF-
responsive genes required to complete the process of cold acclimation (Novillo,
et al., 2004). Upstream of CBF lie both ICE1 (inducer of CBF expression), a
positive regulator of CBF3, and HOS1 (high expression of osmotically
sensitive), a negative regulator of ICE1. The HOS1 product is a RING E3
ligase targeting ICE1 for degradation in the proteasome (Dong, et al., 2006).
Because of the rapid (within a few minutes) induction of CBF transcripts
following plant exposure to low temperature, ICE1 is unlikely to require de-
novo synthesis, but rather is already present in the absence of cold stress and is
only activated when the temperature decreases (Chinnusamy et al., 2003). The
LOS1 (low expression of osmotically responsive genes) product is a translation
elongation factor 2-like protein, which negatively regulates CBF expression.
The likely regulators of CBF1 and CBF2 are bHLH proteins other than ICE1.
To obtain transient expression of CBFs, the levels of CBF1 and CBF3
transcript, after their induction by ICE1, are subsequently lowered by CBF2.
Accordingly, the peak expression of CBF2 in response to low temperature has
been shown to occur about 1 h later than that of either CBF1 or CBF3 (Novillo,
et al., 2004). In addition to ICE1, a further positive regulator of CBF
expression is LOS4, an RNA helicase-like protein (Gong, et al., 2002) .
However, CAX1(cation exchanger), which plays a role in returning cytosolic
Ca2+ concentrations to basal levels following a transient increase in response to
low-temperature stress, is a negative regulator of CBF1, 2 and 3 (Catala, et al.,
2002). The ICE1-CBF pathway provides positive regulation of the expression
22
of certain Zn finger transcriptional repressors and the SFR6 (sensitivity to
freezing) protein, which is required for CBF function. SFR6 may be part of an
adaptor complex required for CBF action, or alternatively, may be involved in
the translation machinery of CBF transcripts and in CBF protein stability
(Chinnusamy, 2006). Further pathways, not under CBF control, are also
involved in the regulation of cold-responsive genes (Van Buskirk &
Thomashow, 2006). In particular, Chinnusamy,2006 have shown that both
HOS9 and HOS10 transcription factors play a role in the regulation of freezing
tolerance in a CBF-independent manner.
Fig. 2. The CBF (C-repeat-binding factor) pathway in plants. ICE1 (inducer of CBF expression) is activated by low temperature and is inhibited by HOS1 (high expression of osmotically sensitive). This triggers the expression of CBF3, which promotes the accumulation of COR (cold regulated)gene products. CBF3 expression also positively regulates the expression of CBF2, which, in turn, leads to the down-regulation of CBF3 and CBF1. LOS4 (low expression of osmotically responsive genes) is a positive regulator of CBF expression, and LOS1 a negative regulator
Knowledge gained from the study of the model plant A.thaliana has
proven to be largely, but not completely, transferable to crop plants.
Unfortunately, many key stress responses are not transferable, and Oh, et al.
(2007) experienced with over-expression of the barley low temperature induced
gene HvCBF4 in rice, which resulted in the up-regulation of a set of genes not
23
predicted from the heterologous expression of AtCBF3 in rice. For cereal
species, this disadvantage is to some extent balanced by the availability of the
rice genome sequence, and its increasing level of annotation, since the cereal
species genomes are all closely related to one another (Moore, et al., 1995). In
barley, the CBF genes HvCBF3, HvCBF4 and HvCBF8 are all components of
the frost resistance quantitative trait locus (QTL) located on chromosome 5H
(Francia, et al., 2004). A contrasting approach has targeted comparisons
between spring- and winter-sown cereal cultivars. Thus, for example, Monroy,
et al., (2007) observed that spring and winter wheats share the same initial
rapid expression of cold-inducible genes, but that their transcriptional profiles
diverge widely during cold acclimation. While in winter cultivars the
expression of cold acclimation genes continues over time, in spring cultivars,
their levels of expression decline and the cold acclimation process is
overridden by the transition from the vegetative to the reproductive stage.
Although the regulation of genes in the CBF-responsive pathway has been only
marginally explored to date in woody plants, there are some indications that
their regulation is more complex than in herbaceous species. For example four
Eucalyptus gunnii CBF1 genes displayed differential expression in response to
cold treatment (Kayal, et al., 2006; Navarro et al., 2009).
Senthil, et al., 2004 (2. 5mM/L NAA) used NAA at different levels to induce
rooting. Few workers reported that the rooting response is also dependent on
the genetic background of explant. Brandt and Hess (1994) observed
differential response for rooting in desi and kabuli cultivars. The highest rate of
rooting shoots (71. 5%) in case of A-1 and 68. 3 per cent in ICCV-2 with
simultaneous good shoot growth was obtained on WH medium supplemented
with 2.5ìM IBA.
Similarly IAA was used by Subhadra et al. (1998) and Batra et al.
(2002) at the concentration of 1 mg/L. The rooting response is also dependent
on the nature of media. Significant differences were noticed between liquid and
solid rooting medium supplemented with NAA, with average rooting of 16.5
per cent and 84.7 per cent in solid and liquid medium respectively Chaturvedi
and Chand, (2001) obtained 50 per cent rooting with 0.8 mg/l NAA in solid
medium and 70 per cent with 1.2 mg/l NAA and 0.04 mg/l IBA. The induced
roots were thicker and longer in liquid medium than in solid medium.
Proliferation of roots was observed only when transferred to liquid plain ½ MS
after the root induction (Kar, et al., 1996). Similarly, Polisetty, et al., (1997)
subcultured plant lets on ¼ th strength MS medium without agar to get good
root growth. Quality of roots can be improved by increasing the agar-agar
concentration in the medium and also helps the normal growth of the plantlets
by reducing the degree of vitrification.
Shoots were rooted in half strength MS under partial dark conditions
(Batra, et al., 2002). Some workers used ¼th strength of MS medium (Singh, et
al., 2002), ½ strength MS medium (Sharma and Amla, 1998) and ½ strength
48
B5 medium (Ramana and Sadanandam, 1997) for root induction. Whereas,
Malik and Saxena, (1992) did not find any difference between media that
contained half or full strength of MS salts for rooting. Singh B. D. (2007) also
highlighted that exogenous carbon source was not needed.
Rooting and transplantation of chickpea is now no more a hurdle for
chickpea transformation. Healthy and strong rooting was achieved by exposing
cut ends of the in vitro raised shoots (3-5 cm) to 5-10 sec pulse treatment with
100 ì moles/ml IBA followed by their transfer to liquid MS basal medium.
Potting-mixture with good aeration and les ser capacity to retain water was
most suitable for successful establishment of plantlets. Garden soil mixed with
sand and bio-manure in equal proportion was most suitable for achieving cent
percent transplantation success. Cent percent of plantlets acclimatized in pots
and showed normal growth, development, flowering, pods and seeds setting. In
this communication, we have shown that shoot length, pulse treatment of cut
ends of shoots with 100 moles /ml IBA (1st report) and aeration of potting
mixture are key factors for rapid micro-propagation and successful
establishment of in vitro raised chickpea plantlets.
Muhammad, et al., 2008 five-day-old in vitro grown seedlings of
cowpea was obtained in MS supplemented with 0.50 mg/l BAP - 0, 0.10, 0.30
and 0.50 mg/l NAA. Regenerated shoots were rooted on MS containing 0.50
mg/l IBA where up to seven adventitous secondary shoots arose from the base
of mother shoot were also recorded. These shoots could also be rooted easily
on the same rooting medium. Rooted plants were adapted at room temperature
in soil mix in pots. All plants flowered and set seeds in the growth room after
three months.
49
In black gram shoot buds elongated on one-third-strength MS (MS1/3)
semisolid medium and plantlets were obtained by transferring the shoots onto
ms1/3 semisolid medium supplemented with indolebutyric acid (1 mg/L, 4.
9mM) for rooting Das( 2002). The rooting development in black gram
observed in half MS medium with NAA, IAA, IBA (-mg/L). (Muruganantham,
2004), The high frequency (100. 0%) of rooting was observed with MS
medium supplemented with 0. 5 mg l-1 IBA. Mony, et al., (2010)
2.8.5 Hardening and Establishment of regenerated plants
Ultimately regenerated plants were establishment in soil. It is
recommended that removal of sugar from the support medium, pre-
conditioning to low relative humidity, high light intensity and high temperature
can ensure higher survival during transfer of plantlets to natural conditions. The
gradual removal of sugar is known to stimulate photosynthetic ability. Neelam,
et al., (1986) used autoclaved soil: sand: compost (1:1:1) mixture, as potting
mixture. Obtained only 30-40 per cent survivability. The mortality was due to
soft, weak stems and roots without good differentiation of vessels and due to
inability to pick the nutrients from soil when transferred from agar medium.
Among the different potting mixtures used, pure vermiculite was the best and
supported the survival of 85.4 per cent plants. Only 20.4 per cent plants
survived when transferred to the vermiculite and perlite (1:1) mixture whereas
the plants transferred to the sand, soil and sawdust mixture (1:1:1) did not
survive (Barna and Wakhlu, 1994).
Surya- Parkash et al., 1992 made attempts to transfer rooted plantlets
to sterilized pots containing soil, sand and vermiculite in 1:1:1 ratio. However,
no success was achieved. Soil rite was used as inert supporting powder. Kumar,
et al., (1994) obtained matured plantlets by adding soil rite to liquid ¼ strength
50
MS medium without sucrose. Polisetty, et al., (1997) irrigated potted plants
with ¼ strength Hoagland�s nutrient medium. Rooted plantlets of black gram
(ca. 5 cm) were transferred to autoclaved vermiculite in 6-cm plastic pots and
covered with a plastic cover to maintain humidity. Two weeks after transfer to
pots, the covers were gradually removed over a period of 7 days, before the
plantlets were finally transferred to soil Das, et al. (2002). Jayanand, et al.,
(2003) derived a comprehensive method for the hardening and transplantation
to glasshouse.
Among the various potting media tried such as black soil, red soil,
smooth sand, coarse sand and vermiculite individually and in combination with
each other, coarse sand showed the best results with about 80 per cent survival.
As the regenerate plantlets have to be adopted not only to soil but to higher
light intensities and lower atmospheric humidity as well, they standardized the
requirement of temperature, humidity, light intensity and photoperiod during
different stages of plant growth (Singh, 2007).
2.9 TRANSFORMATION STUDIES
2.9.1 Efficient techniques for transformation
The development of efficient transformation method is frequently not
straight forward and can take many years of painstaking research with a range of
different methods (Potrykus, 1991). Although several approaches have been tried
successfully for integrative transformation (Potrykus, 1991)only three are widely
used to introduce genes into a wide range of crop plants (Dale et al.,, 1993). These
include (i) Agrobacterium mediated gene transfer, (ii) microprojectile
bombardment with DNA or biolistics and (iii) direct DNA transfer into
isolated protoplasts. Of these techniques the first two approaches have been
more successfully used in many of crops and chickpea in specific.
51
2.9.2 Indirect method of Gene transformation (Agrobacterium mediated)
A. tumefaciens plasmid transfers only T-DNA. In principle, desired
DNA sequence can be intentionally introduced into plant genomes (Zambryski,
et al., 1983; Trieu, et al., 2000). The oncogenic T-DNA region is deleted from
Agrobacterium strain in order to prevent over production of phytohormones,
which interfere regeneration from tissue and with normal plant development.
The strain with disarmed T-DNA has only the �vir� region and is a suitable
system for plant transformation. The T-DNA transfer is mediated by products
encoded by the �vir� (virulence) region of the Ti-plasmid, which is composed
of at least six inducible operons that are activated by signal molecules, mainly
small phenolics, certain class of monosaccharides and acidic pH acts
synergistically with phenolic compounds (Riva, et al., 1998).
Induction of �vir� operons by inclusion of phenolics like
acetosyringone in the co-cultivation medium could therefore enhance T-DNA
transfer to plant cells. Addition of 50ìM acetosyringone to the bacterial re-
suspension medium as well as co-cultivation medium resulted in non-
significant increase in transformation frequency from 88 % in cultures without
acetosyringoneto 95 % with large GUS positive sector(s). Acetosyringone
enhances vir functions during transformation (Stach, et al., 1996) and has been
shown to increase transformation potential of Agrobacterium strain with
moderately virulent vir region in several plant species (Atkinson and Gardner
1991, Janssen and Gardner, 1993:Kaneyoshi, et al., 1994).
Injuries implicated with the help of hypodermic needle, enhanced the
frequency of transient GUS expression, at the regeneration and cotyledons
detachment sites of the cotyledonary nodes up to 98 %. Wounding the plant
material before co-cultivation allows better bacterial penetration into the tissue
52
facilitating the accessibility of plant cells for Agrobacterium or possibly stimulated
the production of potent �vir� gene inducers like phenolic substances such as
acetosyringone and hydroxyl acetosyringone (Stach et al.1996) and enhanced the
plant cell competence for transformation (Binns and Thomashow, 1988).
Wounding the plant material before co-cultivation has also been shown
to increase transformation frequency. Mechanical injury of the meristematic
region probably induces meristem reorganizations promoting formation of
large transgenic sectors and enhanced recovery of transformants. Pre-culture of
explants on regeneration medium prior to inoculation and co-cultivation with
Agrobacterium has been reported to enhance efficiency of transformation in
some grain legumes, e. g. Vigna unguiculata (Muthukumar, et al., 1996) and
Cajanus cajan (Geetha, et al., 1999).
These signal molecules are recognized especially by Agrobacterium to
induce �vir� gene expression and thereby activate T-DNA transfer (Zambryski,
1992). When preculture was combined with mechanical injury the results were
reversed that leads to increase in transient GUS expression up to 100 % and
specifically at the regeneration site of the cotyledonary node explants. This
may be attributed to visually more clear regeneration site on the pre-cultured
explants for mechanical injury as compared to non-pre-cultured and freshly
release of phenolics as a result of mechanical injury. High vigor of pre-cultured
explants was also found to increases the regenerability of mechanically injured
explants. It is concluded that inoculation of pre-cultured and mechanically
injured cotyledonary node explants of V. mungo for 30 min with A. tumefaciens
at a density of 108 cells cm-3 followed by co-culture on SR medium for 3 d has
been found more beneficial and resulted in the production of significant
number of transgenic plants to efficiency of 4.31 %. (Saini, et al., 2007).
53
In Agrobacteria mediated transformation many co-cultivation, culture
and micro environmental condition affected the transformation efficiency. The
length of co-cultivation period required for achieving maximum gene transfer
was found to be 2 - 3 d with no significant difference between them for
cotyledonary node explants of Vigna mungo. Further extension in co-culture
time decreased the transformation frequency resulting in bacterial overgrowth
and had detrimental effect on regeneration potential of explants. A short co-
culture period of 2 or 3 d has also been found to be optimum in other plant
species such as Antirrhinum majus (Holford, et al., 1992), Vigna unguiculata
(Muthukumar, et al., 1996), Vigna radiate (Jaiwal, et al., 2001), Cajanus cajan
(Mohan and Krishnamurthy, 2003), Glycine max (Li ,et al., 2004) and
Nicotiana tabacum (Uranbey, et al., 2005).
The modification in the co-cultivation media for avoiding necrosis
which occurring during incubation for 72 h, due to the Agrobacterium over
infection to plant and hypersensitivity reaction given by the plant against to
Agrobacterium for this modifying LPGM medium with acetosyringone, sodium
thiosulphate, L-cysteine and di-thiothreitol and incubated at 25°C±20C in the
dark given in chickpea genetic transformation with Agrobacterium mediated
protocol. (S. Ignacimuthu and Prakas, 2006)
The improvement in black gram for salt stress tolerance through
Agrobacterium mediated genetic transformation was done by the over-
expression of the Glyoxalase 1 (Gly 1) gene. (Muruganantham, et al., 2007)
reported the herbicide tolerant (Vigna mungo L. Hepper) plants were produced
using cotyledonary-node and shoot-tip explants from seedlings. In vitro
selection was performed with phosphinothricin as the selection agent. Explants
were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring
54
the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the
presence of acetosyringone. Shoot regeneration occurred for 6 wk on
regeneration medium (MS medium with 4.44ìM benzyl adenine, 0.91ìM
thidiazuron, and 81.43ìM adenine sulfate). Transgenic black gram plants were
produced at a 1% transformation. Frequency using kanamycin (75 mg/l) from
cotyledonary-node explants (Saini and Jaiwal, 2003), where chimeras (79%)
were observed as a result of a high level of antibiotic tolerance in the explants.
More than 50% of the putatively transformed mungbean shoots growing in
kanamycin-containing media were escapees after co-cultivation of
cotyledonary-node explants (Jaiwal, et al., 2001). PPT (10 mg/l) proved to be
more efficient than kanamycin for rapid selection, production, and
identification of putative pea transformants.
The potential of genetic engineering to incorporate insect resistance
cholesterol oxidase gene (choM) into mung bean plants through Plasmid
pCAMBIA 1301-choA was transformed into Agrobacterium rhizogenes strain
K599 and A. tumefaciens strain EHA 105 for mungbean transformation. The
two-day-old cotyledons that were co-cultured with hairy root bacteria showed
higher ability to produce branched roots than the others. An average of 10
branched roots was formed on both the wounded axial site and the hypocotyl
cut end. Ten of 75 individual lines (13.25%) showed GUS positive. In addition,
cotyledons that were cut and cultured on MB medium supplemented with
2mg/L BAP for 4 days before co-culture with A. tumefaciens using hairy root
method revealed high transformation ability (31.25%) discovered by(Potjamarn
suraninpong, et al., 2004).
Sita Mahalakshmi, et al.(2006) was reported Transgenic mungbean
plants were developed via primary leaf explants with disarmed Agrobacterium
55
tumefaciens strain C-58 harbouring a binary plasmid, pCAMBIA� 1301
(containing genes for b-glucuronidase (GUS) and hygromycin
phosphotransferase (hpt)]. By using of primary leaf explants (cut at the node)
from four-day old and ten-day-old seedlings.
Jaiwal.,2007 first time reported the normal and fertile transgenic plants
of mungbean with two transgenes, bar and á-amylase inhibitor, Cotyledonary
node explants were transformed by co-cultivation with Agrobacterium
tumefaciens strain EHA105 harboring a binary vector pKSB that carried
bialaphos resistance (bar) gene and Phaseolus vulgaris á-amylase inhibitor-1
(áAI-1) gene. Green transformed shoots were regenerated and rooted on
medium containing phosphinothricin (PPT).
Islam and Islam (2010) used two different types of explants, namely
cotyledonary leaf and cotyledon attached with embryonic axis (CAEA) were
used in different experiments. CAEA and cotyledonary leaf explants were
infected with A.tumefaciens strain LBA4404 harboring the binary plasmid
pBI121 containing gusA and Neomycin phosphotransferase (nptII) marker
genes to determine theirtransformation ability. Between two explants, CAEA
showed better response towards transformation than the cotyledonary leaf.
Maximum transformation of CAEA explants was obtained following 45 min of
infection with Agrobacterium suspension having an OD of 1.3 at 600 nm and
72 hours of co-cultivation as judged by transient GUS assays.
Sushil Kumar Yadav et al. (2012) reported highly efficient protocol for
genetic transformation mediated by Agrobacterium has been established for
green gram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN)
explants were inoculated with Agrobacterium tumefaciens strain LBA 4404
56
harbouring a binary vector pCAMBIA 2301 containing neomycin
phosphotransferase (npt II) gene as selectable marker, ß-glucuronidase (GUS)
as a reporter (uidA) gene and annexin 1bj gene. Important parameters like
optical density of Agrobacterium culture, culture quantity, infection medium,
Infection and co-cultivation time and acetosyringone concentration were
standardized to optimize the transformation frequency. Kanamycin at a
concentration of 100 mg/l was used to select transformed cells. Transient and
stable GUS expressions were studied in transformed explants and regenerated
putative plants, respectively. Transformed shoot were produced on regeneration
medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted
on ½ MS medium. Transient and constitutive GUS expression was observed in
DCN explants and different tissues of T0and T1 plants.
Yellisetty Varalaxmi et al.(2013) used Agrobacterium tumefaciens
strain LBA4404 harbouring binary vector pCAMBIA 2301, which contains a
neomycin phosphotransferase gene (nptII) and a -glucuronidase (GUS) gene
(uid A) for transformation of Vigna mungo cotyledon derived calli. Wounding
of explants before infection, osmotic effects of infection and co-cultivation
media had an effect on the competence of the tissue as well as transforming
ability of Agrobacterium cells. Transient GUS expression studies revealed
that a cell.
Density of 108cells/ml, 100 µM acetosyringone and 330 µM cysteine
were effective in increasing the transformation frequency and obtaining stable
transformants with a3.8% transformation efficiency. IBA pulse treatment was
effective in root induction of kanamycin selected putative transformants.
Molecular analysis using polymerase chain reaction (PCR) of nptII gene
confirmed the transgenic nature of T0 transformants.
57
2.9.3 Direct DNA transfer
Physical as well as chemical methods have been developed to facilitate
DNA delivery across the plasma membrane, which lead to both stable and
transient gene expression. The first report on direct delivery of DNA molecules
into plant protoplasts was documented by Davey, et al. (2009). However, in
Blackgram there are no reports on direct DNA transformation.
Microinjection, sonication, electroporation and biolistic mediated transfer are the
main procedures to let desired DNA molecules enter any living cell; plant, animal,
or microbial. Microinjection involves immobilization of protoplasts and micro
injecting DNA directly into the nucleus. Miki, et al. (1987) reported
transformation efficiencies of 12-66 per cent in tobacco. In the sonication process,
ultra sound waves are used to facilitate uptake of nucleic acids into plant cells and
protoplasts. Joersbo and Brunstedt (1992) used mild sonication (20 KHz) to
facilitate uptake of chloramphenicol transferase (cat gene) in tobacco and achieved
a maximum of 81per cent transient expression with no significant loss of viability.
The electroporation method was originally developed to introduce
DNA in prokaryotic cell (Fromm et al., 1985). This has been successfully
used for transformation of wide range of crop species including pigeonpea
(Christou et al., 1987)
2.9.4 Microprojectile bombardment with DNA or biolistic
Acceleration of heavy microprojectile (0.5-5.0 µ m diameter
tungsten or gold particles) coated with DNA has been developed into a
technique that carries genes into virtually every type of cell and tissue
(Klein, et al., 1988; Sanford, et al.,1990; Taylor and Vasil, 1991). This
method allows the transport of genes into many cells at nearly any desired
58
position in a plant. The technology basically involves loading tiny
tungsten or gold particles with vector DNA and then spreading the
particles on the surface of a mobile plate.
Then, under a partial vacuum, the microprojectile is fired
against a retaining plate or mesh by a shock wave caused by helium
under pressure achieving speeds of one to several hundred meters per
second. The particles are capable of penetrating several layers of cells and
allow the transformation of cells within tissue explants (Register, et al.,
1994). This technique, although not as efficient as the Agrobacterium-
mediated gene transfer, has a distinct advantage in that virtually any type
of meristematic totipotent cells, tissues, organs and monocots that are not readily
amenable to agro-infection can be used with a reasonable success rate. Another
major advantage lies in its application in transient gene expression studies in
differentiated tissues (Fitch et al., 1990).
2.9.5 Selectable markers
The genetic transformation of plants requires �marker� genes
that allow the recognition of the transformed cells in the background of
untransformed ones. These genes are dominant, usually of microbial origin
and placed under the control of strong, constitutive, eukaryotic promoters,
often of viral origin (Birch, 1997). The most popular selectable marker
genes used in plant transformation vectors include constructs providing
resistance to antibiotics such as kanamycin and hygromycin and genes that
allow growth in the presence of herbicides such as phosphinothricin,
glyphosate, bialaphos and several other chemicals. For successful
selection, the target plant cells must be susceptible to relatively low
concentrations of the antibiotic or herbicide. The utility of any particular
59
gene construct as a transformation marker varies depending on the plant
species and explant involved (Li, et al., 2002).
Kanamycin has proven to be the most widely applicable selective agent but
the concentration is species specific. Species like Vigna mungo L., Cicer arietinum
(Fontana, et al.,1993; Sanyal, et al., 2003; Sarmah, et al.,2004), Kanamycin has
been used for selection in most of the chickpea transformation studies
reported (Fontana et al.,1993; Kar et al.,1996, ; Krishamurthy, et al.,2000) are
selected at low concentration of kanamycin (15-100 mg/l). However, recently
Tewari-Singh, et al., (2004) used a desensitized aspartate kinase (AK) gene as a
non-antibiotic selection marker for production of transgenic chickpea, which was
found to be a better selection marker than kanamycin as transgenic plants could be
identified more easily and rapidly using the marker.
2.14.6 Molecular and genetic characterization of transgenic plants
The integration of the target gene is confirmed by technique
�polymerase chain reaction� (PCR) routinely, which screens putative transgenic
and classify either as positive or negative (Edwards et al.,1991). Another
technique could be used to detect the presence of a given sequence of DNA or
RNA in the non-fractionated (not subjected to electrophoresis) DNA is �Dot
blot�, where sample DNA�s from several individuals can be tested in a single
test run. Dot blots are useful in detecting presence of the sequence being
transferred in a number of suspected transgenic individuals and a presence of
specific mRNA in several such individuals or in different tissues of a single
individual. Southern blotting or southern hybridization (Southern, 1975) is used
to demonstrate the presence of the gene in question in transgenics, where
detection of DNA fragments which are complementary to given DNA is critical
(Sambrook and Russell, 2001). This is the common method to confirm the
60
stable integration of DNA in the genome and also to know the number of
copies integrated. Few examples of use of the Southern blotting to know the
presence of transgene are available in chickpea (Krishnamurthy, et al.,, 2000;
Kar, et al.,, 1996; Tewari-Singh, et al.,, 2004 and Sarmah, et al.,, 2004),
pigeonpea (Geetha, et al.,, 1999; Lawrence and Koundal, 2001). The number of
copies of a transgene construct inserted is variable for all transformation
methods. Data from several different transgenic dicotyledonous species showed
an average of three T-DNA inserts, with occasionally upto 20-50 copies in
some plants. Kar, et al., (1996) noted multiple gene inserts in all transgenic
chickpea plants while, Krishnamurth, et al. (2000) and Tewari-Singh, et al.
(2004) found 50 per cent single insert and 50 per cent multiple (4-6) gene
inserts. However, single copy insertion of the target gene based on strong
signal generated by hybridization of GUS and npt II specific homologous
probes was reported by (Sarmah, et al., 2004; Sanyal, et al., 2003).
The expression of transgenes can vary considerably between different
independently transformed plants (Hobbs, et al.,1990; Jefferson, et al., 1990;
Blundy, et al., 1991). In some instances there is a positive association between
transgene expression and copy number, but other studies have shown no
association or even a negative one (Hobbs, et al.,, 1990).
Transgene expression many a times be unstable or may decline over
generations. Several techniques have been used for detection of expression of inserted
gene such as northern hybridization, immunoblotting and western blotting
(Sambrook, et al.,1989; Saghai-Mahroof, et al.1984). Unlike Southern hybridization,
northern hybridization detects transcription of DNA sequence that is used as a probe.
This technique has been used to know the expression of cowpea protease inhibitor at
mRNA level in transgenic pigeon pea (Lawrence and Koundal, 2001).
61
Bhattacharya et al., (2002), used Southern confirmed plants for northern
blotting of Bt (cry IA(b)) transformants in cabbage and reported differences in the
level of transgene expression. Western blotting is used to detect proteins of a
particular specificity. When a transferred gene expresses in transformed cells, the
translated product in from of protein can be identified by this technique. Sarmah, et
al., (2004) analyzed PCR positive transgenic lines through western blotting and
compared with bean seed protein. The chickpea a-AII polypeptides detected by
western blotting had similar size to those found in bean seeds. This indicated that
the primary translation product, which is proteolytically processed in bean seeds,
was similarly processed in chickpea seeds. They also used this assay to estimate the
level of a-A II in the T1 seeds of transgenic lines.
The immunoblotting technique is based on antigen-antibody reaction. In
this method total protein from plants is fractioned on SDS-PAGE (10% polyacryl
amide) and transferred to PVDF and detected by antiserum of specific antigen.
Bhattacharya et al., (2002) detected Bt cry1Ab proteins using the rabbit anti cry
IA(b) serum and a goat antirabbit IgG coupled to alkaline phosphtase as secondary
antibody. They could detect 81.3 kDa cry protein through this method which was
further tested to know viability of second instar larvae of Plutella xylostella. They
observed high reduction in growth rate and mortality. Sarmah, et al., (2004) used
this technique to confirm the expression of the bean a- amylase inhibitor gene
against Callesobruchus maculatus in putative transgenic chickpea.
Sanyal et al., (2005) reported the expression of Cry1Ac in chickpea by
elisa method and he observed that there was variation in protein content in all
transgenic plants and it is because of site of its integration and number of
copies integrated into plants. There could be several reasons for non-expression
62
or low expression of the transgene in transgenic plants (Finnegan and
Mc Elroy, 1994; Matzke and Matzke, 1995).
These include pleiotropic effects from transgenes, somaclonal
variations in the regenerated transgenic plants or environmental effects on the
promoters driving the transgenes (Senthil et al., 2004)reported partial
suppression of Uid gene due to presence of two inserts in a tandem array which
tend to become inactivated as result of gene silencing. Although the CaMV35S
promoter is considered constitutive, its level of expression is found to change
with respect of cell cycle or the various tissues (Nagata et al., 1987).
Molecular characterization on the expression of Cry1Ac gene in chickpea
plants was carried out by Indurkar, et al. (2007) and reported the level of protein
expression in transgenic plants showing variation found 6-20 ng/ mg. The practical
way of avoiding problems associated with variation in transgene expression and
stability and somaclonal variation is to produce a large number of independently
transformed plants (often>100) and to select those with a desirable phenotype
(Birch, 1997). Except for vegetatively propagated crop plants, it is usually desirable
to identify genotypes 1with single inserts of the transgene construct which will have
simpler inheritance patterns and are likely to have more predictable transgene
expression levels in subsequent segregating populations. Following initial analysis,
the transgenic plants need to be moved into a containment glasshouse for further
phenotypic and genotypic analysis using the original non-transgenic genotype as a
control. Further evaluation of transgenic plants is done under agronomic conditions
by carrying out field assessment studies.
63
MATERIALS AND METHODS
The present work was carried out on �Studies on genetic
transformation of black gram (Vigna mungo L.) with cold induced
transcriptome gene (ICE- 1) for abiotic stress tolerance� during 2011-2013 at
the Department of Agricultural Biotechnology, College of Agriculture, Orissa
University of Agriculture and Technology, Bhubaneswar-751003 (Odisha).
The details of materials used and the experimental techniques adopted during
the entire course of investigation are presented in this chapter.
3.1 MATERIALS
3.1.1 Genotypes
The cultivar used in the present investigation is Type-9�(T-9) which is
released from Agriculture University, Kanpur. It is popular, high yielding
(8q/ha) and suitable for different agro- climatic situations of entire Orissa as
well as throughout India. The seeds of these varieties were collected from State
Seed Corporation, Odisha.
3.1.2 Explant source
Different types of explants from four days old sterile seedlings of cultivars
�T-9� were used for in vitro regeneration and transformation studies. The explants
used were Cotyledonary node and Cotyledonary node with Shoot tip.
3.1.3 Plant nutrient medium
For in vitro culture experiments Murashige and Skoog (1962) basal
salts were used (Appendix-I).
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64
3.1.4 Plant growth regulators
The following different plant growth regulators at different
concentrations in different experiments (Appendix-III) were used.
Auxins
Indole Butyric Acid (IBA)
Naphthalene Acetic Acid (NAA)
Cytokinins
Benzylamino purine (BAP)
Thidiazuron (TDZ)
Kinetin (Kn)
Gibberellin
Gibberllic acid (GA3)
3.1.5 Agrobacterium strain and plasmid vector
The disarmed Agrobacterium strain EHA105 harboring binary vector
pCAMBIA2301 was used for in vitro transformation. pCAMBIA2301 contains
ICE1 gene, nptII marker and GUS reporter gene linked to CaMV35S promoter
and nos terminator.
3.2 METHODOLOGY
3.2.1 Sterilization of Glassware and Media
Sterilization refers to any process that effectively kills or eliminates
transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) from a
working surface, equipment, article of food or medication, or biological culture
medium. Sterilization can be achieved through application of heat, chemicals,
irradiation, high pressure or filtration.
65
Plate . 2 Explant source of regeneration and transformation A. Seeds of black gram cultivar �T-9� B. sterile explants culture of 4 days C. Shoot tip with Cotyledonary node D. shoot tip
66
Fig -3 Schematic map of vector pCAMBIA2301
Fig- 4 Linear map of T-DNA region of vector pCAMBIA2301
67
3.2.2 Steam sterilization
A widely-used method for heat sterilization is the autoclave.
Autoclaves commonly use steam heated to 121°C or 134°C. To achieve
sterility, a holding time of at least 15 minutes at 121°C or 3 minutes at 134 °C
is required. Additional sterilizing time is usually required for liquids and
instruments packed in layers of cloth, as they may take longer to reach the
required temperature. After sterilization, autoclaved liquids must be cooled
slowly to avoid boiling over when the pressure is released.
3.2.3 Sterilization by filters
Hormones that would be damaged by heat, irradiation or chemical
sterilization can be sterilized by mechanical filtration. This method is
commonly used for sensitive pharmaceuticals and protein solutions in
biological research.
A filter with pore size 0.2 µm will effectively remove bacteria. If
viruses must also be removed, a much smaller pore size around 20 nm is
needed. Solutions filter slowly through membranes with smaller pore
diameters. Prions are not removed by filtration. The filtration equipment and
the filters themselves may be purchased as presterilized disposable units in
sealed packaging, or must be sterilized by the user, generally by autoclaving at
a temperature that does not damage the fragile filter membranes.
3.2.4 Glasswares and chemicals:
Glasswares like culture tubes, conical flasks, petriplates, beakers etc.,
are of Borosil make. All the chemicals and plant growth regulators are of
analytical grade and are procured from standard chemical manufacturing
companies.
68
3.2.5 Cleaning of glasswares
Glasswares were rinsed in water and then soaked in 0.15% chromic
acid overnight. The chromic acid were drained out and the glasswares were
washed with clean soap solution. The thoroughly washed glasswares are rinsed
in distilled water and dried in a hot air oven. The instruments like forceps,
scalpels etc., were also cleaned and dried.
3.2.6 Sterilization of glasswares:
Clean glasswares were rinsed in double distilled water and dried in
oven at 80C and sealed with aluminum foil, petriplates placed in autoclavable
covers, instruments like scalpel, forceps, and blade holders wrapped in
aluminum foil were autoclaved at 121 C and at 15 lbs. pressure for 15
minutes. The glasswares were then transferred to sterile inoculation chambers.
3.2.7 Sterilization of laminar- flow chamber:
All steps in this experiment like the sterilization, preparation,
inoculation of the explants, sub culturing were conducted under aseptic
condition in the laminar- flow cabinet. Before the laminar flow cabinet was
used, the working surface of the chamber is sterilized by swabbing with 70%
alcohol. The chamber was then exposed to UV light for 15 minutes. The walls
of the chamber were also swabbed with 70% alcohol to ensure total sterility.
Before taking the materials into cabinet, they were swabbed with 70% alcohol.
In case of glasswares, the mouth of the bottles, flasks etc. are flamed before
and after use. Also, before starting the experiment, the hands are swabbed well
with alcohol.
69
3.3 PREPARATION OF EXPLANTS
Seed were surface sterilized with 70% ethanol (v/v) for 1 minute
followed by washing twice with sterilized distilled water for 2-3 times. Seeds were
treated with 0.2% HgCl2 (w/v) solution for 3 minutes followed by thorough
washing for 4 to 5 times with sterile distilled water to remove all the traces of
HgCl2 and blot dried on sterilized filter paper. Sterilized seeds were germinated
under aseptic conditions on MS medium with 2 mg/l BAP for 4 days.
The following explants were taken from 4 day old seedlings under
sterile conditions using scalpel and blade.
Cotyledonary node
Cotyledonary node with Shoot tip
3.4. PREPARATION OF MEDIA
MS basal salts supplemented with different concentrations of different
growth regulators were used depending on the purpose of the individual
experiment. Separate stock solutions of macronutrients, micronutrients, iron
source and organic supplements (except Myoinositol) were prepared (Appendix
I). The medium was prepared by adding appropriate quantities of the stock
solutions and correct volume was made up with distilled water. Sucrose 30 g/l
and Myoinositol 100 mg/l were added freshly to the medium. The pH of the
medium was adjusted to 5.6 to 5.8 using 0.1N HCL or 0.1N NaOH. Then agar
7.5 g/l was added and dissolved by warming the medium. Then appropriate
quantities of medium were poured in test tube (150x25 mm diameter) or bottles
(300 ml capacity) before it solidify. The test tubes were then plugged with non-
absorbent cotton plugs or glass bottles with suitable caps. The media was
autoclaved at 1210C for 15 minutes and kept for solidification. Medium, where
70
thermolabile compounds had to be added, filter sterilized solutions were added
to warm (40-500c) autoclaved medium and then dispensed in pre-autoclaved
containers.
Table-5 Amount of stock solutions added to the media
Sl. no. Stock solution Strength Amount to be
added (ml)
1 Macronutrients 20X 50
2 Micronutrients 1000X 1
3 Iron source 200X 5
4 Organic supplements 1000X 1
Agar (8g/l) and Myoinositol(100mg/l) were added separately
3.5 PHYSICAL FACTORS FOR IN VITRO CULTURE
All the in vitro culture work was carried out aseptically in a laminar
airflow chamber and the plant tissue culture experiments were conducted under
defined conditions of the culture room maintained at 25±20C, uniform light
(1000 lux) was provided by fluorescent tubes over a photoperiod of 16/8 hours.
3.6 PREPARATION OF ANTIBIOTIC STOCKS
Kanamycin, cefotaxime and carbenicillin stocks of 100 mg/l were
prepared in sterile double distilled water and rifampicin stock of 100 mg/l was
prepared in DMSO. All the stocks were filter sterilized and stored at 40C for
future work (Appendix- IV).
71
3.7 STANDARDIZATION OF REGENERATION PROTOCOL
3.7.1 Callus induction
The different explants were cultured on MS medium supplemented
with different growth regulators and coconut water and incubated in culture
room for callus induction. Observations were recorded on days to callus
initiation, number of explants responding, type of callus, and color of callus,
visual callus quality and percentage of callus induction. MS medium with
different combinations of growth regulators were used for callus induction.
Response of explants to callus initiation was assessed by calculating
number of explants responded for callus initiation and expressed in percentage.
Percent callus induction ∶= No. of explants with callus initiation Total no. of explants cultured × 100
3.7.2 Multiple shoots induction
Different explants like cotyledonary node and cotyledonary node with
shoot tip were cultured on MS medium supplemented with different levels of
BAP, TDZ, IBA, and kinetin for induction of multiple shoots. Observations
were recorded on number of days for shoot induction. Shoot percentage and
number of shoots. Shoots were subcultured on different medium for elongation.
Shoot induction at the end of culture period was assessed by
calculating number of explants responded for multiple shoot induction and
expressed in percentage.
Percent shoot induction = No. of explants with multiple shoots Total no. of explants cultured X 100
72
3.7.3 Rooting and hardening
Elongated shoots were transferred to MS medium supplemented with
different concentrations of IBA and NAA for rooting. Number of shoots
producing roots and types of roots produced and rooting percentage were
recorded. Rooted shoots were transferred to pots containing soil: sand: FYM
(1:1:1) mixture for hardening. Response of regenerated shoots to rooting was
assessed by calculating number of shoots responded for rooting and expressed
in percentage.
Percent rooting induction = No. of shoots with rooting Total no. of shoots cultured X 100
3.8 TRANSFORMATION
In this study Agrobacterium mediated genetic transformation method
was tried for gene transfer in Blackgram.
3.8.1 Maintenance and growing of Agrobacterium
The Agrobacterium strain EHA105 carrying plasmid vector
pCAMBIA2301 containing ICE1 gene construct was maintained on solid LB
(Appendix-VIII) medium containing 50 mg/l kanamycin and 10mg/l
rifampicin. Sub culturing was done every fortnight in fresh medium. Single
Agrobacterium colony was taken from the plate with the help of sterilized loop
and inoculated into 100ml LB broth containing 25 mg/l kanamycin and was
incubated on shaker for 16 hrs. At room temperature and fresh culture was used
for transformation. The culture having O.D. 0.6 to 0.8 at 620 nm wavelength
was used for bacterial infection.
73
3.8.2 Co-cultivation
Cotyledonary node with shoot tip explants were taken from different
days old seedlings and co-cultivated with Agrobacterium. The overnight grown
culture of Agrobacterium was centrifuged at 6000 rpm for 5 minutes, the
supernatant was discarded and the bacterial pellet was suspended in liquid plant
growth medium (LPGM). Explants were suspended in Agrobacterium
suspension containing 100 µM acetosyringone and kept for different interval of
time on shaker at 100 rpm. Excess bacteria were blot dried and explants were
placed in petriplates lined with blotting paper soaked in LPGM (Appendix II).
Petriplates was wrapped with aluminum foil and kept for different days
intervals in culture room for co-cultivation. Then the explants were washed
with sterile distilled water and cefotaxime 500 mg/l, blot dried and inoculated
on the MS medium containing 2.0 mg/l BAP +0.05mg/l IBA+ 500mg/l
cefotaxime + 80 mg/l kanamycin medium and transformation efficiency
recorded based on observation kanamycin positive selection and GUS positive
selection.
3.8.2 GUS histochemical assay
To conform the presence of transgene, histochemical staining of the
co-cultivated explants, after washing with antibiotics, the explants were
blot dried on sterile blotting paper and analyzed. The best substrate for
localization â-glucuronidase activity in tissue and cell is 5-Bromo 4-Chloro-3
indolylglucronide (x-Gluc) that gives a blue precipitate at the site of enzymatic
activity. The product of â-glucuronidase action is not colored. The derivative
produced must undergo an oxidative dimerization to from the insoluble and
highly coloured blue dye. GUS test was carried out to analyses transient
expression of GUS reporter.
74
Protocol for GUS Histochemical Staining
The explants were washed in phosphate buffer and incubated in
phosphate buffer with 1%Triton X-100 (Appendix VII) at 37˚C for 1
hr.
Then the explants were transferred to X-Glu staining solution (1mM)
and incubated at 37˚C in incubator for 16 to 24 hr.
After incubation explants were washed with alcohol and analyzed for
GUS expression.
The GUS expression cell are detected microscopically by distinct blue
coloration, which is a result of enzymatic cleavage of 5-Bromo 4-chloro
3-indolylglucronide.
3.8.3 Cefotaxime sensitivity test
To know the minimal level of cefotaxime which would completely
eliminate the excess bacteria after co-cultivation and to find out the suitable
concentration of cefotaxime/ carbenicillin to avoid bacterial contamination, this
test was conducted at 0, 100, 200,300, 400 and 500 mg/l cefotaxime..
3.8.4 Kanamycin sensitivity test
Kanamycin sensitivity test was carried out to find out the minimum
concentration of kanamycin required to inhibit the growth of untransformed explants
in order to design the selection medium. The test was carried out by culturing the
explants on regeneration medium along with different levels of kanamycin (0, 20, 40,
60, and 80 mg/l). Putative transformants and percent of putative transformants were
calculated on the basis of kanamycin selection as fallows.
*Each treatment replicated four times. Twenty observations per replication
Out of the twelve different treatments, the medium supplemented with 3
mg/l 2.4.D along with 1mg/l Kinetin resulted highest callus induction from
87.25% explants within 12 days of incubation (Plate-3D). Callus obtained from
leaf and cotyledonary node explant cultured on MS medium supplemented with
2.4.D (3 mg/l) and kinetin (1 mg/l) were found to be loose friable whitish green
callus colour. The time taken for the callus induction is comparatively reduced
in MS medium supplemented with same concentration of Phytohormones along
with organic supplement (Cocoanut water) and callus induction were found to
83
be 87.5% response with average of 11 days to induce callus. Where in plane
MS medium callus proliferation was not observed in leaf as well as shoot tip
explant (Plate-3A). As compared to the former, the callus obtained from inter
cotyledonary node was not better in quality than leaf. Callus induction from
cotyledonary node had taken more time as compared to leaf. But Callus
obtained from the above experiments when cultured on the MS medium
supplemented with various combination of growth regulator for regeneration of
callus failed to regenerate the complete plant.
4.1.3 Induction of Multiple shoot
Both the explants (cotyledonary node and shoot tip) harvested from
four days old in-vitro raised seedlings were tested on MS medium. The
multiple shoot induction response varied with the type of phytohormone and
their combination. In the present study, MS medium supplemented with
different concentrations of BAP, Kinetin, and TDZ and in combination of IBA
was used for multiple shoot induction. Among the various treatments tested
(Table 11, Fig 6 and Plate 5) for multiple shoots induction, the medium SIM-4
(Shoot Induction Medium) supplemented with 3mg/l BAP, 0.05mg/l IBA
(including adenine sulphate (100mg/l) exhibited the best induction of multiple
shoots (Plate 5E) among thirteen treatments. This treatment resulted
development of shoots from cotyledon node in reasonable time frame of 13
days with more than 84.33% of the explants responding with average of 7.17
shoots per explant.
84
PLATE - 3 Effect of plant growth hormone on callus induction from leaf
explant A. MS (control) B. MS+ 1.5 mg/l 2.4. D C. MS+ 2.0 mg/l 2.4. D D. MS+3.0 mg/l 2.4. D +1mg/l Kn E. MS+ 3.0 mg/l 2.4. D+1mg/l Kn+ 0.2% Cocoanut water F. MS+ 3.0 mg/l 2.4. D+1mg/l IAA+ 0.1% Cocoanut water
PLATE NO. 4 Effect of plant growth hormone on callus induction from shoot tip explant
A. MS+ 1.5 mg/l 2.4. D B. MS+ 2.0 mg/l 2.4. D C. MS+3.0 mg/l 2.4. D +1mg/l Kn D. MS+ 3.0 mg/l 2.4. D+1mg/l Kn+ 0.2% Cocoanut water E. MS+ 3.0 mg/l 2.4. D+1mg/l IAA+ 0.1% Cocoanut water
85
Table-11 Effect of Plant growth regulators on shoot multiplication from of shoot tip and cotyledon node
Treatments
No.
Treatment
MS+ growth regulators(mg/l)
Shoot tip with cotyledon node Shoot tip explant No. of days to
*Each treatment replicated four times. Twenty observations per replications
86
Similarly, better shooting percentage was also observed in the medium SIM-7
(fortified with TDZ 0.1 mg/l and 0.05 mg/l IBA) with average shoot induction
of 80.00% with 5.17 shoots per explant (Plate 5D). In case of shoot tip explant
multiple shoots were observed after 15.67 days of incubation with 77.67%
response and 5.50 shoots per explant (Plate 5E). The treatment (with the TDZ
0.1mg/l +IBA 0.05mg/l) showed about 74.33% percentage of shoot induction
with 3.67 shoots per explant. Lowest response (15-18%) and shooting (2-3
shoots per explant) was recorded in the treatment of 1mg/l BAP and 0.05mg/l
IBA. The explant shoot tip with cotyledonary node showed better response
(84.33% response with average multiple shoots 7.17 per explant) than that of
shoot tip explant (77.67% with 5.5 shoots per explant).
4.1.4 Elongation of Multiple shoots
The number of shoots produced per explant was invariably less until
the optimum concentration 3mg/l BAP + 0.05mg/l IBA. Even though 7.17
Shoots per explant were obtained, but shoots were stunted and did respond well
while cultured on fresh media. For elongation of multiples shoots, these were
cultured on the MS medium supplemented with various combination of GA3
and adenine sulphate (100mg/l). Among different medium combination, the
better elongation of shoots was observed with MS medium fortified with
1.0 mg/l GA3 and this medium promoted the maximum elongation of shoots
(11.23 cm) within a week incubation period (Table 12, Fig 7 and Plate 6). The
MS medium supplemented with the 1.2 mg/l GA3 showed elongation about
10.42 cm (Plat 6E) and the lowest elongation (5.6 cm) was observed in MS
medium supplemented with 0.2mg/l GA3.
87
Plate - 5 Effect of the Plant growth regulators on Direct organogenesis : initiation of multiple shoots from cotyledonary node (above) and shoot tip (below) A. MS+1.0 BAP + 0.05 IBA B. MS+0.5 Kinetin +0.05 IBA C. MS+0.5 TDZ D. MS+0.1 TDZ + 0.05 IBA E. MS+3 BAP + 0.05 IBA
88
Fig. 5. Effect of Plant growth regulators (PGR) on callus induction of black gram cv.�T-9�
Fig. 6 Effect of Plant growth regulators on shoot multiplication from
responded for rooting in medium NAA (0.25mg/l) which was comparatively
lower percentage response than the 0.5mg/l NAA but number of roots per
shoots are quite higher(8.40).
Table -13 Effect of Plant growth regulators on rooting of multiple shoots
Treatments No.
Treatments MS + growth regulators(mg/l)
No. of Days to
induction
No. of explants
response(%)
No. of roots/shoots
RIM 0 MS 0 0 0.00
RIM 1 MS+IBA 0.25 10 21.2 2.45
RIM 2 MS+ IBA 0.5 12 32.4 3.60
RIM 3 MS+ IBA 1 14 55.5 2.80
RIM 4 MS+ IBA 2 15 59.2 3.60
RIM 5 MS+ IBA 3 10 53.33 4.00
RIM 6 MS+ IBA 4 15 63.4 4.20
RIM 7 MS+ IBA 5 16 61.5 3.80
RIM 8 MS +NAA 0.25 12 68.9 8.40
RIM 9 MS+NAA 0.5 10 72.5 6.20
RIM 10 MS+NAA 1 13 59.2 3.80
C.D(p=0.05) 2.68 0.93 2.12
C.V% 9.63 13.56 12.25
*Each treatment replicated four times. Twenty observations per replication
4.1.6 Hardening of rooted plantlets
The pre- hardening was done for the rooted plantlets (about 13-15cm
height) and these plantlets were transferred in to liquid plant growth medium
(LPGM) for 48 hours so as to acclimatize them. Then, the plants were
transferred to pots containing soil: sand: FYM (1:1:1) and kept in green house
at 90% humidity. Seventy percentage of plantlets were survived and
completely established in Green house (Plate-8A &B).
92
Fig. 7 Effect of Plant growth regulators on shoot elongation of multiple shoots
Fig. 8 Effect of Plant growth regulators on rooting of multiple shoots
0
2
4
6
8
10
12
0
10
20
30
40
50
60
70
80
90
EIM 0 EIM 1 EIM 2 EIM 3 EIM 4 EIM 5 EIM 6 EIM 7
Leng
th o
f sho
ots
(cm
)
No.
of d
ays
to in
duct
ion/
No.
of e
xpla
nts
res
pons
e
Treatment
No. of Days to induction No. Of explants response Length Of shoots in cm
0
10
20
30
40
50
60
70
80
RIM 0 RIM 1 RIM 2 RIM 3 RIM 4 RIM 5 RIM 6 RIM 7 RIM 8 RIM 9 RIM 10
Surv
ival
(%
)
Treatment
No. of Days to induction No. of explants response.(%) No. Of roots/shoots
93
Plate - 8 Hardening of plantlets A. Pre-hardening of rooted plantlets B. Hardening of rooted plantlets in soil: sand: vermicompost (1:1:1)
94
4.2 Transformation studies
4.2.1 Kanamycin sensitivity test
To standardize lethal dose of kanamycin, putative transgenics explants
were cultured on MS medium containing the different concentration of
Kanamycin (20.40.60 and 80mg/l). At the 20 and 40 mg/l kanamycin explant
were not inhibited by the antibiotic and were able to grow healthy. The
treatment with 60mg/l kanamycin appear to have a slightly toxic effect over the
explant causing loss of chlorophyll, but the explants were able to overcome this
toxicity in later stages. Kanamycin at 80mg/l had a deleterious effect. (Plate-9E).
The plant become albino at the higher concentration of the kanamycin and caused
immediate death of the explant as shown in the Table- 14
Therefore in the present transformation studies, 80 mg/l kanamycin
was used as a selection agent. The co-cultivated explants that grew uninhibited
on this concentration of kanamycin considered a putative transformants.
Table -14 Detection of lethal concentration of Kanamycin for selection medium
Treatments Kanamycin (mg/l)
No. of explants
inoculated
Explant survival after 20
day
Percentage of survival
Appearance of explant
T1 0 20 19.66 98.00 Dark green
T2 20 20 12.00 60.00 Dark green
T3 40 20 7.33 36.65 Pale green
T4 60 20 2.66 13.33 Yellowish
green
T5 80 20 0 0 White
C.D(p=0.05) 1.29
C.V % 10.43
95
4.5 Optimization of Agrobacterium mediated transformation protocol.
4.3.1 Effect of pre-culture period of explant in the co-cultivation protocol
Pre-culture is one of the important steps for co-cultivation experiment.
Variations in the pre-culture period in co-cultivation were taken at 0 - 96 hours.
The effect of pre-culture on survivability of explants after co-
cultivation is presented in Table 15 and Fig 9. Significant difference was
found in explants pre-cultured for 48 (68.30%) and 72 hours (76.65%) and
GUS expression (about 60, 78.30%) was observed respectively. The significant
survivability (85.00%) with 88.33% GUS expression was recorded in pre-
culture for 96 hours. Compared with all treatment, it showed highest
survivability and GUS expression percentage after co-cultivation. Therefore
it was concluded that 96 hours is the best pre-culture period in order to
obtain maximum survivability and also found to be suitable for co-
cultivation found based on the percentage Gus expression.
Table -15 Effect of pre-culture period on co-cultivation
Treatment Duration of
pre-culture in hours
Average no. explant alive after co-
cultivation
Percent survival
GUS expressio
n %
T1 0 9.00 45.00 36.65
T2 24 12.33 61.45 40
T3 48 13.67 68.30 60
T4 72 15.33 76.65 78.3
T5 96 17.00 85.00 88.3
C.D(p=0.05) 1.29 2.77
C.V% 5.42 12.85
*Each treatment replicated four times. Twenty observations per replication
96
4.3.2 Co-cultivation period
Explants were immersed with Agrobacterium suspension under
shaking condition for 20 to 30 minutes. Then the explants were taken out,
blot dried and co-cultivated on liquid MS medium (without sucrose)
supplemented with BAP (2mg/l) for different periods (24 to 96 hours) in order
to determine the optimum co-cultivation period to get maximum survivability.
The effect of different co-cultivation period on survivability of explants is
presented in Table 16 and Fig 10. Significant survivability and GUS expression
were found from the explants co-cultivated for 72 hours (78.35 and 75%
respectively). GUS expression (88.33%) was recorded in co-cultivation period
of 96 hours but survivability percentage was reduced to 51.65%. Hence 72
hours found to be suitable for co-cultivation.
Table-16 Effect of Duration of co-cultivation
Treatment Duration of co-culture in
hours
Average no.explant alive after co-cultivation
GUS expression %
Bacterium growth
T1 24 14.33 35 +
T2 48 14.67 55 +
T3 72 15.67 75 +
T4 96 10.33 88.3 +++
C.D(p=0.05) 1.54 1.43
C.V% 6.15 5.9
+ Slight growth , +++ Prominent growth of Agrobacterium, *20 explants used for the experiment with 4 replication was done.
97
Fig. 9 Effect of pre-culture period on co-cultivation
Fig. 10 Effect of duration of co-cultivation
0
10
20
30
40
50
60
70
80
90
100
0
20
40
60
80
100
120
T1 T2 T3 T4 T5
Perc
enta
ge o
f sur
viva
l
Dur
atio
nof p
re-c
ultu
re in
hou
rs/A
vera
ge n
o. o
f ex
plan
t al
ive
afte
r co
-cul
ture
Treatment
Duration of pre-culture in hours
Average no. explant alive after co-cultivation
Percent survival
GUS expression %
0
10
20
30
40
50
60
70
80
90
100
0
20
40
60
80
100
120
T1 T2 T3 T4
Perc
enta
ge o
f su
rviv
al
Dur
atio
nof p
re-c
ultu
re in
hou
rs/A
vera
ge n
o. o
f exp
lant
al
ive
afte
r co
-cul
tiva
tion
Treatment
Duration of co-culture in hours
Average no.explant alive after co-cultivation
GUS expression %
98
4.3.3 Optimization of antibiotic concentration for inhibition of Agrobacterium tumefaciens growth
Sensitivity of Agrobacterium tumefaciens to various levels of
antibiotics was determined by culturing the co-cultivated explants on MS
medium containing 2.0 mg/l BAP and different concentrations of
cefotaxime (0 to 500 mg/l). The effect of different concentrations of
antibiotics on growth of Agrobacterium is presented in Table 17.
The percentage of reappearance of Agrobacterium was t h e
maximum (100 %) on growth medium without antibiotic and it was found
to be gradually decreased with increase in concentration of antibiotics from
100 to 500 mg/l. The growth of Agrobacterium was totally inhibited at 500
mg/l concentration of both cefotaxime. So 500 mg/l cefotaxime was
added in the selection medium for getting contamination free
transformants.
Table-17 Determine the sensitivity of Agrobacterium to various level of cefotaxime
Treatment Concentration of cefotaxime
mg/L
Bacterium growth
Reappearance
Bacterium growth
reappearance percentage
Bacterium growth
T1 0 20 100 ++++
T2 100 15.75 78.75 +++
T3 200 13.5 67.5 +++
T4 300 9.75 48.5 ++
T5 400 3.5 17.5 +
T6 500 0 0 -
C.D(p=0.05) 1.46
C.V% 7.62
- No growth,+ little growth , ++ moderate ,+++ Prominent growth , ++++ over growth of Agrobacterium, *20 explants used for the experiment with 4 replication was done.
99
4.3.4 Transformation efficiency of Agrobacterium on selection of putative transformants on Kanamycin medium.
The cotyledonary nodes and shoot tips were co-cultivated with strain
EHA-105 containing the gene construct ICE -I. Four days pre-cultured shoot
tips were dipped in the bacterial suspension and then resuspended in LPGM
medium containing Acetosyringone (100µM) for 20 min, Then cotyledonary
nodes and shoot tips were incubated for 72 hours and subsequently cultured in
the shooting medium without selection pressure and grown for the next 3 days
followed by sub culturing on selection medium. Inclusion of the selective
agent, Kanamycin (80 mg/l) in the selection medium allowed transformed cells
to proliferate (Plate-10H) however, with a few albino shoots. These explants
were then sub-cultured after every 2 weeks on selection medium. A total of 130
explants of cv. �T-9� were obtained after co-cultivation. Out of these 12
explants of �T-9� were survived on selection medium containing 80 mg/l
Kanamycin. A transformation frequency of 9.23%.was achieved (Table-19).
The growth of the resistance shoots was rapid. Kanamycin- resistant shoots
were subjected to further high selection pressure and were maintained for
evaluation of transgenic black gram.
100
Plate - 9 Determination of Kanamycin sensitivity A. control (0mg/l) B. 20mg/l C. 40mg/l D. 60mg/l E. 80mg/l
�Plate - 10 Kanamycin based selection of transformants
F. control G. a week culture on selection medium H. two week old culture on selection medium
101
4.3.5 Gus assay of transformants
The different tissue of transformants was stained in X-gal. High GUS
expression was detected in the leaf (Plate-11 A & B) and cotyledonary node. The
relative expression of GUS was analyzed as blue colour development (Plate-11).
About 95% percentage of transient GUS expression was recorded (Table-18). The
explant which were not inoculated with Agrobacterium, showed no GUS activity.
The cross section of GUS positive (Plate-11 E&F) as well as negative explants
were made (Plate-11C&D) and observed in microscope at 10X and 20X
magnification.
Table-18 Transient GUS expression percentage
Total no of explant assayed
No. putative GUS +ve
Transformation efficiency in %
20 19 95
*Each treatment replicated four times. Twenty observations per replication
4.3.6 Molecular analysis of putative transformants
Putative transformants were screened by the PCR for presence of nptII
genes. For this purpose total genomic DNA was isolated from the explants
survived on the selection medium (Kanamycin 80 mg/l) and control. Then the
PCR reaction was carried out using primers specific for nptII gene and plasmid
DNA used as a positive control. The PCR amplified product was electrophoresed
on 1.2% agarose gel (Plate-12). Expected band of about 700 bp was obtained
only in two samples (out 10 samples). The transformation efficiency found to be
about 1.53% (Table-19).
Table 19 Transformation efficiency based on kanamycin selection and PCR analysis
Total no of explant assayed
No. of shoots obtained after co-
cultivation
No. shoots selected based on Kanamycin
selection
Transformation efficiency (%)
based on Kanamycin
selection
No. plants selected based on
PCR analysis
Transformation
efficiency (%) based on PCR
165 130 12 9.23 02 1.53
102
Plate� 11 GUS histochemical assay A. Control B. Transformants (+ve for GUS) C. T.S of Untransformed cells (10X) D. L.S of Untransformed cells (10X) E. T.S of transformed cells (GUS +ve )(20X) F. callus showing GUS +ve
103
Plate - 12 PCR analysis of putative transformants using nptII as primer M. 20 bp ladder
1. Positive control 2. negative control 3. untransformed 4-11. putative transformants
104
DISCUSSION
Transformation of crop plants with desired genes is the focus of many
plant genetic engineering programs. The stable introduction of foreign genes
into plants is one of the significant advances in crop improvement programme,
which would help in transforming desirable genes into local adopted varieties,
thus supplement conventional breeding programme. Among the several
methods used for transformation of plants, Agrobacterium tumefaciens
mediated transformation is preferred in many cases because of several distinct
advantages over other methods. These include single copy integration, greater
precision with excellent stability. The rate of transformation is influenced by
various factors like plant genotype, Agrobacterium strains, plasmid vectors,
temperature etc. Along with these factors, like Agro-inoculum treatment
duration and co-cultivation period also influences transformation rate.
5.1 IN- VITRO REGENERATION
A highly in-vitro plant regeneration system is very essential for carrying
out genetic transformation work. In case of legume crops, direct organogenesis
of shoots from cotyledonary nodes, shoot apices, leaflets and embryo axes is
the most common regeneration pathway.
5.1.1 Effect of surface sterilants
In present study 0.2 % HgCl2 was used as surface sterilants for
sterilization of black gram seeds. Among the various treatments, the best
result was observed in treatment of seeds with of 0.2% HgCl2 for a period four
minutes. In this treatment 91.25 % aseptic culture and 88.75 %
survivability of explants were recorded. The present result is in congruent with
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the result reported by Saini, et al., 2007, Yadav, et al., 2010 and Varalaxmi, et
al., 2007 where 0.1% of HgCl2 treatment for 5 � 8 minute duration also
resulted higher aseptic culture with greater survival percentage.
5.1.2 Indirect regeneration
A very rapid and efficient regeneration method of Vigna mungo L. has
been established using liquid culture (Das, et al., 2002) by using a leaf explant
in liquid culture medium. The callus induction and plant regeneration of Indian
soybean (Glycine max L.) via half seed explant culture was also carried out
(Radhakrishnan and Ranjithakumari, 2007). Protocol for callus induction and
plant regeneration was established by using half seed explant on B 5 media of
Vigna mungo (L.) Hepper. The callus was induced in almost all media
combinations supplemented with varying levels of phytohormones. In the
present investigation among all the media and phytohormone combination
tested, the MS medium supplemented with 2.4.D (3.0 mg/l) and kinetin
(0.1mg/l) along with coconut water had induced profuse callusing in both types
of explants like leaf and inter cotyledonary node. The calli so developed were
loose, friable, whitish green coloured and of good quality as compared to calli
produced on other media combinations. Harisaranraj (2008) reported the best
callus development in MS medium supplemented with 2.0 mg/l BAP and 0.5
mg/l NAA. Further attempt to convert callus into plants was not successful.
Successful regeneration of calli of black gram to whole plant had not been
reported yet by any researchers.
5.1.3 Multiple shoot induction
The efficiency of in vitro multiple shoot formation and regeneration
was found to be dependent on various parameters, viz. explant size, age of
explant donor seedling, explant type, genotype, media composition, and growth
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regulators (Gulati and Jaiwal,1992). For in vitro regeneration of black gram
various explants viz. stem, epicotyl, cotyledonary node explants (Gill et
al.,1987), excised cotyledonary and hypocotyl segments (Sen and Guha, 1998),
cotyledons and embryo axes (Ignacimuthu and Franklin,1997) were utilized
for in vitro multiple shoot induction. In the present investigation shoot tips and
cotyledonary nodes were used for efficient regeneration. Multiple shoots (7.17
shoots/ explant) were achieved from the inoculated explant excised from 4 days
old germinating seedling when cultured on half-strength MS medium
supplemented with 3mg/l BAP, 0.05mg/l IBA. Yadav et al., 2010 used MS B5
media for induction of multiple shoots. Cotyledonary node with embryonic
axis were used by Sumita et al., 2012 for induction of the of multiple shoot.
They reported that MS medium fortified with BAP (2mg/l),Kinetin (2mg/l)
was found to be only 3.24 shoots per explant in the �T-9� cultivar which is
comparatively lower percentage of multiple shoot induction as compared to our
present result. In present investigation similarly, better shooting percentage
was also observed in the medium SIM-7 (fortified with Thidiazuron (TDZ )0.1
mg/l and 0.05 mg/l IBA) with average shoot induction of 80.00% with 5.17
shoots per explant obtained. The TDZ has been reported to induce the
adventitious shoots. Mean multiple shoots per explant decrease with the
increase (up to 3mg/l) of TDZ and also causes shunted shoots with abnormal
morphology. Similar results were reported when TDZ used longer time in the
media (Jayanaand, et al., 2003).
5.1.4 Elongation of Multiple shoots
The explants with multiple shoots from various experiments were
sequentially sub-cultured on to shoot elongation media containing low
concentrations of BAP and Kinetin for production of healthy elongated shoots.
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In the Blackgram cv. Co-5, the highest (10.45) mean numbers of healthy
elongated shoots per explant was obtained in medium supplemented with 0.5
µM of BAP and 0.5 µM of Kinetin which significantly differed from the mean
number of elongated Shoots per explant in all the other media tested. The mean
number of healthy elongated multiple shoots per explant in the cultivar T-9 and
local market collection was found 10.10 and 9.75, respectively (Sumita, et
al.,2012). A positive effect of a low concentration of the cytokinins group of
growth hormones (BAP, Kinetin, etc.) on multiple shoot elongation was also
observed in other legumes such as V. raditata (Chandra and Pal 1995; Gulati
and Jaiwal 1994), cowpea (V. unguiculata) (Muthukumar, et al. 1996), and
chickpea (Cicer aritenum) (Chakraborti, et al., 2006; Das, et al., 2004; Sarmah,
et al., 2004). In the present work medium supplemented with various
combination of GA3 and adenine sulphate (100mg/l) were used for elongation
of multiple shoots. Among different medium combination, the better elongation
of shoots was observed with MS medium fortified with 1.0 mg/l GA3 and this
medium promoted the maximum elongation of shoots (11.23 cm) within a
week incubation period. The MS medium supplemented with the 1.2 mg/l GA3
showed elongation about 10.42 cm. The phytohormone GA3 showed greater
elongation as compared to the result reported by Sumita, et al., 2012.
5.1.5 Rhizogenes of Multiple shoots
The rooting of shoots was significantly affected by the auxins
concentration. MS medium supplemented with 0.25 mg/l IBA and 0.2 mg/l
NAA performed better rooting within least number of days (8.33) for rooting.
Khawar and Özcan (2002) reported that MS medium containing 0.25 mg/l. IBA
performed best and required four weeks for rooting. Geetha, et al. (1999)
reported that roots emerged within 15 days. A higher percentage of rooting
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(100%) was found with 0.5 mg/l IBA in the study. Raman, et al. (2004)
reported that efficient rooting (100%) of the shoots on medium containing half
MS salts, full MS vitamins and IBA (2.5 ìM). Khawar and Özcan (2002)
reported that medium containing 0.25 mg/l IBA obtained only 25% roots and
Geetha, et al. (1999) reported that medium containing 3 mg/l IBA showed
78.3% of rooting. The maximum number of roots (14.33) per shoot was
recorded in medium containing 0.5 mg/l NAA. Geetha, et al. (1999) reported
that medium containing 3.0 mg/l IBA produced 14.5 roots/plants. It was clear
from the above discussion that 0.5 mg/l IBA was better for root formation than
any other treatments. Das, et al. (2002) and Geetha, et al. (1999) reported that
IBA was effective for rooting of black gram, while Roy et al. (2007) reported
that NAA was effective for rooting.
In our study we found higher percentage of rooting in MS medium
supplemented with NAA (0.5 mg/l) and in this treatment about 72.5% of shoots
responded well for rooting with an average of 6.2 roots per shoots. Similarly,
68.9% explant responded for rooting in medium containing NAA (0.25mg/l)
which was comparatively lower percentage response than the 0.5mg/l NAA but
number of roots per shoots are quite higher (8.40). in vitro raised healthy
plantlets of 13-15 cm in height were planted in a mixture of soil: sand: FYM
present in the ratio of 1:1:1. Survival rate of the transplanted plantlets was
recorded to be 70%.
5.2 Transformation studies
Agrobacterium-mediated transformation involves interaction between
two biological systems and is affected by various physiological conditions.
Therefore optimization of some of the aspects that enhance the virulence of
Agrobacterium for T-DNA transfer and factors that improve survival and
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regeneration of transformed cells is crucial for a recalcitrant crop like
black gram. Transient GUS expression provides an easy and clear indication of
the expression of transferred genes and can be used to assess the frequency of
transformation. In the present study, sub-culturing of explants prior to agro
infection facilitated entry of Agrobacterium cells and production of �vir� gene
inducers. Compromise between the efficiency of infection and survival rate
after transfer.
Inclusion of synthetic phenolic compound acetosyringone in co-
cultivation medium enhanced transient expression of GUS when used at a final
concentration of 100 µM. Acetsyringone enhances �vir� functions during
transformation (Stachel, et al., 1985) and has been shown to increase
transformation potential of Agrobacterium strain with moderately virulent �vir�
region in several plant species (Atkinson and Gardner, 1991; Janssen and
Gardner, 1993; Kaneyoshi et al., 1994). Also the acidic pH (5.2) used in the
infection medium acts synergistically with acetosyringone for increasing the
transformation efficiency.
The blue coloration in the infected cotyledon explants upon histochemical
assay suggested the introduction and expression of GUS gene. Based on molecular
characterization using PCR, the integration of nptII gene was confirmed in
transgenics. The untransformed control did not show any amplification with these
primers. Two transgenic lines expressing the nptII. The transformation frequency
increased with increase in concentration of Agrobacterium cells up to 108 and,
thereafter, decreased with further increase in number of Agrobacterium cells.
Similar results were mobtained in Nicotiana tabacum and Arabidopsis thaliana
and in most of the grain legumes (Bean, et al. 1997).
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The length of co-cultivation period required for achieving maximum
gene transfer was found to be 3 days. Further extension in co-culture time
decreased the transformation frequency resulting in bacterial overgrowth and
had detrimental effect on regeneration potential of explants. A short co-culture
period of 2 or 3days has also been found to be optimum in other plant species
such as Antirrhinum majus (Holford, et al.,1992), Vigna unguiculata
(Muthukumar, et al., 1996), Vigna radiate (Jaiwal, et al., 2001), Cajanus cajan
(Mohan and Krishnamurthy, 2003), and Glycine max (Li, et al. 2004)
Wounding the plant material before co-cultivation allows better bacterial
penetration into the tissue facilitating the accessibility of plant cells for
Agrobacterium or possibly stimulated the production of potent �vir� gene
inducers like phenolic substances such as acetosyringone and
hydroxyacetosyringone (Stachel, et al.,1985) and enhanced the plant cell
competence for transformation (Binns and Thomashow,1988). Wounding the
plant material before co-cultivation has also been shown to increase
transformation frequency. Mechanical injury of the meristematic region
probably induces meristem reorganizations promoting formation of large
transgenic sectors and enhanced recovery of transformants.
Pre-culture of explants on regeneration medium prior to inoculation and co-
cultivation with Agrobacterium has been reported to enhance efficiency of
transformation in some grain legumes, e.g. Vigna unguiculata (Muthukumar, et al.
1996) and Cajanus cajan (Geetha, et al. 1999). However, in present study, no
such results were obtained. This may be due to the specificity of species to pre-
culture. In contrast, pre-culture (0 - 3 days) of cotyledonary node explants prior
to co-culture with bacteria reduced the frequency of transient GUS expression
high. This may be due to the healing of the wounding site, because wounding is
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a prerequisite for Agrobacterium-mediated transformation. The reduction may
be attributed to the secretion of compounds that inhibit �vir� gene induction or
dilution of the �vir� gene inducing signal molecules released as result of
wounding. Wounding induced division and production of phenolic compounds
such as acetosyringone and hydroxyacetosyringone. These signal molecules are
recognized especially by Agrobacterium to induce �vir� gene expression and
thereby activate T-DNA transfer (Zambryski, 1983). Pre-culture was found to
reduce transformation efficiency in other plant species also such as kiwifruit
(Janssen and Gardner, 1993) had no effect in peanut transformation (Sharma
and Anjaiah, 2000). When preculture was combined with mechanical injury the
results were reversed that leads to increase in transient GUS expression and
was found to be up to 95 %. This may be attributed to visually more clear
regeneration site on the pre-cultured explants for mechanical injury as
compared to non-pre-cultured and freshly release of phenolics as a result of
mechanical injury. High vigour of pre-cultured explants was also found to
increases the regenerability of mechanically injured explants. These optimized
transformation factors were used for the stable genetic transformation of Vigna
mungo. One hundred sixty five cotyledonary node explants co-cultured with
Agrobacterium produced a total of 12 shoots on kanamycin selection medium.
The green shoots (2 to 3 cm) were subjected to a second round of selection at
the rooting stage. These plantlets were subsequently transferred to soil. The
plant genomic DNA was isolated for PCR analyses. PCR results showed
amplification of a 700 kb band corresponding to the coding region of nptII
gene, indicating the presence of transgene in 2 out of 10 putatively transformed
plants established in soil, with an overall transformation frequency of 1.53%.
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SUMMARY AND CONCLUSION
Black gram (Vigna mungo L. Hepper) is popularly known as Urad in
Hindi, Biri in Odia and Uddu in Kannada. It belongs to the family Fabaceae. It
is diploid in nature with 2n=2x=22. It has a small genome size containing 574
million base pairs. It is the third most important pulse crop in India after
chickpea and pigeon pea and grown mostly as a fallow crop in rotation with
cereals. In India total area under cultivation is about 31.0 lakh hectares with the
average production and productivity of 14.0 lakh tones and 451.61 kg/ha
respectively. The total area under black gram in Odisha is about 1.50 lakh
hectares with total production 0.42 lakh tones. But the productivity of black
gram is 280.0 kg/ha which is far below than the national average productivity
(451.61 kg/ha). Its yield is highly affected by a number of abiotic stresses
particularly cold and moisture stress. Abiotic stress causes the crop loss more
than 50% from their potential yield (Wang et al., 2006). Varietal improvement
against abiotic stress tolerance is the major remedy for increasing productivity
and production of black gram. Varietal improvement of black gram through
genetic engineering of plant for tolerance to abiotic stress could be achieved by
the regulated expression of a large number of stress responsive genes. Among
available genes, ICE-1 gene has been identified as one of the potential genes
conferring resistance to abiotic stress. This gene had been isolated from the
model plant Arabidopsis thaliana and well characterised. It is an upstream
transcription factor and is a positive regulator of CBF-3 and plays a critical role
in cold tolerance. The present research aimed at developing an efficient
regeneration and transformation system for genetic improvement of black
gram cv. �T-9�.
For development of an efficient in vitro regeneration protocol, different
explant like leaf, shoot tips, shoot tips with cotyledon nodes were tested for
callus initiation. The leaf was found to be more responsive for callus initiation
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than other explants. The MS medium supplemented with 2.4.D (3 mg/l) and
kinetin at (1mg/l) was found to be the most responsive in the callus formation.
Callus induction was achieved in 87.25% explants cultured in this medium
after 12 days of incubation. Further conversion of callus to whole plants could
not be achieved in spite of different media, phytohormone combination and
organic supplements The direct multiple shoots induction from different
explants was observed in medium SIM-4 (Shoot Induction Medium)
supplemented with 3mg/l BAP, 0.05mg/l IBA (including adenine sulphate
(100mg/l). This treatment resulted development of shoots from cotyledon node
in reasonable time frame of 13 days with more than 84.33% of the explants
responding with average of 7.17 shoots per explant. The explants shoot tip with
cotyledonary node showed better response than that of shoot tip. About 84.33%
response of direct regeneration were recorded with average multiple shoots of
7.17 per explant. During sub-culturing MS medium fortified with 1.0 mg/l GA3
promoted the maximum elongation of shoots (11.23 cm) within a week
incubation period. About 68.9% explant responded for rooting in medium
NAA (0.25mg/l) which was comparatively lower percentage response than the
0.5mg/l NAA but number of roots per shoots are quite higher (8.40).
Transformation of regenerated shoots was achieved by employing