Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR

i

Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera

AND MOLECULAR ANALYSES OF SELECTED EVENTS

MAHALE BARKU MANOHAR

DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD

UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005

JUNE, 2014

ii

Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera

AND MOLECULAR ANALYSES OF SELECTED EVENTS

Thesis submitted to the University of Agricultural Sciences, Dharwad

In partial fulfillment of the requirements for the

Degree of

Doctor of Philosophy

in

Molecular Biology and Biotechnology

By

MAHALE BARKU MANOHAR


UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005

JUNE, 2014

iii


UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD

CERTIFICATE

This is to certify that the thesis entitled “Agrobacterium tumefaciens MEDIATED

TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac,

cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera AND MOLECULAR

ANALYSES OF SELECTED EVENTS” submitted by Mr. MAHALE BARKU MANOHAR.,

for the degree of DOCTOR OF PHILOSOPHY in Molecular Biology and Biotechnology, to

the University of Agricultural Sciences, Dharwad, is a record of research work done by him

during the period of his study in this University under my guidance and the thesis has not

previously formed the basis for the award of any degree, diploma, associateship,

fellowship or other similar titles.

DHARWAD

JUNE, 2014 (B. FAKRUDIN)

CHAIRMAN

Approved by :

Chairman :

Members : 1.

2.

3.

(RAMESH BHAT)

(NARAYAN MOGER)

(B. M. KHADI)

(B. FAKRUDIN)

(S. S. UDIKERI)

4.

iv

Acknowledgement

One would not achieve whatever he is now, without all help, encouragement and

the well wishes of the near and dear ones. Teachers, parents, relatives, friends and all

well-wishers are an integral part of this. I owe them a lot and it is always a difficult task to

express the sense of gratitude for them in words.

First and foremost, I would like to express my deep sense of gratitude to

Dr. B. FAKRIDUN, Professor Dept. of Biotechnology, College of Agriculture, Dharwad

and the esteemed chairman of Advisory Committee for his inspiring and peerless

guidance, scholarly advice, thought provoking suggestions, sparing his time amidst busy

schedule and sustained encouragement throughout the course of investigation.

I record with sincerity my profound sense of gratitude to members of my Advisory

Committee Dr. B. M. KHADI, Directorate of Research, University of Agricultural

Sciences, Dharwad, , Dr. S. S. UDIKERI, Professor, Dept. of Agricultural Entomology,

College of Agriculture, Bijapur, UAS, Dharwad, Dr. RAMESH BHAT, Professor,

Department of Biotechnology, Dr. NARAYAN MOGER, Associate Professor, Department

of Biotechnology, for their encouragement and valuable suggestions during the course of

my investigation and evaluation of the manuscript.

I am grateful to my teachers Dr. P. U. Krishnaraj, Dr. Sumangla Bhat, Dr. S. K.

Prashanti and Dr. K. M. Sumesh, Department Biotechnology, for their cooperation

extended to me during the course of my study.

My untold heartfelt gratitude and profound indebtness to my beloved my

mother Smt. Suman and father Sri. Manohar whose prayers, unfailing love and affection

has upholded me at every moment of tension, difficulty and achievement. It is an

immense pleasure to express my sincere gratitude to my brother Mr. Mahesh, for their

sacrifice, co-operation, love and affection in every aspects of my studies and research

work. Without their support, I would never have come through this far.

I express my deep sense of gratitude to my seniors Shivu, Omkar, Siji, Rajkumar,

Abid, Suvarna, Dadakhalandar, Nagaraj and my friends Vaibhav, Sambhaji, for their

needful and timely help, moral support during my research period.

v

Life is not work all the time, one necessarily has to have some diversion once in

awhile, to be able to return to work with renewed enthusiasm and vigour. On a personal

note, I am very glad to mention sincere mental support, word of encouragement,

boundless love, selfless sacrifices of my friends Abhijit, Gourav, Bhabesh, Sandeep,

Neha, Diksha, Divya, Suhasini, Navya, Rose, Anil, Shrinvas, Dnyaneshwar,

Padmabhushan, Ramesh, Yogesh who have played an excellent role by showing friendly

attitude, love, generous hospitality right guidance and encouragement and all the trouble

they took for my sake, well association with them is truly of inestimable value. But, then

one does not need to thank the true friends.

I thank Lab maintainer Kallappa, Vijay, Santosh, Malik, Ravi, Basu and green

house maintainer Chanappa and Manju and other non-teaching faculty in the

Department of Biotechnology for their co-operation and help during the period of my

research work.

I sincerely acknowledge the necessary facilities provided by the concerned

authorities at U.A.S., Dharwad for smooth carrying out of my Ph. D. Programme. The

financial support of Indian Council for Agriculture Research (ICAR) under the framework

of National Fund for Basic Strategic Research in Agriculture (NFBSFARA) is gratefully

acknowledged.

Last but not the least, I express a special word of thanks to Mr. Kalmesh and Mr.

Arjun (Arjun Computers) for neat and timely printing of this manuscript.

Any omission in this acknowledgement does not mean lack of gratitude……….

DHARWAD

JUNE, 2014 (BARKU M. MAHALE)

vi

Affectionately Dedicated To

The Indian Farmers

vii

CONTENTS

Sl. No. Chapter Particulars

CERTIFICATE

ACKNOWLEDGEMENT

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATES

LIST OF APPENDICES

1. INTRODUCTION

2. REVIEW OF LITERATURE

2.1 Pigeonpea: Worlds important pulse crop

2.2 Major threats to pigeonpea production

2.3 Crop improvement strategies for insect pest resistance in pigeonpea

2.4 Pigeonpea pod borer, Helicoverpa armigera life cycle and damaging stages

2.5 Bacillus thuringiensis delta-endotoxin

2.6 Insecticidal proteins other than delta-endotoxin

2.7 Tissue culture studies in pigeonpea

2.8 Plant transformation studies

2.9 Selection marker

2.10 Southern Blotting

2.11 Immunoassay

2.12 Real-Time PCR

2.13 Insect bioassay

3. MATERIAL AND METHODS

3.1 Plant material and in vitro plantlet regeneration in pigeonpea

3.2 Maintenance of Agrobacterium tumefaciens strains and in vitro transformation of pigeonpea

3.3 Explant preparation and improvisation of in planta transformation protocol in pigeonpea

3.4 Identification of putative transformants

3.5 Transgene segregation analysis of T2 generation progenies

viii

Sl. No. Chapter Particulars


3.7 Insect culture and transgene bioefficacy analysis

3.8 Quantitative estimation of Cry protein using ELISA assay

3.9 Absolute real time qRT-PCR for cry transcript analysis

3.10 Genomic Southern blot analysis

3.11 Northern blot analysis

3.12 Recovering the site of transgene integration by TAIL PCR

3.13 The plant growth parameters

3.14 Statistical analysis

4. EXPERIMENTAL RESULTS

4.1

Effect of cytokinins and their concentration regimes on multiple shoot induction and plantlet regeneration in pigeonpea

4.2 Effect of different treatments on in planta transformation in pigeonpea

4.3 Generation of transgenic pigeonpea conferring expression of cry1Ac gene

4.4 Generation of transgenic pigeonpea conferring expression of cry2Aa gene

4.5 Generation of transgenic pigeonpea conferring expression of cry1F gene

4.6 Generation of transgenic pigeonpea conferring expression of cry1Acm gene

4.7 The plant growth parameters comparison between parental genotypes and transgenic lines developed

5 DISCUSSION

5.1 Improvisation of multiple shoot induction and plantlet regeneration in pigeonpea

5.2 Improvisation of in planta transformation protocol in pigeonpea

5.3 Generation of transgenic pigeonpea carrying different cry genes for pod borer resistance

5.4 Molecular characterization of pigeonpea transgenic lines expressing different cry genes

6 SUMMARY AND CONCLUSIONS

REFERENCES

APPENDICES

ix

LIST OF TABLES

Table No.

Title

1

Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from cotyledonary node with cotyledons explant of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture (50 explants)

2

Number of shoot bud induced in response to different concentration regimes of cytokinins (benzylamino purine, thidiazuron and zeatin) from cotyledonary node with cotyledons and embryo discs with half cotyledons explants.

3

Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from embryo discs with half cotyledon explants of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 12 days of culture (50 explants)

4

Effect of indole butyric acid (IBA) concentration regimes on root induction and number of root induced per shoot in pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture

5 The effect of targeting embryonic axis attached to single cotyledon for successful Agrobacterium tumefaciens infection and successful transformation.

6 The effect of targeting embryonic axis attached to single cotyledon with tobacco extract added in overnight grown Agrobacterium culture on plant transformation.

7 The effect of targeting embryonic axis attached to single cotyledon with air evacuation to increase the proximity between Agrobacterium tumefaciens and embryonic axis on transformation

8 Effect of different treatments used in in planta transformation of pigeonpea on explant survival, explants responded and transformation efficacy presented in per cent.

9 Summary of transformation carried out using cry1Ac gene in pigeonpea

10a Summary of transformation work being carried out using cry1Ac gene following in vitro kanamycin selection method in pigeonpea

10b Identification of T1 plants progenies of 10 putative transformants carrying cry1Ac gene. Testing for the presence of gene was done through gene specific PCR assay

11 The transgene segregation pattern in eighty eight transformants carrying cry1Ac in T2 generation revealed by gene specific PCR assay

x

12a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eighty eight putative transformants carrying cry1Ac gene in T2 generation

12b Comparison of H. armigera mortality in transgenic lines of

ICPL87119 and BSMR736 (by unpaired ‘t’-test at α=0.05)

12c Per cent corrected mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving in vitro generated putative transformants carrying cry1Ac gene in T1 generation

13a The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as

revealed by ELISA assay (α=0.05)

13b The Cry1Ac protein level detected in in vitro generated transgenic pigeonpea plants of T1 generation as revealed by ELISA assay in

leaf, flower and pod tissues (α=0.05).

13c The correlation analysis of insect mortality levels and estimated Cry1Ac protein in leaf, flower and pod tissues of T2 generation

plants (α=0.01)

14 Transgene segregation pattern in eight transgenic lines carrying cry1Ac gene in T3 generation

15a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eight putative transformants carrying cry1Ac gene in T3 generation.

15b The ‘t’-test analysis of Helicoverpa armigera mortality levels from T2

and T3 generation of eight cry1Ac transgenic lines (α=0.05)

16 The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay

17a The cry1Ac transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

17b The correlation analysis of insect mortality levels, estimated Cry1Ac protein and cry1Ac transcript in leaf, flower and pod tissues of T3

generation plants (α=0.01).

18 Juncture region analysis of cry1Ac cassette in AC20-2, AC20-3 and AC29-1 transgenic lines as revealed by TAIL-PCR analysis

19 Summary of transformation carried out using cry2Aa gene in pigeonpea

20 The transgene segregation pattern in sixty five transformants carrying cry2Aa in T2 generation revealed by gene specific PCR assay

21 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving sixty five putative transformants carrying cry2Aa gene in T2 generation

xi

22a The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as

revealed by ELISA assay (α=0.05)

22b The correlation analysis of insect mortality levels and estimated Cry2Aa protein in leaf, flower and pod tissues of T2 generation

plants (α=0.01)

23 Transgene segregation pattern in fifteen transgenic lines carrying cry2Aa gene in T3 generation

24a Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fifteen putative transformants carrying cry2Aa gene in T3 generation

24b The ‘t’-test analysis of mortality levels from T2 and T3 generation of

fifteen cry2Aa transgenic lines (α=0.05)

25 The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay

26a The cry2Aa transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

26b The correlation analysis of insect mortality levels, estimated Cry2Aa protein and cry2Aa transcript in leaf, flower and pod tissues of T3

generation plants (α=0.01)

27 Juncture region analysis of cry2Aa cassette in selected transgenic lines as revealed by TAIL PCR analysis

28 Summary of transformation carried out using cry1F gene in pigeonpea

29 The transgene segregation pattern in fourteen transformants carrying cry1F in T2 generation revealed by gene specific PCR assay

30 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fourteen putative transformants carrying cry1F gene in T2 generation

31 The Cry1F protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay

32a The cry1F transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

32b The correlation analysis of insect mortality levels, estimated Cry1F protein and cry1F transcript in leaf, flower and pod tissues of T2

generation plants (α=0.05; 0.01)

33

Summary of transformation carried out using cry1Acm gene in pigeonpea

xii

34 The transgene segregation pattern in eleven transformants carrying cry1Acm in T2 generation revealed by gene specific PCR assay

35 Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eleven putative transformants carrying cry1Acm gene in T2 generation

36 The Cry1Acm protein level detected in leaf, flower and pod tissues developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay

37a The cry1Acm transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

37b The correlation analysis of insect mortality levels, estimated Cry1Acm protein and cry1Acm transcript in leaf, flower and pod

tissues of T2 generation plants (α=0.01).

38 The per cent recovery of parental phenotypes for tested traits in transgenic plants carrying different cry genes

xiii

LIST OF FIGURES

Figure No.

Title

1a Construct map of the binary vector pBinBt3

1b Construct map of the binary vector pBinAR

1c Construct map of the binary vector pBinAR

1d Construct map of the binary vector pMKK1708. T-DNA carrying cry1Acm gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator

2 Response pattern of explants cultured on medium augmented with different cytokinins for multiple shoot induction in ICPL 87119 and BSMR 736 pigeonpea genotypes

3 Multiple shoot bud induced per explant cultured on medium supplemented with different cytokinins in ICPL 87119 and BSMR 736 pigeonpea genotypes

4 Effect of MS nutrient media strength and different IBA concentration regimes on rooting

5 Effect of different treatments on in planta transformation in pigeonpea.

6 Frequency distribution analyses of transgenic plants

7 Correlation analysis between insect mortality, Cry1Ac protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation

8. Correlation analysis between insect mortality, Cry1Ac protein and transcript levels in leaf tissues of developed transgenic pigeonpea plants in T3 generation

9. Frequency distribution analyses of transgenic plants.

10 Correlation analysis between insect mortality, Cry2Aa protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation.

11 Correlation analysis between insect mortality, Cry2Aa protein and transcript levels in leaf tissues of developed transgenic pigeonpea plants in T3 generation.

xiv


13 Correlation analysis between insect mortality, Cry1F protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation


15 Correlation analysis between insect mortality, Cry1Acm protein and gene segregation pattern in developed transgenic pigeonpea plants in T2 generation.

16 The per cent deviation observed between transgenic plants and parental genotypes for selected morphological traits

xv

LIST OF PLATES

Plate No.

Title

1. Direct multiple shoot bud induction and plantlet regeneration from cotyledonary node with cotyledons, embryo discs with half cotyledon explants of pigeonpea

2 Establishment of primary and putative transformants carrying cry1Ac gene in transgenic containment facility

3 Gene specific PCR assay carried out for identification putative of transformants carrying cry1Ac gene and transgene segregation analysis in T2 and T3 generations.

4 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants.

5 Estimation of Cry1Ac protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay.

6 Absolut real-time quantification of cry1Ac gene transcript in transgenic pigeonpea plants

7 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 600bp cry1Ac gene fragment

8 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 600bp cry1Ac gene fragment

9 TAIL-PCR assays (tertiary reaction) conducted in T3 generation plants of AC20-2, AC20-3 and 29-1 transgenic lines carrying cry1Ac gene

10 Establishment of primary and putative transformants carrying cry2Aa gene in transgenic containment facility

11 Gene specific PCR assay carried out for identification putative transformants carrying cry2Aa gene and transgene segregation analysis in T2 and T3 generations

12 Comparative feeding pattern of Helicoverpa armigera larvae on leaf, flower and pod tissues of transgenic and non-transgenic plants

13 Estimation of Cry2Aa protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay

xvi

14 Absolut real-time quantification of cry2Aa gene transcript in developed transgenic pigeonpea plants.

15 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 676bp cry2Aa gene fragment.

16 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 676bp cry2Aa gene fragment

17 TAIL-PCR assays (tertiary reaction) conducted for T3 generation plants of 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68 transgenic lines carrying cry2Aa gene.

18 Establishment of primary and putative transformants carrying cry1F gene in transgenic containment facility

19 Gene specific PCR assay carried out for identification putative transformants carrying cry1F gene and transgene segregation analysis in T2 generation


21 Estimation of Cry1F protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay.

22 Absolut real time quantification of cry1F gene transcript in developed transgenic pigeonpea plants

23 Genomic Southern analysis: 15 µg of genomic DNA was digested with EcoRI and probed with Dig labelled 600bp cry1F gene fragment

24 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 600bp cry1F gene fragment

25 Establishment of primary and putative transformants carrying cry1Acm gene in transgenic containment facility

26 Gene specific PCR assay carried out for identification putative transformants carrying cry1Acm gene and transgene segregation analysis in T2 generations


28 Estimation of Cry1Acm protein in different test tissues of developed transgenic pigeonpea plants using ELISA assay

xvii

29 Absolut real-time quantification of cry1Acm gene transcript in developed transgenic pigeonpea plants

30 Genomic Southern analysis: 15 µg of genomic DNA was digested with HindIII and probed with Dig labelled 452 bp cry1Acm gene fragment

31 Northern blot analysis: 10 µg of total RNA was probed with Dig labelled 452 bp cry1Ac gene fragment

32 The comparison of plant growth parameters of developed transgenic plants and non-transgenic control plants.

xviii

LIST OF APPENDICES

Appendix No.

Title

I Appendix I

II Appendix II

III Appendix III

IV Appendix IV

V Appendix V

VI Appendix VI

1. INTRODUCTION

Pigeonpea [Cajanus cajan (L.) Millsp.] belongs to family Fabaceae, is one of the

major pulse crops grown in many countries of Asia, Africa and America. India ranks first

in the world accounting for 3.53 m. ha area with 2.51 million tons of pigeonpea

production (http://faostat3.fao.org). Pigeonpea is consumed as green peas, whole grain

or split peas and is a major source of protein (20 - 22%) to human population

particularly in developing countries (Singh and Eggum, 1984; Varshney et al., 2010).

The production and productivity of pigeonpea is threatened by many biotic and abiotic

stresses. The most important yield constraint in pigeonpea is from the polyphagous

lepidopteran pest, Helicoverpa armigera Hübner (Sharma et al., 2006). In India,

pigeonpea is prone to more than 200 species of insects, among which the gram pod

borer, H. armigera causes enormous losses (46.6 - 63.6%) which persists throughout

the year on one or other crops. In recent years other insect pests such as Maruca

(Maruca vitrata Fabricius), pod fly (Melanagromyza obtuse Malloch) and plume moth

(Exelastis atomosa Walsingham) are becoming economically important inflicting

significant yield losses (Choudhary et al., 2013).

An indiscriminate use of synthetic chemical insecticides has resulted in the

contamination of water and food sources, poisoning of non-target beneficial insects

leading to many serious environmental concerns (Kumar et al., 2008). Increased

understanding about the adverse environmental effects of indiscriminate use of

insecticides has promoted search of altermnative methods form pest control. In nature,

wild species of pigeonpea are rich source of resistance genes against many biotic and

abiotic stresses (Lal and Rathore, 2001). Poor crossing ability of cultivated pigeonpea to

species other than the closest ones, such as Cajanus cajanifolia and C. scaraboides

has limited the use of inter specific crosses in pigeonpea improvement (Varshney et al.,

2010). Conventional breeding approaches for pigeonpea improvement have been in use

for several decades, but have had limited success in overcoming different biotic and

abiotic challenges to sustainable crop production (Varshney et al., 2007; Saxena, 2008).

Hence, insect resistant transgenics expected to be an ideal solution in the interest of the

pigeonpea farmers and the productivity.

2

It has been reported that the global farm income gain due to use of genetically

modified insect resistance technology in maize and cotton have been $6.71 billion and

$5.3 billion respectively during 2012. The genetic engineering technology has been

adopted on large scale i.e. by 17.3 million farmers in 2012, demonstrating the important

economic benefits of this technology (Brookes and Barfoot, 2014). Cumulatively since

1996, the global farm income gain has been $32.3 billion for genetically modified insect

resistant maize and $36.3 billion for that of cotton. Considering the current statistics and

economic benefits from use of genetically modified insect resistant cultivars clearly

points at immense scope for use of genetic engineering technology in crop improvement

programme. Lawrence and Koundal (2001) have successfully transferred the cowpea

protease inhibitor gene (pCPI) in pigeonpea for biotic stress tolerance. Attempts have

been made to develop Spodoptera litura resistant pigeonpea by expressing synthetic

cryIE-C gene using transgenic technology (Surekha et al., 2005). Recently, Ramu et al.

(2012) have reported significant reduction in H. armigera feeding on genetically

modified pigeonpea lines expressing chimeric cry1AcF.

More than 500 different cry genes have been known and classified into 67

groups based on their primary amino acid sequence (Crickmore et al., 2010). Broadly

these cry genes have been divided in to four protein families such as, the family of three

domain Cry toxins (3D), mosquitocidal Cry toxins (Mtx), the binary-like (Bin) and the Cyt

family of toxins (Bravo et al., 2005). The Cry1Ac belong to 3D-Cry group proteins and

commercial Bt crops have been developed for expression of cry1Ac gene itself for the

control of many lepidopteran pests (Christou et al., 2006). Further, a second generation

Bt-cotton has been developed for Cry2Ab besides Cry1Ac as a resistance management

strategy. Bt-corn expressing Cry1Ac has been tested for their effectivity against

lepidopteran pests (Christou et al., 2006). Like any other Cry proteins, the mode of

action of Cry1F also involves the enzymatic cleavage of the protoxin to form core toxin

(Gao et al., 2006). Many studies have determined the activity of Cry1F against

lepidopteran species (Balog et al., 2011; Oppert et al., 2010). The native cry1Ac gene

sequence from Bacillus thuringiensis have been in silico modified and artificially

synthesized to overcome codon bias and other undesirable regulatory coding

sequences for its improved expression in transgenic plants. The study resulted in the

development of modified cry1Acm with increased GC content (Mohan, 2008).

3

There are very few reports on in vitro regeneration of pigeonpea through

organogenesis from unorganised callus (Kumar et al., 1983). Different explants such as,

cotyledons, embryonic axes, cotyledonary node from mature seeds and seedling

petioles have been studied for the multiple shoot production and plantlet regeneration

through direct organogenesis (Franklin et al., 1998; Srinivasan et al., 2004). The effect

of cytokinins such as, benzylamino purine (BAP) and furfurylamino purine (kinetin) and

their varied levels of concentrations on multiple shoot induction have been studied in

pigeonpea (Geetha et al., 1998). Similarly, Kashyap et al. (2011) has analysed the

frequency of multiple shoot bud induction using different cytokinins viz., BAP, kinetin

and TDZ in eleven Indian cultivars of pigeonpea. The type of explant, genotype and

concentrations of cytokinin can influence the frequency of shoot bud regeneration

(Geetha et al., 1998; Kashyap et al., 2011). A variable frequency (20-60 per cent) of

palntlet regeneration has been reported in pigeonpea (Srivastava et al., 2013).

However, there is a need to fine-tune the in vitro regeneration protocols to achieve high

frequency regeneration of plantlets.

The poor in vitro regeneration and low transformation frequency are the major

constrains for development of transgenics in pigeonpea. Efforts have been made for the

development of efficient protocols of Agrobacterium tumefaciens mediated and

microprojectile bombardment-based genetic transformations of pigeonpea (Rao et al.,

2008). Further, the in vitro regeneration conditions so far described are available for

only a few of the many cultivars/genotypes of pigeonpea (Rao et al., 2008). The in

planta plant transformation method has been developed and successfully applied in

wide range of crop plants such as, mulberry, soybean, rice, cotton, fieldbean, sunflower,

groundnut and safflower (Ping et al., 2003; Supartana et al., 2005; Keshamma et al.,

2008; Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b; 2001). In recent

efforts in planta transformation method has also been used for development of

transgenic pigeonpea for expression of a chimeric cry1AcF gene (Ramu et al., 2012).

Similarly, in case of alfalfa, this method has been employed for the development of

marker free transgenic plants (Weeks et al., 2008). Hence, the use of in planta methods

expected to be an important component for future transgenic development programs in

crops like pigeonpea.

4

Different characterization methods viz, insect bioassay, protein quantitation using

ELISA, mRNA quantitation using qRT-PCR assay, Sothern, western and northern blot,

and TAIL-PCR assay for juncture region analysis have been employed for transgenic

plants in many studies (Sharma et al., 2006; Ramu et al., 2012). Bhat and Srinivasan

(2002) has discussed genetic analysis of transgenic plants and indicated that handling

of primary transgenics to obtain progeny generation needs few careful considerations

such as, the breeding behaviour of the plant species, the performance of parent and

progeny etc. The selfing of the T0 plants helps to achieve homozygosity and resultant T1

progenies will have complex mixture of genotypes and their composition depends on

the genetic constitution of the T0 parents. Further, they have said that in case of multiple

copy integration of transgene, determination of exact genotypic constitution of individual

plants might be difficult due to difficulties in distinguishing hemizygotes from

homozygotes by Southern analysis (Bhat and Srinivasan, 2002).

Southern blotting methods have been assisted with gel electrophoresis. Southern

blotting involves a method for separating genomic DNA with exceptional resolving

power (Southern, 2006). Blotting enables the detection of specific molecules among the

mixture separated in the gel. DNA specific sequences are detected in the membrane by

molecular hybridization with labeled nucleic acid probes (Southern, 2006). The method

has been used in several key studies (Kan and Dozy, 1978; Jeffreys and Flavell, 1977).

The original proposal for the genetic mapping of the human genome has been based on

restriction fragment length polymorphisms (RFLPs) detected by blotting (Kan and Dozy,

1978). Introns have been first seen in blots of rabbit genomic DNA hybridized with

probes for the ß-globin gene (Jeffreys and Flavell, 1977). The first DNA fingerprints

have been produced by hybridizing restriction digests of human DNA with minisatellite

probes (Jeffreys et al., 1985). Acharjee et al. (2010) have used Southern blot technique

and showed the integration of cry2Aa gene in eight of the nine transgenic chickpea

lines, with integration of one, two or more copies of the transgene. The southern blot

technique has been used in many transgenic studies to check the presence and copy

number of transgene in transgenic plants (Mehrotra et al., 2011; Rao et al., 2008; Ramu

et al., 2012).

5

Bioassays have been used test the functionality of the transgene product (Bhat

and Srinivasan, 2002). The detached leaf bioassay tests have been undertaken for

transgenic pigeonpea plants against the pest S. litura, wherein study reported 80 per

cent insect mortality in developed transgenic plants (Surekha et al., 2005). Similarly,

many independent studies have performed bioefficacy tests against H. armigera

(Gopalaswamy et al., 2007; Ramu et al., 2012; Acharjee et al., 2010). The transgenic

plants of pigeonpea and chickpea, expressing different insecticidal cry genes have been

analysed for their bioefficacy levels against targeted insects by following bioassay

technique (Sanyal et al., 2005; Gopalaswamy et al., 2007; Ramu et al., 2012; Acharjee

et al., 2010).

An immunoassay technique based on antibodies has been a standard approach

for qualitative and quantitative detection of protein of a known target analyte (Brett et al.,

1999). Both monoclonal and polyclonal antibodies have been used depending on the

specificity of the detection system (Kamle and Ali, 2013). Sharma et al. (2006), using

this technique, have determined the synthetic Cry1Ab protein in developed transgenic

pigeonpea plants. Many parallel studies have reported the detection and estimation of

target protein in test samples using immunoassay technique (Gopalaswamy et al.,

2007; Ramu et al., 2012).

Another characterization method for genetically modified plants involves

transcript analysis of transgene using RT-PCR and northern blot techniques (Bhat and

Srinivasan, 2002). These methods have been used successfully in transgenic studies

for quantification of targeted cDNA molecules (Kamle and Ali, 2013). The real time PCR

has also been used for validating and estimating the number of copies of transgene into

the host genome in several genetically modified crop plants such as maize, cassava,

rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al., 2013; Beltrán et

al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007).

Considaring the severity of pod borer, H. armigera in pigeonpea, the Bt

transgenic approach has been felt essential. Hence, the present study on development

of Bt transgenic pigeonpea has been undertaken through all necessary precise

biotechnological and conventional tools with following objectives;

6

1. To improvise the in vitro plantlet regeneration and in planta transformation protocols

in pigeonpea

2. To develop genetically transformed pigeonpea lines using cry1Ac, cry2Aa, cry1F

and cry1Acm genes separately

3. To assess the bioefficacy and molecular characterization of selected putative

transgenic pigeonpea lines

2. REVIEW OF LITERATURE

Pigeonpea [Cajanus cajan (L.) Millsp.] (Family: Fabaceae) also known as red

gram, is one of the major grain legume (pulse) crops grown in the semi-arid tropics. It is

the second most important food legume in India, where more than 80% of the world

production occurs (FAO, 2008). It is cultivated in about 50 countries of Asia, Africa and

the America for a variety of uses like food, fodder, fuel wood, rearing lac insects,

hedges, wind breaks, soil conservation, green manure and roofing. It is often grown on

marginal soils and intercropped with cereals.

Pigeonpea is a major source of protein (21%) to human population mainly in

developing countries and several Latin American countries (Singh and Eggum, 1984).

Its production and productivity are constrained by several biotic and abiotic stresses,

whose levels of resistance in world germplasm accessions are low to moderate.

Breeding incompatibility problems associated with wild species warrant the exploration

of alternative approaches. Genetic engineering technology plays a significant role as an

additional tool for the introduction of agronomically useful traits in a high yield

background. The most important yield constraint in pigeonpea is from the lepidopteran

pest, Helicoverpa armigera. Insect resistant transgenics would be an ideal solution in

the interest of the pigeonpea farmers and the productivity. Among the many potential

genes, cry genes are generally effective against Lepidoptera, and are likely to be

species specific. Hence, genetic transformation of pigeonpea holds greater promise to

manage pod borer pest. A brief review on various aspects of present study is presented

below.

2.1 Pigeonpea: World’s important pulse crop

Pigeonpea is an important food legume crop, cultivation predominantly takes

place in tropical and subtropical regions of the world. It is a diploid crop with

2n = 22 and a genome size of 808 Mbp. Pigeonpea is a naturally drought tolerant crop

with large variation for days to maturity that ranges from 90 to 300 days duration.

Pigeonpea is cultivated as a sole crop as well as a mixed crop with cereals and

legumes. Globally pigeonpea is cultivated on 4.64 Mha, with an annual production of

3.43 million tons and productivity of 780 kg/ha. In pigeonpea growing countries, India

8

ranks first in the world accounting for 3.53 Mha area with 2.51 million tons of production

(Varshney et al., 2010). Pigeonpea consumed as green peas, whole grain or split peas

and contains 20-22 per cent protein. The seed, pod husks, dry branches and stems are

also used as a quality feed and also serve as domestic fuel. In field, pigeonpea plants

harbours nitrogen fixing bacteria, enriching soil fertility by symbiotic nitrogen fixation.

India alone occupies three-fourth of global harvested area and contributes almost

a similar share in production. After chickpea, pigeonpea is the second most important

pulse crop in India. Pigeonpea is mainly grown in Maharashtra, Karnataka and Andhra

Pradesh states (>60% area in India) with 60 per cent of production (1.4 million tons)

from these three states. Although there is increase in demand for pigeonpea, in last five

years, the area and production of pigeonpea has remained constant over a time period.

Despite its importance in subsistence and sustainable agriculture, the average global

productivity of pigeonpea has remained static over the last three decades. The yield gap

observed between the potential yield and on-farm yield is mainly due to prevalence of

various abiotic (Choudhary et al., 2011) and biotic factors together with the cultivation of

pigeonpea in marginal lands with low input supply and lack of efficient management

practices (Varshney et al., 2012). Wide yearly fluctuations have been observed in total

production mainly due to attack by insect pests including disease-causing pathogens.

2.2 Major threats to pigeonpea production

Pigeonpea plant growth and reproductive stage both are influenced by

environmental conditions. It has made crop breeding more complex in pigeonpea. In

nature, wild species of pigeonpea are rich source of resistance genes against many

biotic and abiotic stresses, the genes responsible for yield components such as, pods

per plant, length of fruiting branches, and number of primary branches per plant etc.

Due to the poor crossing ability of cultivated pigeonpea to species other than the closest

species, such as Cajanus cajanifolia and C. scaraboides has limited the use of inter

specifics in pigeonpea improvement. Use of biotechnological approaches viz., in vitro

rescue and propagation of wide cross hybrids, in conjunction with the use of bridge

crosses, has enabled the transfer of novel genes from a wider range of germplasm

within and outside the genus Cajanus (Varshney et al., 2010).

9

Although, pigeonpea is important crop in semi-arid regions of the world, less

concentration has been directed towards pigeonpea crop improvement. The important

factors responsible for poor productivity in pigeonpea are lack of improved cultivars,

poor crop husbandry, pests and diseases. Major disease and pests that have

threatened pigeonpea cultivation are fusarium wilt (Fusarium udum Butler), sterility

mosaic disease (Sterility mosaic virus) and phytophthora blight (Phytophthora

drechsleri), gram pod borer (Helicoverpa armigera), Maruca (Maruca vitrata), pod fly

(Melanagromyza obtusa), plume moth (Exelastis atomosa) and mite (Aceria cajani).

Besides, abiotic stresses such as water-logging, common in rain fed crop during early

stages, and stress from low water conditions in the later stages, and salinity has also

contributed significantly to reduce pigeonpea production (Varshney et al., 2010).

2.2.1 Major disease in pigeonpea

Among diseases, fusarium wilt (Fusarium udum) in Central and Southern states

followed by sterility mosaic (virus transmitted by eriophyid mite Aceria cajani), and SMD

and Phytophthora blight (Phytophthora drechsleri f.sp.cajani) diseases in the northeast

plain (Uttar Pradesh) cause substantial yield losses to the crop. Unchecked weeds also

cause 21-97% yield loss in pigeonpea. Fusarium wilt is one of the major diseases in

pigeonpea caused by Fusarium udum f. sp. cajani. This disease is characterized by

yellowing and slow withering of plant. The infected plants show symptoms of yellowing

followed by dropping and finally the whole plant dries up. Level of incidence is

significantly influenced by pathogen surviving saprophytically on the crop residue.

Phytopthora organisms are commonly referred to as water molds. The disease is

caused by the fungus P. drechsleri var. cajani and the disease is commonly known as

stem rot of pigeonpea. Pigeonpea seedlings are highly prone to infection and can

become infected as soon as they emerge (Sharma et al., 2010).

Bacterial Leaf Spot and Stem canker were first reported from India in 1950.

There is no specific period and the disease can occur anytime in the crop. Symptoms

on leaves are characterized by the appearance of minute, brown lesions surrounded by

a yellow halo. These lesions often coalesce and form larger ones. On the main stem

and branches, rough, cankerous dark brown lesions of various shapes and size appear.

The disease is caused by bacteria of Xanthomonas type belonging to the family of plant

10

pathogenic yellow pigmented pseudomonades. It is a species of Gram-negative rod-

shape flagellated bacteria known for being a common plant pathogen. Xanthomonas is

dependent on the type III protein secretion system, which relies on transport proteins, it

secrets hypersensitive and degradable exo-polysaccarides (Xanthan), causing an

interaction with the plant. X. campestris pv cajani is specific to pigeonpea and is seed

borne in nature (Sharma et al., 2010).

Sterility mosaic disease (SMD) was first described in 1931 from Pusa (Bihar) and

subsequently from rest of India and other countries. Patches of bushy pale and green

plants with smaller leaves, without flowers or pods are the common field symptoms and

is a serious disease in India. The disease is of a major concern in UP, Bihar, Gujarat,

Tamil Nadu and Karnataka. The disease is transmitted by mites (Aceria cajani).

Infection at an early stage (<45-day-old plants) results in a 95 to 100% loss in yield

while losses from late infection (>45-day-old plants) depend on the level of infection (i.e.

number of affected branches per plant) and range from 26 to 97% (Kannaiyan et al.,

1984).

Cercospora leaf spots is caused by a fungus Cercospora indica, causing several

types of spots on the leaves and petioles of affected plants. Initially small, circular,

necrotic spots forming typical concentric rings appear on the leaves during Alternaria

blight/leaf spot disease (Sharma et al., 2010). Collar rot /soft rot (Sclerotium rolfsii

Saccardo) disease appears within a month of sowing and is usually scattered over the

field. Seedlings turn slightly chlorotic before drying. Rotting in the collar region is often

covered with white mycelial growth accompanied with small mustard like bodies

(Sharma et al., 2010). Characteristic symptoms of Foliar Rust include dark brown raised

pustules (full of uredia) on the lower leaf surfaces. When the disease is severe, it

causes extensive defoliation. The disease is known to occur in Uttar Pradesh,

Maharashtra and Tamil Nadu (Gahukar and Peshney, 1985; Kannaiyan et al., 1984).

The powdery mildew symptoms appear mostly on older leaves, however, in severe

cases even young buds and pods also get infected (Sharma et al., 2010).

2.2.2 Major insect pests in pigeonpea

More than 200 species of insects have been identified, which feed on pigeonpea

from germination stage till harvest. Many of the insects attacking the crop are not

11

actually responsible for significant loss in production (Rangarao and Shanower, 1999).

Among the variety of insects feeding on pigeonpea, the pod borer (Helicoverpa

armigera Hubner) is the most damaging pest worldwide. Its frequent occurrence often

results in complete crop failure and cause losses of more than US $300 million annually

(Choudhary et al., 2013). The wide host range, high degree of migration, indiscriminate

pesticide application by farmers and innate ability of the insect to quickly develop

resistance to applied insecticides have made it attain the status of the key pest

(Vishwadhar et al., 2008). The pod fly infestation causes 2.5 to 86.8 per cent of grain

losses in different parts of the country. On other hand, the estimates of losses due to

pod borer have been recorded to be 30.2 per cent.

Nearly 30 species of Lepidoptera have been reported to feed on the reproductive

structures of pigeonpea. Most of these insects occur at low densities and are only

occasional pests or are of local importance. There are two most important species, viz.,

Helicoverpa armigera (Noctuidae) and Maruca vitrata (Dtestulalis) (Pyralidae). H.

armigera is the major biotic constraint to pigeonpea production. It is the key pest due to

the larval preference for feeding on nitrogen rich plant parts such as reproductive

structures and growing tips (Fitt, 1989). These plant parts are also the most suitable for

larval development. Besides its preference for feeding on reproductive structures, the

four important features of H. armigera life history that made it one of the most serious

and widespread insect pests are high fecundity, extensive polyphagy, strong flying

ability and a facultative diapause (Fitt, 1989). Other important pests of pigeonpea, M.

vitrata, is distributed throughout tropical and subtropical regions world-wide and has a

wide host range but is restricted to legumes (Atachi and Djihou, 1994). The M. vitrata

larvae are known to feed from inside a webbed mass of leaves, flowers and pods, and

are a serious pest of pigeonpea in India, Sri Lanka and Africa. Maruca is an important

pest of pigeonpea in early stage of growth and cause yield losses ranged up to 100 per

cent (Choudhary et al., 2013).

Another group of insects that belongs to Hemiptera involves mainly pests from

the families Alydidae, Coreidae, and Pentatomidae. They feed on pigeonpea and are

commonly referred to as pod-sucking bugs. The most important pests from this group

are the coreids Anoplecnemis spp., Clavigralla spp. (Acanthomia spp.) and Riptortus

spp. Both, adults and nymphs of Clavigralla spp. are known to feed on pigeonpea by

12

piercing the pod wall and extracting nutrients from the developing seeds (Bindra, 1965).

Damaged seeds appear dark and shriveled, and they are difficult to distinguish from

those damaged by drought.

Two Dipteran and one Hymenopteran insects feed on developing seeds of

pigeonpea within the pod. Among them the most important is Melanagromyza obtuse

Malloch (Diptera: Agromyzidae), the pigeonpea pod fly, which is observed mainly in

Asia. Pod fly damage has been reported from several countries. In India, the pod fly is a

more serious pest in northern and central areas than in other parts of the country

(Lateef and Reed, 1983). Damage levels in farmers’ fields range from 10 to 50 per cent

(Lal et al., 1992).

2.3 Crop improvement strategies for insect pest resistance in pigeonpea

The breeding strategies and methodologies for crop improvement have been

developed after understanding the important role of genetic inheritance of yield and

related traits. Relatively less effort has been made to understand the genetics of

important traits in pigeonpea, when compared with other economically important crops.

Saxena and Sharma (1990) have reported the importance of both additive effects and

dominant non-additive effects in determining yield, plant height and protein content. The

features like pleiotropic effects of genes, physiological changes and highly sensitive

nature of pigeonpea towards the environmental changes has made the interpretation of

yield and associated characters in pigeonpea difficult (Byth et al., 1981). Other than

yield related parameters, restoration of male fertility in cytoplasmic-genetic male-sterility

(CGMS) based hybrids has been critical and important trait in pigeonpea. Conventional

breeding approaches for pigeonpea improvement have been in use for several decades

but have had limited success in overcoming different biotic and abiotic challenges to

sustainable crop production (Varshney et al., 2007; Saxena, 2008).

2.3.1 Conventional breeding

Identification and utilization of resistant cultivars against these insects expected

to provide an environment friendly solution and sustainable management of these insect

pests. Many efforts have been made for the identification of sources of resistance to

various insects in pigeonpea by screening the pigeonpea genotypes for insect

13

resistance. The large variation in maturity period has been reported as a cause for

differential infestation specificity by insects. Wherein, the extra short-duration varieties,

harvested during October-November, and the medium-duration genotypes, harvested in

December-January, are more severely damaged (90 per cent) by pod borer. On other

hand, the long-duration genotypes maturing from February onwards shows lower pod

borer damage. Determinate types have heavier damage than the indeterminate types

(Choudhary et al., 2013). The pigeonpea genotype, UPAS 120, is fairly tolerant,

whereas ICPL 151 is highly susceptible. Lateef (1992) screened more than 14,000

pigeonpea germplasm accessions for identification of the sources of resistance to pod

borer. The study resulted in identification of only a few accessions with low levels of

resistance, wherein among the cultivated pigeonpea genotypes, ICPL 332, the resistant

check, was consistently less damaged than the susceptible check ICPL 87 (Lateef,

1992). The pigeonpea varieties ICPL87119 has been derived from a cross between C11

and a breeding line, and is medium duration variety with resistance to Fusarium wilt and

pigeonpea sterility mosaic virus (Dharmaraj and Lohithaswa, 2004).

Studies have reported that early maturing genotypes are more susceptible to pod

fly damage when compared with late maturing ones (Lal et al., 1998), and the

determinate types are less susceptible than the indeterminate types (Gupta et al.,

1991). Similarly, it has been revealed that long-duration varieties show heavier damage

than the early maturing types. More than 10,000 germplasm accessions of pigeonpea

have been screened for pod fly resistance (Lateef and Pimbert, 1990). Many

independent studies have identified the genotypes with a high degree of resistance

such as PDA 88-2E, PDA 92-1E, PDA 89-2E, PDA 92-2E, PDA 92-3E and PDA 93-1E

(Durairaj and Ganapathy, 1997; Srivastava et al., 1994; Lal and Rathore, 2001).

Although these lines can be used as the sources of resistances in the breeding

programmes, the field observations have indicated that these lines have small seed

size. Moudgal et al. (2008) have studied pigeonpea genotypes for physico-chemical pod

traits imparting resistance to pod fly under field conditions. The study reported that the

non-determinate genotype GP 75, extra early maturing, and GP 118, early maturing,

and determinate genotype GP 233, extra early maturing and GP 253, early maturing

genotypes recorded significantly lower pod and seed damage as compared to early

maturing checks Prabhat, determinate and Manak, non-determinate. The study also

14

indicated that resistance to pod fly has not been linked to plant growth habit and

maturity period of pigeonpea genotypes.

Furthermore, wild relatives of pigeonpea have been identified as useful sources

of resistance to pod borer (Romeis et al., 1999). Many studies have reported different

wild species especially C. scarabaeoides and C. platycarpus as the potential sources of

resistance to pod borer (Shanower et al., 1998; Mallikarjuna et al., 2011). Sharma et al.

(2009) have reported significant variation in egg laying, numbers of larvae and pod

damage among the wild relatives of pigeonpea under field conditions.

MacFoy et al. (1983) have recorded high concentrations of sugars and amino

acids in the cowpea cultivar Vita-1, which is susceptible to spotted pod borer/Maruca.

Similarly, in another stream of efforts, low amounts of polyphenols have been reported

to be associated with their high susceptibility to pod borer in the cultivated pigeonpea

(Sharma et al., 2009). It has been studied that the low amounts of phenols in pigeonpea

flowers are associated with susceptibility to spotted pod borer, M. testulalis. High

amount of soluble protein content have been reported in the pods of C. Scarabaeoides

when compared with those of ICPL 87, a cultivated variety of pigeonpea. High amounts

of polyphenols have been revealed in the resistant wild species as compared to the

cultivated pigeonpea (Sharma et al., 2009). Smith (1989) documented that the presence

of condensed tannins in plants act as insect growth inhibitors. The pod wall biochemical

traits have been studied for their role in pigeonpea resistance to insects (Moudgal et al.,

2008). Many studies have demonstrated that the pigeonpea pod walls with more wax,

total phenols and less reducing and non-reducing sugars and total amino acids suffers

less pod and grain damage by pod fly.

2.3.2 Molecular breeding

Studies have identified wild relatives of pigeonpea like C. scarabaeoides,

C. sericeus, C. acutifolius, C. albicans and Flemingia bracteata that possesses high

degree of resistance to pod borer, pod fly and pod wasp (Sharma et al., 2003;

Choudhary et al., 2013). Mallikarjuna et al. (2007) have developed the advanced

generation population from a cross utilizing C. acutifolius as the pollen parent and

reported introduction of resistance trait in progenies. Study also reported that some

15

lines having high level of resistance to pod borers, pod fly and bruchids under un-

protected field conditions (Mallikarjuna et al. 2007).

Among available 21 wild species in the tertiary gene pool of pigeonpea,

C. platycarpus has been used for improving insect resistance through inter-specific

hybridization (Mallikarjuna et al., 2006). Sujana et al. (2008) has given the important

trait features of C. platycarpus as extra-early flowering and maturity, photoperiod

insensitivity, prolific flowering and podding, high harvest index, annuality and rapid

seedling growth including resistances to biotic stresses such as pod borer. These

characteristic features have become a major attraction for pigeonpea breeders.

Mallikarjuna et al. (2011) developed back cross progenies and reported a range of

morphological and resistance traits in the progeny lines for pod borer, bruchid, and pod

fly resistance. The study quantified that the presence of C. platycarpus genome in

advance generation progeny lines using Diversity Array Technology (DArT) markers.

The progeny screening for resistance to pod borer, pod fly and bruchids under

unprotected field conditions revealed observation of the damage ranged from 6.85 to

22.84 per cent in BC44F-A derivatives. Majority of the lines indicated less than 15 per

cent damage (Mallikarjuna et al., 2011).

Progeny lines derived from C. platycarpus have shown 0 to 7.44 per cent pod

borer damage when compared with compared with 14.5 per cent damage recorded in

the control lines. Study also reported that some of the lines showed less to no bruchid

damage with significant lower damage by pod borer. Three accessions of C.

platycarpus have been screened and identified for bruchid resistance (Mallikarjuna et

al., 2011). The studies also reported 82 to 91 per cent reduction in eggs hatch. The

minimum seed damage have been recorded on C. platycarpus accession ICPW 66

(14%), while the damage was moderate to medium in other C. platycarpus derivatives

compared with the susceptible check ICPL 85010 (75% damage). In advance

generation interspecific derivative lines, there have been 32.78 to 92 per cent eggs that

failed to hatch. Study also has reported low to moderate resistance for the pod fly in the

BC4F-A derivatives, which ranged between 4 to 22 per cent, with a single derivative

BC4F11-A, indicated a low damage of 3.73 per cent (Mallikarjuna et al., 2011).

16

Substantial progress has been made towards development of large-scale

genomic resources in pigeonpea especially during the last decade. These efforts have

resulted in the development of large-scale molecular markers, construction of

comprehensive genetic maps, identification of various marker-trait associations and

initiation of molecular breeding in this crop. The large numbers of SSR markers (Raju et

al., 2010), DArT markers (Mallikarjuna et al., 2011), SNPs (Dubey et al., 2011) and

ESTs (Raju et al., 2010) hold high promise for improvement of a number of economic

traits in pigeonpea. More than 3000 SSR markers have been developed that are useful

in development of inter as well as intra specific genetic maps using several F mapping

populations (Bohra et al., 2012). This represented the first instance of merging multiple

genetic maps in pigeonpea. Preliminary mapping efforts for trait mapping in pigeonpea

have resulted in mapping of Fusarium wilt resistance (Kotresh et al., 2006), SMD

(Ganapathy et al., 2010).

It has been reported that the presence of genetic variability for resistance to

insects particularly pod borer is not available in the primary gene pool of pigeonpea

(Choudhary et al., 2013). And hence, breeders need to opt wild relatives such as,

C. scarabaeoides, C. platycarpus that are considered reservoirs of superior alleles for

traits imparting resistance to insects that must have lost during domestication and

breeding. On other hand, the transfer of desirable alleles is not so simple because of

difficulty in efficient tracking for desired and non-desired alleles in breeding lines. This

problem can be overcome by advance backcross QTL based breeding. It is most

suitable for introducing novel alleles from wild relatives to the cultivated species

cultivars or varieties in a controlled manner (Tanksley and Nelson, 1996).

2.3.3 Genetic engineering

Few lines of pigeonpea have been identified for resistance to pod borer and pod

fly. Their resistance has been only partial and lines with absolute resistance are not

available in the cultivated germplasm (Grover and Pental, 2003). Attempts have been

made to produce insect resistant genotypes of pigeonpea using conventional breeding

methods with very less success. The reasons for limited success are limited resistance

sources in the crossable germplasm and incompatibility with wild species (Choudhary et

al., 2013). The advent of transgenic technology has provided the best alternative to

17

improve resistance to insect pests. A well defined reproducible regeneration and

transformation protocol have been developed for development of transgenics in

pigeonpea (Rao et al., 2008).

During 2001, transformation technique using Agrobacterium tumefaciens have

been used in pigeonpea (Cajanus cajan L. Millsp.) for expression of cowpea protease

inhibitor gene (pCPI), wherein pigeonpea embryonic axes excised from germinated

seeds on MS basal supplemented with BAP (2 mg/L) used as explants for

transformation (Lawrence and Koundal, 2001). Using embryonal segments as explants

the effective transformation and quick regeneration of transgenic plants of pigeonpea

has been documented by the expression of synthetic cryIE-C gene under a constitutive

35S promoter (Surekha et al., 2005). In their study selection of transformed shoots has

been performed on MS medium supplemented with 2.0 mg/L BAP, 250 mg/L cefotaxime

and 75 mg/L kanamycin and rooted on MS medium supplemented with 1.0 mg/L NAA.

In vitro insect bio-efficacy analysis of developed transgenic pigeonpea plants

expressing cryIE-C reported resistance against Spodoptera litura larvae.

Sharma et al. (2006) have reported novel tissue culture method involving the

direct regeneration of adventitious shoot buds from axillary bud region of in vitro

germinating seedlings through suppression of axillary and primary shoot buds on a

medium containing a high concentration of N 6 -benzyladenine (22.0 mM). Method has

been used for successful transfer and expression of synthetic cry1Ab in pigeonpea with

varied range of expression of gene in different tissues of the whole plant, showing the

highest expression in flowers (0.1 per cent of total soluble protein) and least in the

leaves (0.025 per cent of total soluble protein) (Sharma et al., 2006). It is also important

to develop efficient techniques for evaluation of transgenic plants for resistance to H.

armigera. Therefore, Gopalaswamy et al. (2007) evaluated the usefulness of detached

leaf assay to test the bioefficacy of transgenic pigeonpea carrying Bt-cry1Ab and SBTI

genes for resistance against H. armigera. The method could determine the levels of

Cry1Ab or SBTI (soybean trypsin inbhibitor) proteins in the transgenic pigeonpea plants

as not sufficient to cause significant deterrent effects on leaf feeding, larval survival and

larval weight of H. armigera and found to be quite useful for evaluation of transgenic

pigeonpea plants for resistance to H. armigera. Further, the field evaluation of

transgenic pigeonpea plants of ICPL 88039 and ICPL 87 carrying cry1Ab and soybean

18

trypsin inhibitor (SBTI) genes indicated lack of significant reduction in leaf feeding, larval

survival and larval weight indicated necessity to develop plants with high expression

levels of cry1Ab endotoxin or SBTI which would be sufficient enough to supress insect

attack (Gopalaswamy et al., 2007).

The major problems in development of transgenics in pigeonpea are poor in vitro

regeneration and low frequency transformation. Many efforts have been done to

develop efficient protocols of Agrobacterium and microprojectile bombardment based

genetic transformation of this crop and plant regeneration from in vitro cultured explants

via organogenesis as well as by somatic embryogenesis. Rao et al. (2008) have

proposed non-tissue culture-based method for transgenic pigeonpea using

Agrobacterium Ti plasmid mediated transformation system. The method involved raising

of whole plant transformants (T0 generation) directly from Agrobacterium infected young

seedlings. The plumular and intercotyledonary meristems of the seedling axes have

been targeted for transformation by pricking of the apical and inter-cotyledonary region

of the seedling axes of two day old germinating seedlings with a sewing needle and

infecting with Agrobacterium in Winans’ AB medium containing wounded tobacco leaf

extract. Co-cultivation is performed in the same medium for 1hr and seedlings are

transferred to soilrite for further growth and hardening and subsequent transfer of

seedlings to soil in pots in the greenhouse (Rao et al., 2008).

The merits associated with in planta transformation methods includes, ensured

generation of pigeonpea transgenic plants with considerable ease in a short time;

applicable across different genotypes/cultivars of the crop and offers immense potential

as a supplemental or an alternative protocol for generating transgenic plants of difficult-

to-regenerate pigeonpea. Besides, it also offers the option of doing away with a

selection step in the procedure and so facilitates transformation, which is free of marker

genes (Rao et al., 2008). Recently, this protocol has been used to express chimeric

cry1AcF (encoding cry1Ac and cry1F domains) gene in transgenic pigeonpea (Ramu et

al., 2012). Interestingly, chimeric Cry1AcF levels in developed transgenic plants ranged

3-15 µg/g of fresh tissue weight with the insect mortality ranged 0 to 100 per cent

indicating the potential effect of Cry1AcF against H. armigera (Ramu et al., 2012).

19

2.4 Pigeonpea pod borer, Helicoverpa armigera life cycle and damaging stages

The Helicoverpa armigera is also known with different synonyms such as,

Heliothis armigera (Hübner), Chloridea armigera (Hübner) Heliothis obsoleta Auctorum,

Chloridea obsoleta, Helicoverpa obsoleta Auctorum, Heliothis fusca Cockerell, Heliothis

rama Bhattacherjee and Gupta, Noctua armigera Hübner. It belongs to Kingdom

Animalia, Phylum Arthropoda, Class Insecta, Order Lepidoptera, Family Noctuidae and

Genus Helicoverpa. H. armigera completes its life cycles in 4-6 weeks starting from egg

to adult in summer, and 8-12 weeks in spring or autumn. The different stages in

Helicoverpa life cycle are egg, larva, pupa and adult (moth) (Begemann and Schoeman,

1999).

It has been studied that the adult moth wingspan in case of H. armigera is 30-45

mm; the forewings are brownish or reddish-brown (females) or dull greenish to yellow or

light brown (males); hindwings are pale with a broad, dark outer margin. The moths

have a pale patch near the centre of this dark region and feed on nectar (Shanower et

al., 1997). They live for around 10 days and females lay 1000 eggs in their life span.

The eggs are laid in clusters, on leaves, flower buds, flowers and developing fruits

(Begemann and Schoeman, 1999). Fertile eggs of H. armigera hatch in about three

days during warm weather (25 °C average) and 6 to 10 days in cooler conditions. With

their development the colour of eggs changes from white to brown to a black-head

stage before producing a hatchling. All laid eggs are fertile. At the same time different

physical factors such as temperature also dramatically affect egg survival and larval

establishment (Yin et al., 2009).

The hatched larva (neonate) of 1 to 1.5 mm long makes exit hole by eating

through the eggshell. Initially larvae feeds on tender young foliage, followed with buds,

flowers or young pods, bolls or fruits. There are six growth stages, also called as

instars, in larval development and it becomes fully grown in 2 to 3 weeks in summer or 4

to 6 weeks in spring or autumn. The larval development is noticed more rapid at higher

temperatures. Ninety per cent of all feeding by Helicoverpa is done by larva mainly from

the third instar, small medium larva of 8 to 13 mm long, onwards. Large Helicoverpa

larvae, more than 24 mm long, are the most damaging, because larvae consume about

80 per cent of their overall diet in the fifth and sixth instars (Yin et al., 2009). It indicates

20

the importance of controlling Helicoverpa larvae in early stages of development.

Helicoverpa larvae feed on different plant tissues such as, leaves, flower buds and

flowers, developing pods, fruits and seeds. Pupae are normally developed to produce a

moth in 10 to 16 days. The moth emerges, feeds, mates and the cycle continue.

2.5 Bacillus thuringiensis delta-endotoxin

The soil bacterium Bacillus thuringiensis (Bt) has been a source of insecticidal

toxins produced in commercial transgenic plants (Gatehouse, 2008). The Bt strains

constituted a large reservoir of genes encoding insecticidal proteins that has been

studied for their different specificities of insecticidal activity toward different pests. These

toxins accumulates in the crystalline inclusion bodies produced by the bacterium on

sporulation known as Cry proteins or Cyt proteins; or expressed during bacterial growth

known as Vip proteins (Gatehouse, 2008). The Cry proteins possessing three domains

have been extensively studied. The mechanism of action of Cry protein involved a

proteolytic activation step in insect gut after ingestion, followed by interaction of one or

both of domains II and III with surface cell receptors of the insect gut epithelium. This

interaction results in oligomerization of the protein and domain I forms an open channel

through the cell membrane resulting in breakdown of the gut due to ionic leakage in

cells leading to bacterial proliferation and insect death (Bravo et al., 2007).

More than 500 different cry genes have been classified into 67 groups based on

their primary amino acid sequence (Cry1-Cry67) (Crickmore et al., 2010). Further, these

cry genes have been divided in to four phylogentically non-related protein families such

as, the family of three domain Cry toxins (3D), the family of mosquitocidal Cry toxins

(Mtx), the family of the binary-like (Bin) and the Cyt family of toxins (Bravo et al., 2005).

Among these, the largest Cry family is the 3D-Cry group, which consisted of at least 40

different groups with more than 200 different gene sequences. The 3D-Cry group

proteins possess three domains, of which domain I is implicated in membrane insertion,

toxin oligomerization and pore formation. Domain II of 3D-Cry group proteins is involved

in receptor recognition. Both domain II and III are responsible for insect specificity by

mediating specific interactions with different insect gut proteins (Bravo et al., 2007).

21

Study has identified Cry1Ac binding proteins as V-ATPAse subunit A and actin of

brush border from M. sexta and H. virescens. It has indicated that the mode of action of

Cry toxins involve binding of the toxin with other components of the midgut cells (McNall

and Adang, 2003; Krishnamoorthy et al., 2007). Commercial Bt cotton has been

developed for expression of Cry1Ac protein for the control of Lepidopteran pests.

Further, a second generation Bt-cotton has been produced for Cry2Ab besides Cry1Ac

as a resistance managing mechanism. Bt-corn expressing Cry1Ac has been tested for

their effectively against lepidopteran pests (Christou et al., 2006).

The Cry2Aa belongs to an unusual subset of crystalline proteins. It possessed

broad insect species specificity against larvae from Lepidoptera and Diptera (Liang and

Dean, 1994). It has been reported that the Cry2Aa protoxin is significantly smaller (72

kDa) than those of the Cry1 proteins (~135 kDa). The Cry2Aa is a short protoxins and

are processed primarily at the N-terminal end. Such activated toxin follows complex

sequential binding events with different insect gut Cry-binding proteins and results in

membrane insertion and pore formation (Bravo et al., 2011).

Like any other Cry protein, the Cry1F protein mechanism of action involves the

enzymatic cleavage of the protoxin to form core toxin. During this process, a short

peptide, involved in the formation of crystalline inclusion bodies, are cleaved from both

N-terminal and C-terminal end (Gao et al., 2006). The remaining protein portion is

believed to be responsible for midgut membrane binding. After such a binding, a toxin

molecule forms oligomers and creates pores in the membrane, causing osmotic

destabilization and cell death (Jurat-Fuentes and Adang, 2001). Many studies have

determined the activity of Cry1F against lepidopteran species (Balog et al., 2011;

Oppert et al., 2010). The Cry1F toxin susceptible species include tobacco budworm,

beet armyworm, soybean looper, cotton bollworm, fall armyworm, lesser cornstalk

borer, wax moth and European corn borer (Adamczyk et al., 2008; Blanco et al., 2010;

Buntin, 2008; Tindall et al., 2009).

The native cry1Ac gene sequence from B. thuringiensis have been in silico

modified and artificially synthesized to overcome codon bias and other undesirable

regulatory coding sequences for its improved expression in transgenic plants. Mohan

(2008) targeted only toxic moiety (1.85 kb) for codon optimization and out of 620 codons

22

259 have been altered. The study resulted in the development of modified cry1Acm with

increased GC content to 42.60 per cent by removing the AT-rich regions that were

typical for cry1Ac genes. Study also involved modifications in specific undesirable

eukaryotic regulatory sequences.

Many novel Bt insecticidal proteins have been expressed in transgenic plants.

The Cry34/35 and Vip1/2 toxins have been known for activity against corn rootworm

(Diabrotica virgifera). Moellenbeck et al. (2001) have developed genetically modified

maize expressing the cry34/35 successfully. On other hand, the single chain Vip

proteins, like Vip3, have been studied for their activity against Lepidopteran larvae, with

a broader range of toxicity, which has been further extended by protein engineering

(Fang et al., 2007). Considerable efforts have been undertakes for production of

commercial transgenic plants expressing these proteins (Christou et al., 2006).

It has been hypothesized that combining domains from different insecticidal

proteins could generate active toxins with novel specificities. It has been reported that

the transfer of the carbohydrate binding domain III generated a Cry1Ab-Cry1C hybrid is

highly toxic to armyworm (S. exigua). Further, it has been shown that the presence of

the Cry1Ca domain III was sufficient to confer toxicity toward Spodoptera (de Maagd et

al., 2000). In similar study, Naimov et al. (2003) have reported that the transgenic potato

expressing a hybrid Cry protein, containing domains I and III from Cry1Ba and domain II

of Cry1Ia, conferred resistance to the lepidopteran pest potato tuber moth (Phthorimaea

operculella) and to the coleopteran Colorado potato beetle (Leptinotarsa decemlineata).

The parental Cry proteins in such hybrid proteins are Lepidopteran specific, with no

toxicity toward coleopterans such as the potato beetle, demonstrating the creation of a

novel specificity (Naimov et al., 2003).

The site-directed mutagenesis in Bt protein coding genes has been used as an

alternative to the domain swap approach to increase toxicity toward target pests. It has

been studied that the mutation of Cry1Ab has increased its toxicity toward larvae of

gypsy moth (Lymantria dispar) by 40 fold (Rajamohan et al., 1996). The similar

strategies have been used to increase the toxicity of Cry3A protein toward target

coleopteran pests (Wu et al., 2000). Furthermore, a directed evolution system based on

phage display technology have been used for producing toxins with improved binding to

23

a receptor and increased toxicity (Ishikawa et al., 2007). It has been reported that a

current commercial transgenic maize variety with resistance to corn rootworm,

MON863, expresses a modified version of Bt Cry3Bb1 toxin (Vaughn et al., 2005).

Many variants of the native Cry3Bb have been developed by incorporating a series of

specific mutations that aimed to improve the channel forming ability of the toxin and

have been screened for activity (English et al., 2003).

2.6 Insecticidal proteins other than delta-endotoxin

The α-amylase inhibitors from some legume seeds have been shown to be

effective in imparting resistance of specific varieties of legumes to coleopteran seed

weevils. The bean (Phaseolus vulgaris) α-amylase inhibitor gene has been expressed in

transgenic garden pea (Pisum sativum) and other grain legumes using seed-specific

promoter (Shade et al., 1994). It has been reported that the developed transgenic seeds

showed resistant against stored product pests, such as larvae of bruchid beetles and

field pests, such as larvae of the pea weevil Bruchus pisorum. Lectin genes have been

potentially explored to confer insect resistance in transgenic plants to target hemipteran

plant pests. The Man-specific snowdrop lectin (GNA) has been expressed in transgenic

rice plants using constitutive or phloem-specific promoters (Rao et al., 1998). The

studied reported that the transgenic plants were partially resistant to rice brown

planthopper (Nilaparvata lugens Stal) and other hemipteran pests, with reduction in

insect feeding, development and fertility of survivors up to 50 per cent (Foissac et al.,

2000).

A study incorporating a 90 days feeding trial reported no adverse effects resulting

from consumption of transgenic rice expressing GNA by rats (Poulsen et al., 2007). The

use of a Man-specific lectin from garlic has shown similar partial resistance to

hemipterans (Allium sativum) leaves (ASA-L) in transgenic rice (Saha et al., 2006b).

Further, the transgenic rice plants expressing ASA-L have shown decreased

transmission of Rice tungro virus by its insect vector, as a result of decreased feeding

by the pest (Saha et al., 2006a). Nematodes of Heterorhabditis species having

symbiotic enterobacteria have been widely used for small-scale biological control of

insect pests. It has been reported that the toxins secreted by the bacteria cause cell

death in the insect host, resulting in a lethal septicaemia (Gatehouse, 2008). The P.

24

luminescens, the well-investigated bacterial species have been reported for expressing

a large number of potentially insecticidal components (Ffrench-Constant et al., 2007).

The tcdA has been cloned and expressed with 5’ and 3’ untranslated region

sequences from a tobacco osmotin gene in transgenic Arabidopsis plants (Liu et al.,

2003). The developed transgenic plant has showed complete protection against larvae

of the lepidopteran tobacco hornworm (Manduca sexta). Bacterial cholesterol oxidase

has been studied for their insecticidal activity that is comparable to Bt toxins. Similarly,

avidin has been studied for their insecticidal effect on many insects. The transgenic

maize expressing avidin has been developed and it reported complete resistant to

larvae of three different coleopteran storage pests (Kramer et al., 2000).

2.7 Tissue culture studies in pigeonpea

Attempts have been failed to develop pigeonpea cultivars resistant to legume

pod borer (H. armigera) and fusarium wilt using conventional breeding methods, due to

narrow genetic variability and breeding incompatibility problems. The development of a

somaclonal variant have been reported in pigeonpea, which produces white seeds

exhibits 25 per cent increase in seed size and 30 per cent increase in yield (Saxena,

2005). The efficient plant regeneration protocols are a prerequisite in recombinant

technology. In vitro tissue culture methods have provided an opportunity to

micropropagate elite plant clones by organogenesis and somatic embryogenesis.

Through many studies, now it has become possible to regenerate pigeonpea plants

from differentiated and undifferentiated tissues in culture conditions. It has been

reported that in vitro regeneration of pigeonpea is often genotype-specific (Krishna et

al., 2010).

2.7.1 Organogenesis

The organogenesis has been the extensively used in pigeonpea due to its wider

adaptability among diverse genotypes. Many studies independently have reported

protocols for obtaining stable regenerants through organogenesis from apical meristem,

undifferentiated callus, differentiated nonmeristematic tissues like leaf, and various

seedling explants such as hypocotyls, cotyledons, cotyledonary nodes, epicotyls, and

embryonal axes (Krishna et al., 2010). Although, Villiers et al. (2008) have reported that

25

pigeonpea cultivars with differential crop maturity regenerate plants without any

abnormalities in growth, flowering and seed set, long-duration maturity cultivars of

pigeonpea respond better in tissue culture.

2.7.1.1 Callus mediated organogenesis

A method has been developed for regenerating pigeonpea [Cajanus cajan (L.)

Millsp.] plant through callus obtained from distal cotyledonary segments of mature

seeds (Mohan and Krishnamurthy, 1998). The study has reported the induction of large

number of shoot buds directly from explants of genotypes T-15-15 and GAUT-82-90

when cultured on basal media fortified with N6-benzylaminopurine, kinetin and adenine

sulfate. Seed and seedling explants of pigeonpea have been evaluated for

organogenesis and somatic embryogenesis (George and Eapen, 1994). Study has

involved de novo regeneration of plants through organogenesis using mature

cotyledons, primary leaves and roots of seedlings as explants. Production of multiple

shoots has been reported from the cotyledonary node in cultures of whole seeds on 6-

benzylaminopurine enriched medium. The somatic embryos have been induced from

immature cotyledons and embryonal axes (George and Eapen, 1994).

Three pigeonpea genotypes viz; ICPL 93086, Tanzania–7 and their hybrids have

been tested for whole plant regeneration in different combinations of growth regulators

(Tyagi et. al., 2001). Study has reported significant differences among explants from

leaf, shoot and roots in calli formation. The differential effect of genotype also has been

observed in callus formation (Tyagi et. al., 2001). Further, the largest percentage of calli

has been reported formed by leaf explants in all three genotypes. The calli formation

has been excellent in medium containing 2,4-dicholorophenoxy acetic acid (2,4-D),

Kinetin (KIN), Napthaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP). No

genotype or explant differences have been recorded in shoot and root regeneration

from calli formed from either type of explants in any three genotypes. The medium

containing indole - 3 acetic acid (IAA), 6-(yy-dimethylallylamino) Purine (2IP),

Gibberrellic acid (GA3) and BAP has been reported as the best culture medium for

rooting (Tyagi et al., 2001).

26

Effects on the developmental morphology of Cajanus seeds irradiated with

Cogamma rays have been investigated in vitro (Shamarao and Narayanaswamy, 1975).

Study has demonstrated that the exposure of seeds to 5 kR produced a cluster of

adventitious roots on a callusing medium. While the hypocotyl explants of germinated

seedlings has found stimulated for cell proliferation and abundant callusing on a

medium resulting in shoot buds and plantlets development. Further, it has been

reported that in controls and with higher doses growth have been limited to the

development of a spongy tissue to form a small callus mass from seeds and a restricted

callus proliferation from excised hypocotyls of irradiated seeds (Shamarao and

Narayanaswamy, 1975).

Kumar et al. (1983) have studied the efficiency of callus induction for four

different seedling explants namely leaf, epicotyl, root and cotyledons of pigeonpea with

different media. Study has reported that the Blaydes' medium supplemented with 2,4-

dichlorophenoxyacetic acid (2,4-D) and kinetin have been most effective in inducing

callus from various explants. Among the tested explants leaves have been found to be

most efficient in producing rapidly growing callus. Multiple shoots ranging 5-18 have

been induced from excised cotyledons on Blaydes' medium with 6-benzylaminopurine

(BAP) (Kumar et al., 1983). Shoot buds have been regenerated from cotyledonary

callus with 8-20 per cent frequency in Blaydes' medium with BAP and α-

naphthaleneacetic acid (NAA). The callus obtained from leaf cultures of ICP 7035 have

been regenerated in 14 per cent of the cultures on Blaydes' medium supplemented with

BAP, NAA and gibberellic acid (Kumar et al., 1983). The regenerated shoots in study

have been rooted on the same medium containing either NAA and KN or indole-3-acetic

acid and KN.

Sreenivasu et al. (1998) have reported the efficient plant regeneration via

somatic embryogenesis in pigeonpea. Cotyledon and leaf explants from 10 day old

seedlings have been subjected for development of embryogenic callus and somatic

embryos when cultured on Murashige and Skoog medium supplemented with

thidiazuron (TDZ). Further, subsequent withdrawal of TDZ from the induction medium

has showed induction of maturation and growth of the embryos into plantlets. In another

study, suspension cultures of calli derived from seedling leaf explants of Cajanus cajan

L. var. Vamban-1 have been used for production of somatic embryos (Anbazhagan and

27

Ganapathi, 1999). The highest embryogenic frequency have been recorded on

semisolid MS medium supplemented with 2,4-dichlorophenoxyacetic acid. Similarly, the

maximum frequency of somatic embryogenesis have been observed when this callus

was transferred to MS liquid medium supplemented with 2,4-D.

2.7.1.2 Direct organogenesis

The morphogenetic response of various explants of seven different cultivars of

pigeonpea has been studied. The stimulation and elongation of shoot buds into shoots

derived from the mature embryo axis and intact seed on Murashige and Skoog medium

supplemented with kinetin and benzyladenine have been found to be optimum in

Murashige and Skoog medium supplemented with kinetin, naphthalene acetic acid, and

gibberellic acid. The cotyledon and epicotyl explants of pigeonpea cultivars on the other

hand differentiated directly into four to eight and two to four shoots, respectively,

depending on the media composition and genotype (Naidu et al., 1995). On the other

hand, cotyledons excised from seedlings of Cajanus cajan have been grown on media

containing cytokinins (6-benzyladenine, zeatin, and zeatin riboside) and an allied

compound, thidiazuron (Chandra et al., 2003). It has been reported that with the

exception of zeatin riboside, initial response in terms of induction of organized

structures was very high. Subsequent regeneration of shoots from cotyledon explants

has been very poor. Anatomical studies on the regenerating explants have been

undertaken to study the pattern of morphogenesis. Cytokinins and thidiazuron induced

divisions in the epidermal and sub-epidermal cell layers have been led to the formation

of primary protrusions on the surface. This, further, has been followed by the

development of foci of high meristematic activity either on the surface or within the

primary protrusions. These foci differentiated into embryo like structures or shoot

meristem-like structures (Chandra et al., 2003).

Geetha et al. (1998) have demonstrated an efficient and direct shoot bud

differentiation and multiple shoot induction from seedling explants of pigeonpea. Study

has reported that the frequency of shoot bud regeneration has been influenced by the

type of explant, genotype and concentrations of cytokinin. Explants, such as, epitocotyl,

hypocotyl, leaf, cotyledon and cotyledonary nodal segments from 7 day old seedlings

have been cultured on MS medium augmented with different concentrations of

28

BAP/kinetin. Among the various concentrations tested, 2.0 mg/L BAP or kinetin has

been found to be the best for maximum shoot bud differentiation. Percentage as well as

the number of shoots per explant showing differentiation of shoot buds has been higher

on MS media supplemented with BAP compared to kinetin (Geetha et al., 1998).

Elongation of multiple shoots has been obtained on MS medium fortified with BAP in

combination with NAA and GA3. The combination of 1.0 mg/L BAP with 0.1 mg/L NAA

has resulted in increase number of multiple shoots and shoots elongation. Addition of

GA3 along with BAP and NAA combination has enhanced both multiple shoot

proliferation and shoot elongation in all the explants. Regenerated plants have been

successfully established in soil where 90–95 per cent of them developed into

morphologically normal and fertile plants (Geetha et al., 1998).

Villiers et al. (2008) has evaluated seven varieties of pigeonpea of varying growth

durations and adapted to a wide range of environments across eastern and southern

Africa for their shoot regeneration response in tissue culture. Study has reported that,

on a standardized shoot regeneration medium, the short duration varieties viz., ICPV

88091 and ICPV 86012, generally responded faster and better than the medium

duration viz., ICEAP 00554 and ICEAP 00557, and long duration viz., ICEAP 00020,

ICEAP 00040 and ICEAP 00053 varieties. All the tested varieties produced healthy

rooted plants in vitro that could be transferred to the greenhouse where they exhibited

normal growth, flowering and viable seed set (Villiers et al., 2008). Thidiazuron either

alone or in combination with IAA have been used for induction of high frequency shoot

regeneration from primary leaf segments of three pigeonpea cultivars (Eapen et al.,

1998). Study reported that that transfer of the cultures to medium with reduced

concentration of thidiazuron has been resulted in further development of the shoots.

The regenerated shoots have been subsequently transferred to medium supplemented

with BA, IAA and gibberellic acid where 5-10 per cent of the shoots elongated further.

Rooting of shoots has been obtained on half strength MS medium supplemented with

NAA (Eapen et al., 1998).

2.7.2 Somatic organogenesis

Somatic embryogenesis represents formation of embryos from somatic tissues

such as roots, cotyledons, leaves and stems on liquid and solid medium (Krishnaraj and

29

Vasil, 1995; Merkle et al., 1995; Sharma and Thorpe, 1995). The embryogenic calli

exhibit prolific multiplication and somatic embryo gives rise to plants by eliminating the

tissue culture steps. The regeneration approach through somatic embryogenesis has

been considered as the preferred pathway for transformation (Hansen and Wright,

1999). It has been studied that the meristematic tissues like immature zygotic embryos

and cotyledon explants are highly responsive for the induction of somatic

embryogenesis (Parrott et al., 1992; Sagare et al., 1995; Ahmed et al., 1996).

Patel et al. (1994) was the first to report somatic embryogenesis in pigeonpea.

Subsequently its pathway of regeneration has been reported in three cultivars (Patel et

al., 1994). Many independent studies has reported that the somatic embryos are

regenerated from diverse genotypes using various explant tissues such as mature

seeds, shoot apices, intact seedlings, leaves, petioles, hypocotyls, epicotyls,

cotyledonary nodes, cotyledons, internodes, roots, endosperm, and cell sus-pensions

(Sarangi et al., 1992; George and Eapen, 1994; Nalini et al., 1996; Sreenivasu et al.,

1998; Anbazhagan and Ganapathi, 1999; Mohan and Krishnamurthy, 2002; Singh et al.,

2003). Among all, cotyledon and leaf explants have been used for direct and callus-

mediated somatic embryogenesis, respectively (George and Eapen 1994; Nalini et al.

1996; Sreenivasu et al. 1998; Mohan and Krishnamurthy 2002). Further, the haploid

somatic embryoids have been derived from anthers of pigeonpea flowers when cultured

on MS medium added with KIN (2.0 mg/L) and IAA (4.0 mg/L) (Bajaj et al., 1980).

High somatic embryo regeneration up to 90-97.0 per cent has been observed

from pigeonpea cv. Gaut 82-90, Gaut 82-99 and T15-15 with cotyledon explants (Patel

et al., 1994; Mohan and Krishnamurthy, 2002). A low regeneration frequency of

embryos has been reported using leaf tissue as an explant from pigeonpea cv.

Vamban-1 (Anbazhagan and Ganapathi, 1999), followed by Pusa 852, H 86-5, Pusa

609, Pusa 856, and Pusa 855 (Sreenivasu et al., 1998). The effect of genotype on

somatic embryogenesis has been further evaluated by culturing the cotyledonary nodes

of various pigeon pea cv. Pusa 853, ICPH 8, Pusa 33, ICP 151, RWL 19, UPAS 120,

and ICP 8863 with a globular embryo formation of explant on the same medium (Singh

et al., 2003). It has been reported that the germination frequency from globular embryos

varies in relation to pigeonpea cultivars such as NP (WR) 15 (70%) (Patel et al., 1994)

and ICPL 87 (3%) (Nalini et al., 1996). These studies have suggested that less

30

responsive genotypes of pigeonpea for somatic embryogenesis can still be exploited for

organogenesis mediated regeneration.

Among the explants, better embryogenic frequency up to 74 per cent has been

observed from leaf explants, with 48 average somatic embryos, and a lower one (45 per

cent), with 34 embryos from each cotyledon explants (Sreenivasu et al., 1998).

Similarly, a higher embryo regeneration frequency has been achieved from immature

embryo axes (91 per cent) than with immature cotyledons (23 per cent). On the other

hand, mature cotyledons reported an embryogenesis frequency comparable to that from

immature embryonic axes (Patel et al., 1994). Moreover, Nalini et al. (1996) have

demonstrated somatic embryo formation from cotyledon, epicotyl, leaf and root

explants, of which only cotyledon-raised embryos converted into a plant. Explant tissues

of cotyledon from mature and immature seeds have been primarily used for achieving

somatic embryogenesis. While the leaf and hypocotyls have been less utilized for

somatic embryogenesis.

In many somatic embryo regeneration studies, TDZ has been most widely used

for pigeonpea (Sreenivasu et al., 1998). It is due to its combined activity of both auxin

and cytokinin (Thomas and Katterman, 1986; Saxena et al., 1992), and optimizing the

suitability of TDZ for somatic embryogenesis studies have indicated that the

concentrations of 1.0-2.0 mg/L realized 53-74 per cent frequency with about 38

embryos from each explant (Mohan and Krishnamurthy, 2002). Studies have also

reported that TDZ failed to regenerate somatic embryos and resulted in callus

proliferation or switched to organogenesis pathway directly, at extreme lower and higher

concentrations, with both cotyledons and leaf explants (Sreenivasu et al., 1998; Singh

et al., 2003). Interestingly some pigeonpea genotypes have responded better at higher

concentrations (4.0 mg/L TDZ) (Singh et al., 2003). The medium comprising BAP (1.0

mg/L) has promoted an average of 28 globular embryos from each explant, but

decreased the embryo formation at higher/lower concentrations (Mohan and

Krishnamurthy, 2002).

In pigeonpea, studies have been continued to exploit the function of cytokinins

(KIN) and auxins (IAA, IBA, NAA, and 2,4-D) either alone or in combination to improvise

the embryogenesis protocols. The study has found 2,4-D as most suitable to derive

31

callus mediated somatic embryos. Sucrose in combination with 2,4-D has found to

efficiently induce embryogenesis. On other hand, carbohydrate sources like glucose,

fructose and maltose have been found not optimal or ineffective (Anbazhagan and

Ganapathi, 1999). Further, comparison of the effect of 2,4-D, NAA and picloram on

somatic embryogenesis have revealed greater response of explant to 2,4-D with 91 per

cent frequency from embryonic axes explants. It was followed by picloram and NAA and

picloram showed 36 per cent response with cotyledon explants followed by a decrease

with 2,4-D and NAA (George and Eapen, 1994). These studies emphasize the need of

utilizing appropriate plant growth regulators for different explants to achieve maximum

response. The somatic embryo formation have been observed both on the adaxial and

abaxial surface of mature cotyledon segment on MS medium supplemented with BAP,

KIN and adenine sulphate (Patel et al., 1994). Nalini et al. (1996) have reported 3 per

cent of plant recovery from somatic embryos, regenerated from the surface of cotyledon

explants on MS medium fortified with NAA (50.0, 25.0, 10.0, and 5.0 mg/L) and BAP

(1.0 mg/L). Above studies clearly indicated that the less responsive leaf explants for the

somatic embryogenesis can be exploited by inoculating cotyledon explants on auxins

alone or on the medium augmented with auxin rich and cytokine-deficient medium

(Krishna et al., 2010).

2.8 Plant transformation studies

The plant transformation has started in the early 1980s. Researchers have

harnessed the causative agent of crown gall disease, Agrobacterium tumefaciens, for

introduction of defined fragments of DNA into plant cells (Newell, 2000). Plant

transformation refers to the introduction and integration of foreign DNA in plant cells and

the consequent regeneration of transgenic plants (Newell, 2000). Transfer of DNA into

plant cells results in transient or stable expression of the introduced DNA. Transient

expression, as name suggests, usually remains for few days only. However, it allows

the effects of experimental manipulations to be seen in a short time and hence occupies

a useful niche in such areas as development of transformation methodology or

metabolic studies. On other hand, stable transformation is often a time-consuming

process involving tissue culture techniques that facilitate the growth of whole plants

from treated cells or tissue explants. The introduced DNA through stable transformation

32

is integrated into the host cell DNA and is thereby eligible to be passed on to

succeeding generations.

2.8.1 Agrobacterium mediated method

The Agrobacterium tumefaciens, a Gram-negative soil bacterium, is soil

phytopathogen and has been identified as the causative agent of crown gall disease

(Smith and Towsend, 1907). Binns and Thomashaw (1988) have revealed that A.

tumefaciens is capable of transferring a particular DNA segment; transfer T-DNA of the

large tumor-inducing Ti plasmid into the nucleus of infected cells. Where, it is integrated

into the host genome subsequently resulting in the crown gall phenotype. The studies

have reported that the T-DNA contains oncogenic genes encoding enzymes involved in

synthesis of auxins and cytokinins, and the genes encoding for synthesis of opines

(octapine, nopaline, and agropine) (Zupan and Zambryski, 1995). Agrobacterium

rhizogenes is closely related to A. tumefaciens produces hairy root disease in

dicotyledonous plants. A. rhizogenes have been known to induce the formation of

proliferative multibranched adventitious hairy roots at the site of infection (Chilton et al.,

1982). The virulence plasmid of A. rhizogenes has been known as the Ri plasmid which

shares extensive functional homology with the Ti plasmid (Mehrotra and Goyal, 2012).

The process of Agrobacterium-mediated plant transformation is a highly complex

and depends on the genetic determinants of both the bacterium and the host plant cell

(Mehrotra and Goyal, 2012). Studies have identified the genetic components carried by

A. tumefaciens that are required for plant transformation, include the T-DNA, the Ti

plasmid virulence vir region, which is the master switch for transformation and three

chromosomal virulence loci, essential for transfer process, chvA, chvB, and pscA

(Statchel et al., 1985; Douglas et al., 1985; Thomashow et al., 1987). It has been

reported that the Ri and Ti plasmids share a functional similarity (White and Nester,

1980; Hooykaas et al., 1984; Sinkar et al., 1987). The gene of interest is placed

between the left and right border repeats of Agrobacterium-transferred T-DNA and the

T-DNA region is flanked by 25-bp border sequences in a directly repeated orientation

(Gelvin, 2003). Sheng and Citovsky (1996) have studied the ability of A. tumefaciens to

transfer a portion of its DNA, T-DNA, to the genome of the plant. The vir region includes

eight operons, virA, virB, virC, virD, virE, virF, virG, and virH encoding proteins, and has

33

been identified to regulate the processing and transfer of T-DNA. Study by Stachel et al.

(1986) has demonstrated that plant cells induce the expression of vir genes that are

essential for the process of plant transformation.

Stachel et al. (1986) has reported that Agrobacterium-infected cells excrete low

molecular weight compounds, which are recognized specifically by the Agrobacterium

as signal molecules that induce vir gene expression and thereby activate T-DNA

transfer. Plant proteins have been identified that contributes to Agrobacterium-mediated

transformation. Many proteins such as, BTI1, VIP1, Ku80, CAK2Ms, histones-H2A, H3-

11, H4, SGA1, UDP glucosyltransferase, and GALLS interacting proteins have been

reported to be involved in T-DNA and virulence protein transfer, cytoplasm trafficking,

nuclear targeting, T-DNA integration, stability and expression, and defense responses

(Gelvin, 2010; Magori and Citovsky, 2011; Tenea, 2012).

Agrobacterium-mediated transformation has been used for engineer plants to

produce a wide variety of useful high-value products and high-quality exogenous

proteins safely and effectively (Mehrotra and Goyal, 2012). Recombinant plant proteins

obtained from transgenic plants is an excellent potential source of recombinant

antibodies (Conrad and Friedler, 1994). A wide range of useful products have been

generated in genetically modified plant cell cultures (Marillonnet et al., 2005).

Biodegradable plastics, primary and secondary metabolites, biopharmaceuticals, and

commercially valuable plant traits have been engineered using Agrobacterium-mediated

transformation. Commercially important traits in trees like timber yield and decreased

generation time have also been a focus of Agrobacterium-mediated transformation

(Tzfira et al., 1998).

Recently, Thakur et al. (2012) have developed Agrobacterium-mediated

transformation in Populus and incorporated the silviculturally important traits like quality

paper production. Blanc et al. (2006) have developed efficient Agrobacterium-mediated

transformation method for Hevea brasiliensis. Similarly, Zombori et al. (2011) developed

highly efficient Agrobacterium-mediated transfer system in Brachypodium. On other

hand, Wang et al. (2012) developed an Agrobacterium-mediated method in the

medicinal herb, Bidens pilosa. Ribas et al. (2011) reported that Agrobacterium-mediated

transformation of embryogenic cultures as a viable and useful tool for coffee breeding

34

and functional analysis of agronomically important genes. A. rhizogenes-transformed

roots of coffee have been developed for functional genomic studies of coffee root genes

for coffee breeding and also have been used for the production of plant secondary

metabolites (Alpizar et al., 2008).

Agrobacterium-mediated transformation has been successfully used in

improvement of Brassica (Rafat et al., 2010). Singh et al. (2010) have reported efficient

transformation protocol in mustard using chickpea lectin gene. Bhuiyan et al. (2011)

have developed an efficient Agrobacterium mediated genetic transformation method for

Brassica juncea, wherein cotyledon explants were used as explants for successful gene

transfer. Similarly, Agrobacterium-mediated transformation has also been reported in

the biodiesel plant, Jatropha as well (Mazumdar et al., 2010; Zong et al., 2010).

Trick and Finer (1997) have described an efficient Agrobacterium based

transformation technology, sonication-assisted Agrobacterium-mediated transformation

(SAAT), in soybean. Many studies have demonstrated an efficient method of

transformation in legumes by regeneration of shoots from the cotyledonary node and

other meristematic explants after Agrobacterium infection (Trieu and Harrison, 1996;

Ohloft et al., 2003; Parmesha, et al., 2012). Efficient transformation of cotyledon

explants from Arachis hypogaea by Agrobacterium have been reported as a cost

effective, routine tool of peanut and chickpea transformation (Sharma and Anjaiah,

2000; Bhatnagar-Mathur et al., 2008). Transgenic soybean plants have been produced

by Agrobacterium-meditated T-DNA delivery (Trick and Finer, 1998). Recently, mature

seed embryos of Phaseolus vulgaris have been successfully transformed by

A. tumefaciens (Amugune et al., 2011). Similarly, Sathyanarayna et al. (2012) have

made a successful attempt for efficient and stable genetic transformation and

regeneration of legume Mucuna pruriens.

Agrobacterium-mediated transformation of commercially important monocots has

been initially achieved in rice and maize (Schlappi and Hohn, 1992; Chan et al., 1992).

Further, several monocots such as, barley, maize, rice, sorghum, triticale and wheat

have been transformed (Chan et al., 1993; Gurel et al., 2009; Harwood et al., 2009;

Raja et al., 2010). Enríquez-Obregón et al. (1998) have reported successful

Agrobacterium-mediated transformation in sugarcane meristems. Chan et al. (1993)

35

have reported the successful transfer and expression of a reporter gene driven by

alpha-amylase promoter in japonica rice using Agrobacterium-mediated gene transfer

system. Zhao et al. (2000) have made successful attempt of stably transformed

sorghum plants using Agrobacterium for production. Furthermore, a highly efficient and

reproducible Agrobacterium-mediated gene targeting transformation system has been

developed in rice (Ozawa and Takaiwa, 2010; Ozawa et al., 2012). Recently,

Ziemienowicz et al. (2012) developed a method of transgene delivery into Triticale

plants using the Agrobacterium-transferred DNA-derived nanocomplex. Many

independent studies have reported the Agrobacterium mediated transformation in

millets like Setaria italica, Panicum virgatum, Eleusine coracana and Pennisetum

glaucum (Liu et al., 2007; Wang et al., 2011; Li et al., 2010; Li et al., 2011; Sharma et

al., 2011).

Initially the in planta transformation protocol has been developed in the model

plant, A. thaliana (Feldman and Marks, 1987). Further, the floral dip method developed

by Clough and Bent (1998), which eliminated the step of vaccum infilteration. The

Camelina sativa have been transformed by floral dipping along with vacuum infiltration

(Lu and Kang, 2008). Trieu et al. (2000) have developed floral infilteration method and

seedling infilteration methods in Medicago. Similarly, Weeks et al. (2008) have

developed in planta transformation method in alfalfa for development of marker free

transgenic plants. In this method the seedlings have been cut at the apical node and

vortexed with suspension of Agrobacterium in sterile sand and then seeds were

subsequently grown to maturity. Many studies have reported in planta techniques

successfully applied to obtain transformants in mulberry, soybean, rice, and cotton

wherein Agrobacterium has been targeted to wounded apical meristem of differentiated

seed embryo in which various organs and primodial had been formed (Ping et al., 2003;

Supartana et al., 2005; Keshamma et al., 2008).

Similar strategy was also applied to pigeonpea, fieldbean, sunflower, and

safflower (Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b). The in planta

approach has been effectively used to introduce agronomically important traits in

groundnut (Rohini and Rao, 2001). In planta transformation has also been

demonstrated in other species like maize, rice, and wheat (Mehrotra and Goyal, 2012).

Razzaq et al. (2011) have developed in planta transformation protocol in wheat through

36

apical meristem. Ramu et al. (2012) reported the expression of a chimeric cry1AcF

(encoding cry1Ac and cry1F domains) gene in transgenic pigeonpea developed used in

planta method and studied their resistance towards H. armigera.

The effect of air evacuation has been studied in Arabidopsis thaliana by Clough

and Bent (1998), it has indicated two folds increase in transformation rate when flower

tissues have been air evacuated with Agrobacterium culture in place of flower dip

method. Dehestani et al. (2010) using in planta transformation method in Arabidopsis

has showed that plants infected with Agrobacterium strain GV3850 only reported

highest transformation frequency of 1.54 per cent. Whereas, using of vacuum infiltration

during Agrobacterium infection, transformation efficiency has been improved by 3.0 per

cent (Dehestani et al., 2010). Similarly, Habashi et al. (2012) have studied the effect of

using vacuum infiltration during Agro-inoculation in two pear (Pyrus communis L.)

cultivars viz., Bartlett and Harrow Delight and reported significant increase in

transformation efficiency (10.63 per cent) that that of common Agro-inoculation method

(4.06 per cent).

2.8.2 Direct methods

Through many studies, protocols for the electroporation of cell suspensions have

been worked out for many species such as, tobacco, rice and wheat (Rafsanjani et al.,

2012). Using this method so far the best results have been obtained for maize (Lurquin,

1997). To et al. (1996) reported that the efficiency of electroporation have been found to

be relatively high and 90 transgenic maize plants have been regenerated from 1440

embryos (6.25 per cent) and 31 plants from 55 calli (54.6 per cent). The efficacy of this

method has been fully comparable with the best results obtained for maize after micro

bombardment (Chowrira et al., 1996). Modifications in electroporation such as post

pulse addition of ascorbic acid or ascorbate could significantly increase the efficiency of

process without having any negative influence on cell viability (Lazzeri, 1995). The

electroporation is a simple and effective method, but its application is limited to only few

species. In addition, application of electric current damages the gene leading to

misleading codons and wrong translational end product (Gaertig et al., 1994; Rakoczy-

trojanowska, 2002).

37

Microinjection is another method wherein, DNA is directly injected into plant

protoplasts or cells, specifically into the nucleus or cytoplasm, using fine needle or tip of

0.5-1.0 micrometre diameter made up of glass needle or micropipette (Rafsanjani et al.,

2012). This method of gene transfer has been used to introduce DNA into large cells

such as oocytes and the early embryonic stage (Oard, 1991; Casas et al., 1995;

Neuhaus et al., 1987). In yet another method, the silicon carbide fibres are simply

added to a suspension, containing both plant tissue and the plasmid DNA, and

rigorously mixed so that DNA-coated fibres penetrate the cell wall (Rakoczy-

trojanowska, 2002). Although this approach is easy, fast and economical method that is

applicable to various plants but this methodology have disadvantages like low

transformation efficiency, damage to cells that negatively influence their further

regeneration capability, and the necessity of taking precautions during lab work as

breathing the fibres in, especially asbestos ones, can lead to serious sicknesses (Asad

et al., 2008; Sailaja et al., 2008).

It is well studied that elevated temperature regimes enhances the gene transfer

that is successfully adopted and conformed to animal cell transfection process (Asad et

al., 2008). It has been documented that at temperature above 37 °C, the gene transfer

tendency found getting increased and further rise in ambient temperature to 43 °C with

certain period of time provided greater transient transfection (Rafsanjani et al., 2012).

Many studies have confirmed this concept through the work on interleukin-2 and swine

growth hormone expressions using indirect ELISA (Baron et al., 2001; Kenel et al.,

2010). Dillen et al. (1997) worked on the effect of temperature on Agrobacterium

tumefaciens-mediated gene transfer in plant. They reported the effect of temperature

(15 °C to 29 °C) in biotransformation process with A. tumefaciens involved in co-

cultivation of Phaseolus acutifolius and further in Nicotiana tabacum biotransformation

(Dillen et al., 1997). In both the situations, irrespective of the type of helper plasmid, the

level of transient uidA expression decreased notably when the temperature was raised

above 22 °C, further lowered down in temperature at 27 °C and was undetectable at 29

°C (Iba, 2002).

38

2.9 Selection marker

Genetic engineering involves the delivery, integration and expression of

defined genes into plant cells, which can be grown to generate transformed plants. The

system to select the transformed cells, tissues or organisms from the non-transformed

is important and hence, marker genes are vital to the plant transformation process.

From the development of first transgenic plants during early 1980s and subsequently

after its commercialization worldwide over a decade, antibiotic and herbicide resistance

selectable marker genes have become one among the integral feature of plant genetic

modification (Ramessar et al., 2007).

Many studies have revealed that antibiotics such as, kanamycin or hygromycin

and herbicide such as, phosphinothricin, PPT have been widely used as selection agent

since the early days of plant transformation (Sundar and Sakthivel, 2008). Positive

selection systems are those that allow the growth of transformed plant cells. Based on

their functionality the positive selection systems can be classified into conditional and

non-conditional positive selection system (Sundar and Sakthivel, 2008). A conditional-

positive selection system involves a gene encoding for a protein, usually an enzyme

that confers resistance to a specific substrate that may be toxic to the untransformed

plant cells or that facilitates the growth as well as differentiation of the transformed cells

alone.

Bacterial aminoglycoside 3’-phosphotransferase II (APH [3’] II, E.C 2.7.1.95),

also known as neomycin phosphotransferase II (NPTII) has been used as an effective

selectable marker in mammalian and yeast cells, it was the first to be tested in plants

(Miki and McHugh, 2004). The NPTII catalyses the ATP-dependent phosphorylation of

the 3’-hydroxyl group of the amino-hexose portion of certain amino-glycosides including

neomycin, kanamycin, geneticin (G418), and paramomycin (Miki and McHugh, 2004).

Hygromycin B is an aminocyclitol antibiotic inhibitor of protein synthesis. It has a broad

spectrum activity against prokaryotes and eukaryotes including in plants. The E. coli

gene aphIV (hph, hpt), coding for hygromycin B phosphotransferase (HPT, E.C.

2.7.1.119), has been reported for conferring resistance against bacteria, fungi, animal

cells and plant cells by detoxifying hygromycin B via an ATP-dependent phosphorylation

of a 7’’-hydroxyl group (Waldron et al., 1985; van den Elzen et al., 1985). Chimeric

39

genes have been studied for their effectiveness in diverse plant species, including

dicots, monocots and gymnosperms, and have been used as a selectable marker when

nptII was not found to be effective (Tian et al., 2000; Twyman et al., 2002).

The gene coding for streptomycin phosphotransferase (SPT, APH [3’], E.C.

2.7.1.87) have been derived from the bacterial transposon, Tn5 (Mazodier et al., 1985).

A mutant form of SPT, containing a two amino acid deletion near the carboxy terminus

of the protein, have been placed under the control of the T-DNA transcript promoter and

introduced into N. tabacum (Miki and McHugh, 2004). This marker system has not been

adopted for general use. Another marker gene, the aminoglycoside-N-acetyl

transferases (AAC), is another class of aminoglycoside-modifying enzyme. It potentially

acts as plant selectable marker genes (Nap et al., 1992). Two of such enzymes viz.,

AAC(3)-III and AAC(3)-IV, have been examined in petunia and Arabidopsis under the

control of the 35S promoter and nos 3 sequences (Hayford et al., 1988). The study

reported that the gene was effective in a variety of plants including Brassica napus,

Nicotiana tabacum and tomato. According to Nap et al. (1992) aminoglycoside-O-

nucleotidyl transferases, bacterial aadA gene, codes for the enzyme aminoglycoside-3’-

adenyltransferase represent the third class of enzymes that modify the aminoglycoside

antibiotics and have been used as plant selectable marker genes.

2.10 Southern Blotting

Southern blotting methods are an assisted to gel electrophoresis. It involves a

method for separating DNA with exceptional resolving power. Blotting enables the

detection of specific molecules among the mixture separated in the gel. The major steps

involve transfer of molecules from the gel to a porous membrane, which is achieved by

soaking solution through the gel and the membrane using absorbent paper. For DNA,

specific sequences are detected in the membrane by molecular hybridization with

labeled nucleic acid probes (Southern, 2006). The method has been used in several key

studies. The original proposal for the genetic mapping of the human genome has been

based on restriction fragment length polymorphisms (RFLPs) detected by blotting (Kan

and Dozy, 1978). Introns have been first seen in blots of rabbit genomic DNA hybridized

with probes for the ß-globin gene (Jeffreys and Flavell, 1977). The first DNA fingerprints

have been produced by hybridizing restriction digests of human DNA with minisatellite

40

probes (Jeffreys et al., 1985). Many of the early applications of the method have been

replaced by Sanger sequencing, which gives more information, or methods based on

the polymerase chain reaction (PCR), which is simpler to perform (Southern, 2006).

The transgenic status of regenerated plants has been performed employing PCR

amplification of the marker gene or transgene. Southern analysis is an important

component of transgene analysis to prove the integration of the foreign gene into host

genome (Bhat and Srinivasan, 2002). Southern hybridization helps to assess the

number of independent transgene insertions, which is important to find transformants

with single, unaltered transgene insertions and considered ideal for analysis (Bhat and

Srinivasan, 2002). It is also necessary to check the presence of multicopy tandem

insertions and other rearrangements at the given locus. Surekha et al. (2005) used

Southern blotting analysis for the confermation of 18 pigeonpea transgenic samples,

which showed clear amplification in PCR. The method confirmed the presence of

transgene and number of the inserts, size of the inserts in different plants confirming the

independent origin of the transgenic lines.

Southern blot analysis has been carried out for the genomic DNA from the T1

generation pigeonpea transgenic plants in order to determine the integration pattern and

the copy number (Sharma et al., 2006). The PCR amplicon of cry1Ab fragment have

been used as probe for the Southern hybridization analysis and hybridization signals

detected in eight of the 12 plants analyzed for the integration of Bt gene in the genome

of these plants. The blotting analysis revealed that four of six transgenic plants

possessed single-copy integrations (Sharma et al., 2006). Eleven of the plants, positive

in the dot blot hybridization; have been tested for genomic Southern analysis of nptII

gene (Rao et al., 2008). The results indicated hybridization signal in all the test eleven

plants except in the non-transgenic control plant sample confirming integration of the

transgene in the pigeonpea genome. Further, in similar king of study, Ramu et al.

(2012) studied the integration pattern using genomic Southern analysis, which was

carried out with restrict digested DNA from three selected plants. A strong signal and

the difference in the hybridization pattern in the selected transgenic plants revealed the

single copy integration in these plants, whereas no signal was observed with the DNA of

untransformed plants.

41

The PCR and Southern analysis of the genomic DNA from T1 progenies of

selected transgenic chickpea plants have showed amplification of expected amplicons

of 800 and 995 bp for cry1Ab and cry1Ac genes respectively similar to the positive

controls (Mehrotra et al., 2011). The result of Southern blot hybridization have shown

single DNA fragments of size ranging from 4.16 to 6.57 kb hybridizing with 1.845 kb

radiolabelled cry1Ab/ c probe in T1 transgenic plants. Results of Southern hybridization

also indicated that the insertion of cry1Ab and cry1Ac genes as single copy integrations

(Mehrotra et al., 2011). In similar study, Acharjee et al. (2010) Southern blotting of DNA

from pooled progeny of nine independent transgenic chickpea lines have been

performed and showed the presence and integration of the cry2Aa gene in eight of the

nine lines. Among all, few lines showed integration of one copy of the transgene into the

genome, few contained two or more copies of the transgene (Acharjee et al., 2010).

2.11 Immunoassay

An immunoassay technique based on antibodies is a standard approach for

qualitative and quantitative detection of protein of a known target analyte (Brett et al.,

1999). Both monoclonal, which is highly specific, and polyclonal, which is often more

sensitive, antibodies have been used depending on the specificity of the detection

system (Kamle and Ali, 2013). On the basis of typical concentrations of a transgenic

material in plant tissues, the limit of detection (LOD) of a protein immunoassay can

predict the presence of recombinant protein in genetically modified plant tissue (Stave,

2002).

The ELISA has shown a significant advantage for protein analysis in transgenic

plants. A sandwich ELISA is the preferable immunoassay used for the detection of Bt

protein, where an analyte is sandwiched in between the two antibodies; a capture

antibody and the detector antibody. In sandwich ELISA protein concentration is directly

proportional to the colour intensity (Kamle and Ali, 2013). ELISA has been successfully

used for the detection of protein encoded by cp4-epsps gene in a RR soybean (Rogan,

1999). Similarly, monoclonal antibodies have been used for the development of

sensitive and single epitope specific immunoassays for the detection of Bt proteins like

Cry1Ac and Cry1Ab (Vázquez-Padrón, 2000). For the detection of Cry1Ab, a capillary

electrophoresis competitive immunoassay and a highly sensitive quanti-dot based

42

fluorescence linked emmuno sorbant assay have been developed (Giovannoli et al.,

2008; Zhu et al., 2011). Similarly, a monoclonal antibody based sandwich immunoassay

having a 100 ng/g LOD for Cry1Ac and a 1 pg/g LOD for Cry2Ab in cotton seed/leaf

samples have been reported (Kamle et al., 2011, 2013; Shan et al., 2007).

A nitrocellulose-strip rather than microtiter wells have been developed and used,

which has resulted in the development of lateral flow strip/ dipstick/immuno-strip

technology (Kamle and Ali, 2013). Immobilized double antibodies, specific to recognize

expressed protein have been conjugated to a colour reactant (gold nano-particles) and

incorporated into a nitrocellulose strip. This nitrocellulose strip when dipped in the

protein extract of plant tissue harboring a GM protein, leads to an antibody reaction

releasing colour. This red coloured gold conjugated complex flows to the other end of

the strip through capillary movement to a porous membrane that has two captured

antibody zones. The immuno-strips can give results as either ‘Yes’ or ‘No’ within 5 to 10

min. The immuno-strip is an economical, easy and field tractable detection method.

These immuno-strips are commercially available to detect Cry1Ab, Cry1Ac, Cry2Ab and

CP4-EPSPS (Lipton et al., 2000; Fagan et al., 2001). The method has been used and

revealed that chimeric Cry1AcF levels in developed transgenic pigeonpea plants ranged

3-15 µg/g of fresh tissue weight (Ramu et al., 2012).

An immunoassay technique has been used to estimate the Cry1Ac protein levels

in developed transgenic chickpea plants and it ranged from 14.5 to 23.5 mg 1-1

extractable protein with high levels of toxicity in insect feeding bioassay with larvae of

pod borer insect H. armigera (Sanyal et al., 2005). Mehrotra et al. (2011) has used

modified cry1Ab and cry1Ac insecticidal genes to introduce into chickpea by

Agrobacterium-mediated transformation of pre-conditioned cotyledonary nodes.

Interestingly, the pyramided transgenic plants having moderate expression levels (15-20

ng/mg) showed high-level of resistance against pod borer larvae of H. armigera when

compared with high level expression of a single individual Cry toxin.

2.12 Real-Time PCR

Many studies have used real-time PCR to quantify a targeted cDNA molecules

(Kamle and Ali, 2013). Real-time PCR has shown great value in validating and

estimating the number of copies of inserted genes into the host genome (Zhang et al.,

43

2003). The method has been reported for several genetically modified crops such as

maize, cassava, rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al.,

2013; Beltrán et al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007).

Furthermore, a sensitive loop mediated isothermal amplication method employed for the

detection of three GM rice events has been reported (Chen et al., 2012; Kiddle et al.,

2012).

The 5′ event-specific/hmg-taxon gene real-time polymerase chain reaction (PCR)

protocol coupled to 2−∆∆CT analysis have been used to determine the MON 810 insert

copy number per haploid genome across 26 genetically modified commercial maize

varieties (Aguilera et al., 2008). The end-point and real-time polymerase chain reaction

methods have been used to detect EE-1 brinjal (Ballari et al., 2013). The study reported

the limits of detection and quantification for SYBR-based real-time PCR assay, which

were 10 and 100 copies respectively. Beltrán et al. (2009) developed real-time

polymerase chain reaction-based methods for the primary scrutiny of putative

transgenic plants. They tested for the presence of transgenes, estimated copy number,

and quantified messenger RNA (mRNA) levels of genes introduced through

Agrobacterium. Copy numbers for the genes ß-glucuronidase and hygromycin

phosphortransferase have been estimated in 15 transgenic lines. The study indicated

that although real-time PCR has been efficient for classifying transgenic lines with one

or more transgenes inserted, for conclusive analysis of gene copy number, i.e., in a

potential breeding line, the Southern blot may still be required (Beltrán et al., 2009).

Further, high, medium and low levels of mRNA expression have been detected but

study revealed that no direct relationship between copy number and expression level of

transgenes was obvious, suggesting that factors like position effects or DNA

rearrangements led to differential expression.

The quantitative real-time PCR have been used to determine transgene copy

number in transgenic wheat (Li et al., 2004). A conserved wheat housekeeping gene,

puroindoline-b, have been used as an internal control to calculate transgene copy

number. Estimated copy number in transgenic lines using real-time quantitative PCR

has been correlated with actual copy number based on Southern blot analysis in

transgenic wheat lines and it has been reported that the real-time PCR is an efficient

method for estimating copy number in transgenic wheat (Li et al., 2004).

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2.13 Insect bioassay

Surekha et al. (2005) performed detached leaf feeding tests on T1 and T2

generation pigeonpea plants for insect resistance using the 1st and 2nd instar larvae of

the pest S. litura. Study reported significant variation between transgenic samples and

the control wild-type plants. The highest mortality of the larvae found in the transgenic

plants was 80 per cent. Further, the plant that showed high mortality rate or larval

damage was more than another plant. The effect of transgenic pigeonpea carrying

cry1Ab and SBTI genes on the growth and development of H. armigera have been

studied for three successive generations using detached leaf bioassay (Gopalaswamy

et al., 2007). Plant numbers Bt 6.1 (1.7%), Bt 1.2 (2.0%), SBTI 2.2 (2.0%), Bt 6.2

(2.2%), Bt 3.6 (2.3%), Bt 9.2 (2.3%), SBTI 1.4 (2.3%), Bt 3.2 (2.7%), SBTI 4.3 (2.7%) Bt

2.1 (3.0%), Bt 6.6 (3.0%), and SBTI 2.5 (3.0%) showed lower leaf feeding compared to

the non-transgenic plants of ICPL 88039 (4.5). whereas, plants Bt 6.1 (10.0%), SBTI 1.4

(13.3%), Bt 3.2 (16.7%), and Bt 6.2 (16.7%) also showed significantly less larval

survival than the non-transgenic control, ICPL 88039 (30.0%). Further, they have

reported that the larval weights in test samples were lower on Bt 2.1 (0.517 mg), Bt 8.1

(0.542 mg), Bt 3.2 (0.567 mg), Bt 7.2 (0.597 mg), Bt 1.2 (0.600 mg), Bt 6.2 (0.622 mg),

SBTI 4.3 (0.628 mg), SBTI 2.5 (0.633 mg), SBTI 1.2 (0.650 mg), and SBTI 7.5 (0.733

mg) as compared to the non-transgenic plants of the respective genotypes.

Ramu et al. (2012) tested the bioefficacy of the plants against first and second

instar Helicoverpa revealed significant variability in larval mortality and damage in both

the pods and leaves. Study indicated that the both the damage and the mortality in

insects varied from 0 to 100 per cent among the putative transformants. The effect of

the cry1AcF gene has been seen on the larva as there was a considerable difference in

the size of the larva that fed on the transgenics and wild type. The transgenic plants that

showed high mortality exhibited less damage in leaves and pods (Ramu et al., 2012).

Selected transgenic chickpea lines, BS2A, BS5A and BS6H have been tested in

insect bioassays using neonate H. armigera and compared to the non-transgenic cv

ICCV 89314 and cv. Semsen (Acharjee et al., 2010). Study reported significantly

greater larval mortality among the larvae receiving transgenic leaves than those

receiving leaves from the controls. Larval mortality was highest on the BS6H transgenic

45

line where almost all larvae died during the assays, wherein larval death on leaves from

BS6H was 100 per cent (Acharjee, et al., 2010).

The toxicity of T0 and T1 plants expressing Cry1Ac protein have been tested

using either isolated or whole plants in feeding assays with second or third instar

neonate larvae of H. armigera (Sanyal et al., 2005). The larvae ceased feeding after 2

days on leaves of A3, A4, B5, B8, B10, C6, C7, D2 and D3 chickpea plants expressing

high level of toxin and exhibited significant reduction in their weight decreased to 40 to

90 per cent and finally showed high mortality. Similarly, the high correlation between

feeding damage caused by H. armigera larvae on the transgenic chickpea leaves has

been reported (Lawo et al., 2008). For the susceptible H. armigera strain, leaf damage

has been significantly higher for control leaves than for Bt chickpea leaves after 24 hr of

feeding.

46

3. MATERIAL AND METHODS

The present study was undertaken at Department of Biotechnology, UAS,

Dharwad for development and molecular characterization of transgenic pigeonpea

plants expressing different cry genes viz., cry1Ac, cry2Aa, cry1F and cry1Acm

separately. The in vitro plantlet regeneration and in planta transformation protocols

were improvised and used for transformation of pigeonpea cultivars viz., ICPL 87119

(Asha) and BSMR 736. The details on material used and methods adopted are as

follows:

3.1. Plant material and in vitro plantlet regeneration in pigeonpea

Two pigeonpea varieties viz., ICPL 87119 (Asha), a moderately resistant variety

for fusarium wilt, sterility mosaic disease and BSMR 736, a sterility mosaic disease

resistant variety were used for present experiment as a plant material. Seeds of these

cultivars were obtained from Agriculture Research Station (ARS), Gulburga, which

were maintained by the breeders with purity. Healthy seeds with uniform size, shape

and colour were surface sterilized with 70% ethanol treatment for one min followed by

two rinses with sterile distilled water for two min each. Further, seeds were subjected to

0.1% (W/V) aqueous mercuric chloride solution treatment for 5 min followed by two

rinses with sterile distilled water for 2 min each. The surface sterilized seeds were used

for inoculation on Murashige and Skoog’s (MS) medium having different cytokinins and

their consecration regimes.

3.1.1 Culture media and conditions

The culture medium was that of MS with 3% (W/V) sucrose. For multiple shoot

bud induction, the culture medium was augmented with 1, 2, 3, 4, 6, 8 and 10 mg/L

BAP, TDZ and zeatin, separately. The pH of the medium was adjusted to 5.8 prior to

adding 8 g/L agar-agar. Initially, MS medium was sterilized at 121 °C for 15 min and

filter sterilized growth hormones was added. Cultures were maintained at 25±2 °C

under light intensity of 1000 lux with 16 hr/day photoperiod.

47

3.1.2 The effect of cytokinins on shoot bud induction

The experiment was designed to compare the effect of three cytokinins viz.,

BAP, TDZ and zeatin with their concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) on

shoot bud induction. The explants used for study were cotyledonary node with

cotyledons and embryo discs with half cotyledon. For cotyledonary node with

cotyledons as explants, the surface sterilized seeds were inoculated on basal MS

supplemented with increasing concentrations of growth hormones. In case of embryo

discs with half cotyledon as explants, the surface sterilized seeds were soaked

overnight in sterile distilled water; seed coat along with half cotyledon was detached

using sterile forceps retaining embryo discs with other half part of cotyledon. After 8-10

days of culture, the number of explants forming shoots buds and shoot buds per

explant were counted. The cotyledonary node with induced multiple shoot buds were

sub-cultured on the MS medium with respective growth hormone augmentations for

shoot bud elongation up to 10-12 days. Experiment was repeated twice using a total of

50 explants for each treatment. The growth hormone solutions were prepared viz., BAP

in 1N NaOH; TDZ in DMSO and Zeatin in 1N NaOH, and filter sterilized before using in

culture medium. The explants cultured on MS medium devoid of growth hormone

fortification were maintained as experimental controls and the effect of different

cytokinins and their concentration regimes was analyzed.

3.1.3 Rooting of elongated shoots

The shoots (~3 cm) devoid of roots were transferred to MS basal media fortified

with (0-2 mg/L) IBA. The treatments maintained for rooting were culture medium MS +

(0-2 mg/L) IBA; ½ MS and ½ MS + 0.50 mg/L IBA. A set of fifty shoots per rooting

medium treatment was maintained for this experiment and the experiment was

repeated twice. After 10-15 days of incubation in rooting media, individual shoots were

observed for root induction and the number of roots induced per shoot.

3.2 Maintenance of Agrobacterium tumefaciens strains and in vitro transformation of

pigeonpea

The A. tumefaciens strains carrying constructs with different cry genes were

grown and maintained on YEMA media (Appendix I) supplemented with respective

48

selection pressures. Such actively growing A. tumefaciens cultures were used in in

vitro transformation process.

3.2.1 Culture and maintenance of A. tumefaciens strains

The freshly prepared YEMA media was sterilized by autoclaving. The media

was allow to cool up to 50 to 60 °C and respective filter sterilized selection

pressures/antibiotics were added and thoroughly mixed before pouring it in UV

sterilized polystyrene petri dishes (Fisher Scientific Pvt. Ltd.). The antibiotics used

were, for A. tumefaciens strain with cry1Ac: Kan100; A. tumefaciens strain with cry2Aa:

Kan50 + Rif10; A. tumefaciens strain with cry1F: Kan50 and A. tumefaciens strain with

cry1Acm: Kan50 + Rif25 (Fig. 1a to 1d). Freshly inoculated culture was grown over night

at room temperature and stored at 4 °C. The cultures were revived after every 20 to 25

days. The cry1Ac, cry2Aa and cry1F gene constructs were provided by Dr. P. Ananda

Kumar, NRC on Plant Biotechnology, IARI, New Delhi as a part of ICAR-NFBSFARA

funded pulse project.

3.2.2 In vitro transformation of pigeonpea

The binary plant transformation vector carrying cry gene was used for

pigeonpea transformation. The pigeonpea seeds were surface sterilized with 70%

ethanol and allowed to germinate overnight in distilled water. The seed coat from

germinated seeds were removed and embryonic axis attached to single cotyledon were

surface sterilized using 1.2% (w/v) aqueous sodium hypochlorite solution for 2 min.

The embryonic axis of germinating seed was injured and infected with Agrobacterium

tumefaciens harbouring binary vector for 30 min. The explants were co-cultivated on

basal MS media for two days at 28 °C in dark. Co-cultivated explants were washed

repeatedly in sterile distilled water containing 300 mg/L cefotaxime and transferred to

selection medium containing MS basal + 2 mg/L BAP + 200 mg/L Kanamycin + 300

mg/L Cefotaxime media in plant growth chamber maintained at 25 ± 2 °C, light intensity

of 1000 lux with 16/8 hr light-dark duration. The survived explants were again sub-

cultured onto Kanamycin containing media for an additional round of testing. Well-

developed shoots were excised and transferred to half MS containing 0.5 mg/L IAA for

rooting. The rooted plants were hardened to establish in transgenic containment

facility.

49

Fig. 1a: Construct map of the binary vector pBinBt3. A. Construct map of the binary vector pBinBt3 carrying cry1Ac and nptII marker gene. B. T-DNA carrying cry1Ac gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator.

A

B

50

A

B

Fig. 1b: Construct map of the binary vector pBinAR. A. Construct map of the binary vector pBinAR carrying cry2Aa and nptII marker gene. B. T-DNA carrying cry2Aa gene with 35S promoter and ocsA terminator; nptII marker genes with nos promoter and terminator.

51

Fig. 1c: Construct map of the binary vector pBinAR. A. Construct map of the

binary vector pBinAR carrying cry1F and nptII marker gene. B. T-DNA carrying cry1F gene with 35S promoter and ocsA terminator; nptII marker genes with nos promoter and terminator.

A

B

52

nos

P

nptII

nos

T

35S

AMV

cry1Acm

uidA

nptII

nos

T

Xba I Bam HI

Fig. 1d: Construct map of the binary vector pMKK1708. T-DNA carrying cry1Acm gene with 35S promoter and nos terminator; nptII marker genes with nos promoter and terminator.

53

3.3 Explant preparation and improvisation of in planta transformation protocol in

pigeonpea

The in planta transformation method reported by Ramu et al. (2012) for pigeonpea

using seeds (embryonic axis) as a source of explants was further optimized to increase

the transformation efficiency. The seeds were surface sterilized with 70% ethanol and

allowed to germinate overnight in sterile distilled water. The seed coat from germinated

seeds were removed and embryonic axis attached to single cotyledon were injured using

fine needle and infected with Agrobacterium tumefaciens culture harbouring binary

plasmid for 30 min. Different treatments such as, (a) A. tumefaciens infection alone, (b) A.

tumefaciens infection using A. tumefaciens culture with tobacco extract added in

overnight grown culture (1.0 g of tobacco leaf tissue per 100 ml of A. tumefaciens culture)

and (c) A. tumefaciens infection with air evacuation to increase the proximity between A.

tumefaciens and embryonic axis (vacuum infiltration for 10 min at 450 mm Hg). A total of

200 explants in four batches (50 explants per batch) were subjected for transformation

using above mentioned treatments. The explants were kept in dark for two days at 25 ± 2

°C for co-cultivation on moist germination paper to avoid the drying of explants. After two

days of co-cultivation explants were allowed to grow at 16/8 hr light-dark period for seven

days on moist germination paper. Well responding seedlings, of seven days old, were

transferred to plastic trays containing sterile coco peat and hardened in green house

conditions for one week period. The well grown healthy seedlings, also called as primary

transformants (T0 generation), were transplanted in earthen pots carrying media mixture

of soil and FYM, and allowed to grow in transgenic containment facility till harvesting. The

per cent explants survived, per cent explants responded and per cent transformation

efficiency were calculated using following formulae;

Explants responded (%) = No. of explants responded

x 100 No. of explants infected

,

Explants survived (%) = No. of explants survived

x 100 No. of explants responded

Transformation efficiency (%) = No. of PCR +ve plants identified in T1

x 100 No. of explants infected

54

3.4 Identification of putative transformants

The putative transformants were identified by sowing the harvested seeds from

each primary transformant (T0) in plant to row progeny manner (all available seeds from

each T0 plants, if more seeds were available, up to 60 seeds). The progenies were tested

using cry and nptII gene specific PCR assay and putative transgenic plants (T1

generation) were identified.

3.4.1 Collection of plant samples and genomic DNA preparation

DNA was extracted from individual plants of primary or putative transformants and

non-transgenic plants. Leaves from 2-3 weeks old plants were collected, surface sterilized

with 70 per cent alcohol and frozen in liquid nitrogen. The leaf samples were stored at -80

°C until further use. The genomic DNA was prepared following the method of Krishna and

Jawali (1997) with a few minor modifications. Frozen tissue sample (2.0 g) was ground

into fine powder in liquid nitrogen, using autoclaved mortar and pestle and immediately

transferred to 2.0 ml Eppendorf tube containing 900 µl of extraction buffer and 90 µl of 20

per cent sodium dodesyl sulphate (SDS). The grounded tissue was not allowed to thaw;

the contents were mixed well and incubated at 65 °C for 10 min with intermittent shaking

for proper mixing of contents. The contents were then cooled on ice for 10 min and

potassium acetate 3 M (300 µl) was added and mixed thoroughly. Further, the contents

were spun for 20 min at 13000 rpm at 4 °C and about 600 µl of supernatant was

transferred to fresh tube and the remaining was discarded along with the tube. About 600

µl of isopropanol-ammonium acetate mixture was added to supernatant to precipitate

nucleic acids. The contents were mixed thoroughly and centrifuged for 20 min at 13000

rpm to pellet the nucleic acids. The supernatant was discarded and the pellet was washed

with 70 per cent alcohol, tubes were inverted on blotting paper to dry the pellet and the

supernatant was carefully drained to avoid damage/slippage of nucleic acid pellet at this

step. Finally, pellet was dissolved in 200 µl of T10E1.

The RNAase (5 µl) (10 mg/ml) was added to each tube. DNA was re-dissolved by

tapping the pellet suspended in RNAase and incubated at room temperature (37 °C) for

30 min. The DNA was precipitated by adding 1/10th volume of 3 M sodium acetate and

2.5 volumes of absolute ethanol. The contents were mixed gently and incubated at 4 °C

for 30 min. The contents were centrifuged for one min at 3000 rpm. The DNA pellet was

55

rinsed with 70 per cent ethanol twice and centrifuged at 3000 rpm for one min.

Supernatant was discarded and the tubes were put upside down on paper towel to

eliminate excess ethanol. Care was taken not to allow the DNA to over dry. Pellet was

dissolved in 100 µl of T10E1 and stored in -20 °C untill further use.

3.4.2 Purification and quantification of extracted genomic DNA

Equal volume (100 µl) of phenol: chloroform: isoamylalcohol (25:24:1) mixture was

added to each tube and the contents were mixed by inverting. The contents were spun at

2500 rpm for 10 min and supernatant was transferred to fresh tubes, 10 µl of 3 M sodium

acetate (1/10 volume of aqueous layer) and 2.5 volumes of chilled absolute ethanol was

added to supernatant and mixed gently and incubated at -20 °C for 15-20 min. The DNA

was spooled in 1.5 ml eppendorf tubes using a glass hook, washed with 70 per cent

alcohol and dried. The DNA was dissolved in 100 µl T10E1 and kept at -20 °C till its further

use. The amount of DNA in each sample was quantified by taking the readings at 260 nm

and 280 nm in the Nano Drop (ND1000-UV/Vis Spectrophotometer, METUS, USA).

Initialization of the instrument was done with nanopure water. The instrument was set

blank with the help of 3 µl T10E1. The quantity of DNA was measured by loading 1-2 µl

DNA sample on Nano-Drop spectrophotometer pedestal. The DNA quantity in ng/µl and

OD value for each sample was noted. The ratio between the readings at 260 and 280 nm

(OD 260/OD 280) was used as an estimate of the purity of the DNA samples. Pure

preparations of DNA have 260 nm/ 280 nm OD ratio between 1.7 and 1.8 (Sambrook and

Russel, 2001). Computed OD values were used to dilute the DNA samples to the working

concentrations of 100 ng/µl for PCR. The DNA working solutions were stored at -20 °C till

further use.

3.4.3 PCR analysis of transformants

Total genomic DNA isolated from young leaves of putative transformants and non-

transgenic plant was used for PCR analysis. The cry and nptII gene fragments were

amplified using PCR assay with gene specific primers. The PCR reaction mixture (20 µl)

contained 0.3 µl (1 U) Taq DNA polymerase (New England Biolabs Pvt. Ltd., UK), 1µl

(1X) assay buffer, 0.5 µl (200 µM) of each dNTP, 1µl (5 µM) of each forward and reverse

primer (Sigma Aldrich Pvt. Ltd., USA) and 1µl (100 ng) template DNA. The DNA extracted

from non-transgenic plants was used as a negative control and the construct carrying cry

56

gene as a positive control. The PCR reaction profile comprised of 38 cycles, with initial

denaturation at 94.0 °C for 3 min followed by cycle denaturation at 94 °C for one min,

annealing at 55.0-64.0 °C (for cry gene specific primers) and 60.0 °C (for nptII gene

specific primers) for 30 sec and extension at 72.0 °C for one min with final extension for

10 min at 72.0 °C. The amplified products were electrophoresed on a 1.2% agarose gel

and visualized under ultraviolet light (Appendix II) (Sambrook and Russel, 2001).

List of gene specific primer pairs used for transgene analysis.

Primer Nucleotide Sequence (5’-3’) Annealing

temperature

cry1Ac Forward: 5’-ACCCAAACATGAAGGAATGC-3’

55.0 °C Revers: 5’-CGGATAGGGTAGGTTCTGGAG-3’

cry2Aa Forward: 5’-GTGGATGGAGTGGAAGAG-3’

64.0 °C Revers: 5’-GAAGAGGGACCAGATGG-3’

cry1F Forward: 5’-CTGCCAATTTGCATCTCTC -3’

56.0 °C Revers: 5’-CCCAGACAGTTTGAGACC -3’

cry1Acm Forward: 5’-TACGACTCAAGGCGATACCC-3’

58.0 °C Revers: 5’-GTGCTGGGAAGATTGGTTGT-3’

nptII Forward: 5’-GAGGCTATTCGGCTATGACTG-3’

60.0 °C Revers: 5’-ATCGGGAGCGGCGATACCGTA-3’


The putative transgenic plants (T1) were maintained in greenhouse condition and

harvested seeds, from each putative transformants, were sown in plant to row progeny

manner. The progenies (T2 generation) were subjected to gene specific PCR assay using

cry and nptII gene specific primer pairs and gene segregation pattern was analysed

based on number of PCR positive and negative plants. The chi-square test was used to

57

study the significance of observed gene segregation pattern with expected 3:1 monogenic

gene segregation pattern.


The putative T2 generation transgenic plants were maintained in greenhouse

condition and harvested seeds, from each putative transformants, were sown in plant to

row progeny manner. The progenies (T3 generation) were subjected to gene specific PCR

assay using cry and nptII gene specific primer pairs and gene segregation pattern was

analysed based on number of PCR positive and negative plants. The chi-square test was

used to study the significance of observed gene segregation pattern with expected 3:1

monogenic gene segregation pattern.

3.7 Insect culture and transgene bioefficacy analysis

The Helicoverpa armigera insect culture was maintained at 26-28 °C and 60%

relative humidity in insect culture room. The chickpea artificial diet was used to feed the

different growth stages of H. armigera. The insect bioassay was carried out in insect

culture room for five days.

3.7.1 Maintenance of insect culture

The oviposition jars for adult moths were prepared by placing moth food petri

plates inside the oviposition jars. Soaked absorbent cotton wad in 50% honey solution

was placed as adult food. Fifteen to twenty moths were released per oviposition jar and it

was covered with black cotton cloth, secured with rubber bands. The eggs were collected

from 2nd day and it was ended on 5th day from each jar. After 2-3 days of egg collection,

hatched neonates were reared on artificial medium. The artificial medium for neonate

larvae was consisted of Part A that was blended with 200 ml of distilled water for 3-5 min,

retaining 200 ml of water (Appendix III). Agar-agar was boiled (Part B) in 400 ml of water

till the agar dissolves completely by intermittent shaking and allowed to cool for 4-5 min

(Appendix III). The molten agar was poured into the blender containing Part A. The steam

was allowed to escape and another 150 ml of water was added in to it. The mixture was

blended for five min and allowed to cool down to 60 °C for 2-3 min. The mixture of sorbic

acid, streptomycin, bavistin, Wesson’s salt mixture and other micro-nutrients (Part C) was

58

mixed completely by rinsing it with 50 ml of water and blended for 2-3 min (Appendix III).

The prepared diet was poured into trays when it is still hot. The diet was cooled at room

temperature for 30 min and used to feed the larvae.

3.7.2 Insect bioassay

The transgenic plants confirmed for the integration of transgene were subjected to

insect bioassay to assess their resistance to H. armigera. Fully expanded leaves from 30-

35 days old plants and 5 days old flowers and pods were excised and leaf petiole/ flower

stalk was inserted into water agar gel (2%) block in the disposable plastic petri plates.

Different plant tissues such as tender pods, flowers and leaves from untransformed plants

served as negative control. On each tissue sample ten first instar neonate larvae (one day

old) were released. Larval mortality and per cent leaf damage was recorded at 24, 48, 72,

96 and 120 hr. The larvae, which failed to show any movement, were considered as

dead. The mortality in transformed plant tissues was corrected with Abbott’s formula

(Abbott, 1925). Two biological and two technical replications were maintained throughout

the experiment and standard deviation was calculated. The experimental design used for

statistical analysis was completely randomized block design and means were compared

at five per cent level of significance using Duncan’s multiple range and Tukey’s test.

3.8 Quantitative estimation of Cry protein using ELISA assay

The quantitative estimation of Cry protein in putative transgenic lines was carried

out using commercially available Quanti-ELISA plates pre-coated with specific Cry

antibody from Envirologix Pvt. Ltd., Hyderabad, India. The sandwich ELISA was carried

out according to the manufacturer’s instructions.

Different tissues samples viz., leaf, flower and pod tissues from transgenic and

non-transgenic plants were collected in disposable 1.5 ml tube. The weight of samples

were taken and used in estimating the Cry protein concentration in µg/g of fresh leaf

tissue. The collected tissues samples were ground by rotating the pestle against the sides

of tube. The process was continued for 20-30 sec and 0.5 ml of 1X extraction buffer

provided with kit was added in to tube along with ground samples. The grinding step was

repeated to mix the tissue samples in extraction buffer. All reagents were allowed to

reach room temperature prior to use. The 100 µl of negative control, 100 µl of each

59

calibrator and 100 µl of each sample extract were added to in two replicates in their

respective places. The content of the well was mixed well by moving the strip holder in

rapid circular motion on the bench top for a full 20-30 sec. Care was taken to avoid cross-

contamination.

Further, the wells in plate were covered with aluminium foil to prevent evaporation

and incubated at ambient temperature for 15 min in shaking condition at 200 rpm on

orbital shaker. The 100 µl of Cry protein specific Cry-enzyme conjugate was added to

each well and mixed thoroughly. Care was taken to avoid cross-contamination. The plate

was covered with aluminium foil to prevent evaporation and incubated at room

temperature for one hour on orbital shaker at 200 rpm. After incubation, the covering was

removed carefully, the content of the well were vigorously shaken and decanted into sink.

The wells were flooded with wash buffer. The washing step was repeated three times.

The plate was inverted and slapped on paper towel to remove as much water as possible.

The 100 µl of substrate was added to each well, the content of well was thoroughly mixed

and covered with aluminium foil. The plate was incubated for 30 min at ambient

temperature in dark condition. The plate was observed for colour development and the

reaction was stopped by adding 100 µl stop solution give with kit.

The spectrophotometric measurements were taken by setting microtiter plate

reader to 450 nm. Based on the slope obtained from slandered graph generated from

calibrators the Cry protein concentrations were calculated by using following formula

given below.

Cry protein conc. (g/mg)

=

(Abs. in test sample – Abs. in negative sample x

Slop x 5

Weight of sample (mg)

3.9 Absolute real time qRT-PCR for cry transcript analysis

The absolute quantification of cry mRNA levels was carried out using real time

qRT-PCR assay. The cry transcript levels were analyzed in three different tissue type viz.,

leaf, flower and pod tissues from selected transgenic events carrying different cry genes

and non-transgenic plant tissue samples.

60

3.9.1 Tissue collection

The selected transgenic pigeonpea plants/events were used to collect leaf, flower

and pod tissues. Normal non-transgenic pigeonpea plants of same genotype were used

as controls. The leaf, flower and pod tissues were harvested from each transgenic plant

and non-transgenic control, separately and carefully labelled. The tissue samples were

collected in such a way to have two biological and two technical replications and stored

immediately in liquid nitrogen, shifted to -80 °C.

3.9.2 Preparation of RNase free water

The RNasa free water was prepared by treatment of diethyl pyrocarbonate (DEPC)

(Sigma Aldrich, USA) to the millipure water and used for preparation of all the solutions

required for RNA isolation. The 0.1% DEPC (one ml) was added to required quantity of

water (999 ml) and kept overnight with gentle shaking. Overnight DEPC treated water

was autoclaved next day in order to degrade the DEPC and this DEPC free water was

used for the preparation of reagents. The utensils and materials used for RNA isolation

was treated with DEPC treated un-autoclaved water for overnight and autoclaved before

used to make them RNase free.

3.9.3 Isolation and purification of total RNA

Total RNA was isolated from leaf, flower and pod tissues samples from selected

transgenic events expressing different cry genes and non-transgenic control pigeonpea

plants using TRIzol reagent (Sigma-Aldrich Pvt. Ltd. USA) as following protocol.

Frozen tissue sample approximately 1.0 g of tissue was grounded into fine powder

in a pre-chilled mortar and pestle and the ground tissue was transferred into a pre-chilled

50 ml conical tube and 5-10 ml of TRIzol reagent (one ml of TRIzol/100 mg of tissue) was

added. Contents were mixed well with vortex and one ml of the mixture was transferred

into labelled, RNase-free 1.5 ml tubes to incubate for 5 min at room temperature.

Chloroform 200 l per one ml of TRIzol was added and vortexed for 20 sec and

incubated the content for 10 min at room temperature and centrifuged at 13,000 rpm for

10 min at 4 °C. The aqueous phase was carefully transferred into a new RNase-free 1.5

61

ml tube without disturbing the other phases and the tube was placed on ice as soon as

transferred. Equal volume of isopropanol was added and mixed gently by inverting 2-3

times and incubated it on ice for 30 min and centrifuged at 13,000 rpm for 20 min at 4 °C.

A very small pellet was visible at the bottom of the tube that was total RNA. The

supernatant was decanted and allowed to stand upside down on kimwipes (Kim tech

science, Canada) for 5 min. The pellet was washed with 75 per cent ethanol (500 l). The

liquid was decanted and the inside of the tube was wiped to dry with a clean Kimwipe,

without touching the pellet. The pellet was re-suspended in 500 l of RNase-free water

and was incubated on ice for at least an hour and pipetted occasionally for dissolving the

pellet. The content was centrifuged at 13,000 rpm for 20 min at 4 °C and the supernatant

was transferred into a new RNase-free 1.5 ml tube. It was precipitated with 10 per cent

3M sodium acetate and equal volume of isopropanol. Contents were incubated on ice for

one hr or overnight at -80 °C and centrifuged at 13,000 rpm for 20 min at 4 °C. The final

pellet was re-suspended into 10 l of RNase-free water and was stored at -80 °C.

The total RNA was treated to remove DNA using Turbo DNA-freeTMkit (cat#

AM1907 Ambion, USA) as per the manufacturer’s instruction. In order to eliminate the

genomic DNA, 20 g of total RNA was digested with RNase-free DNase I and finally the

total RNA was precipitated into desired volume of water. The quantity and quality of total

RNA was checked using NanoDrop ND-1000 spectrophotometer (NanoDrop

Technologies, USA). The total RNA was subjected to Nanodrop ND-1000 using RNase-

free water as blank: absorbance was recorded at 260/280 and 260/230. Further, the

sample was fractionated over a formaldehyde agarose gel (Appendix IV). Absence of

genomic contamination was subsequently confirmed by PCR with total RNA as template

(Caldana et al., 2007).

3.9.4 Preparation of cDNA

Single stranded cDNA was prepared by using High Capacity cDNA Reverse

Transcription kit (cat# 4368814, Ambion, USA) as per the manufacturer’s protocol. All

reagents were thawed and stored on ice in RNase and DNase free work environment. All

individual reagents were mixed thoroughly and spin down and pipetted. About two g of

total RNA in a single 20 l reaction was quantitatively converted to single-stranded cDNA

using standard thermal condition mentioned below.

62

Composition of components used for the cDNA synthesis reaction

Sl. No. Components Volume/reaction (ml)

1 10X RT buffer 2.00

2 25X dNTP (100mM) 0.80

3 10X RT random primers 2.00

4 MultiScribeTM Reverse Transcriptase 1.00

5 RNase inhibitor 1.00

6 Nuclease free water 3.20

7 Total RNA (2 mg) 10.00

Total volume 20.00

Thermal condition for single-stranded cDNA conversion

Step 1 Step 2 Step3 Step 4

Temperature 25.0 °C 37.0 °C 85.0 °C 4.0 °C

Time 10 min 120 min 5 sec Hold

3.9.5 Standardization of real-time PCR condition

The primer concentration is one of the key factors in real-time quantitation of any

gene expression. Primer concentrations ranging from 150 nM, 200 nM, 250 nM, 300 nM,

350 nM, and 400 nM were used to optimize the amplification. Primer at 200 nM

concentration gave single melting curve, low Ct value, high fluorescence value and no

primer dimer when loaded on gel. Thus, this concentration of primer was used for rest of

the experiment. Similarly, it is also essential to determine optimal annealing temperature

63

for each primer pair before their use. All primers designed for cry genes were tittered for

optimal annealing temperature ranges from 55 to 60 °C.

3.9.6 qRT-PCR reaction

The master mix of different components of real-time PCR was prepared fresh to

avoid handling errors. The reaction mixture of 10 l containing 1.0 ng cDNA, 200 nM of

each gene-specific primer and 5 l of 2X SYBR green reagents (Cat. #4367659, Ambion,

USA) were used in the experiment. Individual components of reaction mixture were

standardized for 10 l reaction volume. An Eppendorf mastercycler ep realplex

instrument (Eppendorf Pvt. Ltd., Germany) was used for all real-time PCR amplifications.

Two biological and two technical replications were maintained for each treatment. An

optimal PCR conditions used for amplification are given as follows.

Real-time PCR amplification conditions

Stage Step Temperature

(°C) Duration No. of cycles

1 Initial denaturation 95.0 °C 10 min 1

2 Denaturation 95.0 °C 15 sec

40 3 Annealing (vary with

primer)

55.0-58.0 °C 20 sec

4 Extension 60.0 °C 20 sec.

5 Melting curve 95.0 °C 10 min 1

64

List of primer used for real time qPCR analysis.

Primer Nucleotide Sequence (5’-3’) Annealing

temperature

rt1Ac Forward: 5’-ACGAAATCCCACCACAGAAC-3’

58.0 °C Revers: 5’-ACGGAACTGTTGCTGAATCC-3’

rt2Aa Forward: 5’-CCGCTCCATTACAACCAGAT-3’

56.5 °C Revers: 5’-ATGGTGAAGCCGGTGTAGTC-3’

rt1F Forward: 5’-TACTGGGGCTTAGGGGAGT -3’

55.0 °C Revers: 5’-GCGGACAAAGGTAACGTGAT-3’

rt1Acm Forward: 5’- TGCCTCCCTTACAACCAATC-3’

56.0 °C Revers: 5’- CGCTATTATCTTGGGGTGGA-3’

3.9.7 Absolute quantification of mRNA levels

Absolute quantification of target cry mRNA was performed based on the standard

curve. The cry gene fragments were PCR amplified, eluted and purified. The purified PCR

products were quantified using Nanodrop ND-1000 and absorbance was recorded at

260/280 and 260/230. The purified product was diluted to prepare standards of 1.0 ng/µl,

10.0 ng/µl and 100.0 ng/µl and used as calibrators during real time PCR reaction. The

observed absorbance for prepared standards was plotted against the known

concentration of template and slop was calculated. Based on the slop, the cry mRNA

levels in test samples was quantified in ng/µl.

3.10 Genomic Southern blot analysis

Genomic Southern blot analysis was carried out using the DIG-High Prime DNA

Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany, cat. no.

11585614910). Genomic DNA was isolated from the young leaves of T2/T3 generation

plants and blotting was carried out following standard protocol (Sambrook et al., 1989).

The 15 µg of high molecular weight genomic DNA was digested with restriction

65

endonuclease viz., EcoRI, HindIII and BamHI separately overnight at 37 °C. The total

volume of the digestion reaction was set to 20 µl. One µl of each sample was checked for

digestion in 0.8% (w/v) agarose gel stained with ethidium bromide and visualized under

UV light. The digested DNA was heated in boiling water for 10 min and quickly chilled on

ice. Such DNA samples were electrophoresed at 40 V 6-8 hr on 0.8% (w/v) agarose gel

followed by denaturation solution treatment (Appendix V) for 30 min and neutralization

solution treatment (Appendix V) for 30 min.

The DNA fragments were transferred from the gel to the Biodyne® B, 0.45 µm

Positively-charged Nylon 6,6 Transfer Membrane (PALL Life Sciences) by capillary

movement of transfer solution (10X Sodium-sodium citrate; SSC) through agarose gel in

to transfer membrane kept overnight. Further, the membrane was exposed to UV light for

UV crosslinking for 10 min. The membrane was pre-hybridized for at least two hr in 10 ml

of hybridization buffer (Appendix V) without probe. The cry gene specific PCR-amplified

fragments from cry gene construct were random primed labelled with DIG-11-dUTP and

were used as hybridization probes. Probe hybridization was performed overnight at 48 °C

in hybridization chamber and the membranes were washed three times each in wash

buffer I (Appendix V) for 10 min, followed by wash buffer II (Appendix V) for 10 min at 68

°C, and wash buffer III (Annexure V) for 10 min. The membrane was treated with blocking

solution (Appendix V) for 30 min followed with antibody treatment (Appendix V) for 30 min

and washed twice with wash buffer III, 15 min each.

Next, the membrane was incubated in freshly prepared substrate solution

(Appendix V) in an appropriate container in the dark without disturbing. Reaction was

stopped by washing the membrane for five minutes with 20 ml sterile water. The

membrane was dried and results were documented by scanning the blot.

3.11 Northern blot analysis

Northern blot analysis was carried out using the DIG-High Prime DNA labelling and

Detection Kit (Roche Diagnostics, Mannheim, Germany, cat. no. 11585614910). Total

RNA was isolated from the young leaves, flower and pod tissues of transgenic and non-

transgenic control plants and blotted following standard protocol (Sambrook et al., 1989).

The 10 µg of total RNA was mixed with 10 µl of 5X sample buffer (one ml glycerol, one ml

10X MOPS and one pinch of bromophenol blue), heated at 65 °C for 15 min, put on ice,

66

then loaded into gel wells and electrophoresed at 40 V for 2 hr on 1.0% (w/v)

formaldehyde agarose gel (Appendix IV) followed by denaturation solution treatment for

30 min and neutralization solution treatment for 30 min.

The RNA strands were transferred from the gel to the Biodyne® B, 0.45 µm

Positively-charged Nylon 6,6 Transfer Membrane (PALL Life Sciences) by capillary

movement of transfer solution (10X SSC) from agarose gel in to transfer membrane, kept

overnight. Further the membrane was exposed to UV light for UV crosslinking for 10 min.

The membrane was pre-hybridized for 2 hr in 10 ml of hybridization buffer (Appendix V)

without probe. Specific PCR-amplified fragments from respective cry construct were

random primed labeled with DIG-11-dUTP and were used as hybridization probes. Probe

hybridization was performed overnight at 48 °C in hybridization chamber and the

membranes were washed three times each in wash buffer I (Appendix V) for 10 min,

followed by wash buffer II (Appendix V) for 10 min at 68 °C, and wash buffer III (Appendix

V) for 10 min. The membrane was treated with blocking solution (Appendix V) for 30 min

followed with antibody treatment (Appendix V) for 30 min and washed twice with wash

buffer III, 15 min each. Then the membrane was incubated in freshly prepared substrate

solution (Appendix V) in an appropriate container in the dark without disturbing. Reaction

was stopped by washing the membrane for 5 minutes with 20 ml sterile water. The

membrane was dried and results were documented by scanning the blot.

3.12 Recovering the site of transgene integration by TAIL PCR

Selected transgenic (T3 generation) plants expressing cry gene were analyzed for

site of integration of T-DNA using TAIL-PCR. T-DNA specific primers, 35S Reverse (5’-

GATAGTGGGATTGTGCGTCA-3’) and arbitrary degenerate AD1 (5’- NTCGAS

TWTSGW-3’), AD2 (5’-NGTCGASWGANAWGAA-3’) and AD3 (5’-AGWGNAGW ANCA-

3’) primers were used to recover the flanking genomic region. The TAIL-PCR included

primary, secondary and tertiary amplification reactions. The primary PCR product was

diluted to 1:29 ratio using nano-pure water and used as template for secondary reaction.

The product of secondary reaction was diluted to 1:9 ratio using nano-pure water and

proceed to tertiary reaction. The primary, secondary and tertiary products were run in

adjacent lanes on a 1.0% low melting agarose gel. The specificity of the products was

confirmed by the expected size change between the secondary and tertiary products.

67

Higher most band from tertiary PCR was eluted and taken for transformation. The eluted

product was ligated into the pTZ vector by using InsTAcloneTMPCR Cloning Kit as per

manufactures instruction.

Components of primary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction

Sl. No. Reaction components Quantity (µl)

1 Water 12.8

2 Buffer 2.0

3 dNTP’s 2.0

4 Specific primer 0.6

5 Arbitrary degenerate primer 0.6

6 Taq polymerase 1.0

7 DNA 1.0

Total 20

Components of secondary and tertiary Thermal Asymmetric Inter Laced - Polymerase

Chain Reaction

Sl. No. Reaction components Quantity (µl)

1 Water 16.7

2 Buffer 2.5

3 dNTP’s 2.0

4 Specific primer 1.0

5 Arbitrary degenerate primer 1.0

6 Taq polymerase 0.8

7 DNA 1.0

Total 25.0

68

Primary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction programme

Particulars No. of cycles

95.0 °C, 3 min; 95.0 °C, 1 min

94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 5

94.0 °C, 30 sec; 25.0 °C, 2 min; 72.0 °C, 3 min 1

94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 15 each

94.0 °C, 30 sec; 41.6 °C, 1 min; 72.0 °C, 2 min

72.0 °C, 15 min

Secondary and tertiary Thermal Asymmetric Inter Laced -Polymerase Chain Reaction

programme

Particulars No. of cycles

94.0 °C, 1 min

94.0 °C, 30 sec; 54.0 °C, 1 min; 72.0 °C, 2 min 2 cycles

94.0 °C, 30 sec; 41.6 °C, 1 min; 72.0 °C, 2 min 20 cycles

72.0 °C, 30 min

3.12.1 Preparation of competent cells

The competent cells were prepared using E. coli DH5α culture by following the

protocol given by Sambrook et al. (1989) with minor modification as described below. An

isolated colony from E. coli DH5α plate was inoculated into 5 ml Luria broth (LB)

(Appendix VI) and incubated at 37 °C overnight on shaking condition at 200 rpm. The

culture was diluted to 1:100 using LB i.e., 0.5 ml of culture was added to 50 ml of LB. The

69

culture was incubated for 2 to 3 hr till it attained an OD of 0.3 to 0.4 at 600 nm. The

culture was chilled in ice for 30 min and 25 ml of culture was dispensed into two

centrifuge tubes of capacity 50 ml. The cells were pelleted at 6000 rpm for 5 min, the

supernatant was discarded and pellet was suspended in 12.5 ml of ice-cold 0.1M calcium

chloride. The centrifuge tubes were again incubated in ice for 45 min and later centrifuged

at 4000 rpm for 10 min. The pellet was dispensed in one ml of 0.1M CaCl2 and to this 88

µl of dimethyl sulfoxide (DMSO) was added. About 200 µl of cells were distributed to each

chilled 1.5 ml micro centrifuge tubes and immediately used.

3.12.2 Transformation of E. coli DH 5α

A freshly prepared competent cell stock (about 100 µl) was taken in a chilled

centrifuge tube and of ligated mixture (10 µl) was added into the tube and was mixed

gently. The mixture was chilled in ice bath for 45 min. Heat shock was given by shifting

the chilled mixture to preheated 42 °C water bath for exactly two min and then shifted to

ice to chill for 5 min. About 800 µl of Luria broth was added to the mixture and incubated

at 37 °C at 200 rpm for 45 min. The culture was centrifuged at 13,000 rpm for one min

and about 700 µl of supernatant was discarded. The pellet was dissolved in remaining

supernatant and spread on the plates having Luria agar with Amp100, X-gal and IPTG,

and incubated overnight at 37 °C. The plates were observed for growth of colonies and

the white colonies were selected and re-streaked on Luria agar plate with Amp100, X-gal

and IPTG.

3.12.3 Colony PCR

The PCR was performed by using selected colonies: loop of colony was taken and

inoculated in the reaction mixture. The reactions were carried out using a master thermal

cycler. The amplified products were assayed by electrophoresis on 1.2% agarose gels.

The colonies, which showed the expected size of the amplified fragment by M13 forward

(5’-GTTGTAAAACGACGGCCAGT-3’) and M13 reverse (5’-CAGGAAACAGCTATGACC-

3’) primers were selected for further confirmation of the transformed colonies.

70

Components used in colony polymerase chain reaction

Sl. No. Components Volume/reaction (µl)

1 Standard Taq reaction buffer (10X) 2.50

2 dNTP’s (1mM) 0.50

3 Forward primers (5 pmol/µl) 1.00

4 Reverse primers (5 pmol/µl) 1.00

5 Template (colony) 1.00

6 Taq DNA polymerase 0.50

7 Water 18.5

Total volume 25.00

Colony polymerase chain reaction programme

Particulars Duration No. of cycles

94.0 °C 4 min 1

94.0 °C 1 min

35 54.0 °C 1 min

72.0 °C 1 min

72.0 °C 10 min 1

4.0 °C ∞ 1

3.12.4 Plasmid isolation

White colonies were inoculated to 10 ml LB carrying ampicillin (100 µg/ml) as a

selection pressure and incubated overnight at 37 °C on shaker at 200 rpm. Overnight

grown culture was centrifuged at 5000 rpm for 2 min in 2.0 ml micro centrifuge tubes. The

71

supernatant was removed and pellet was washed with STET buffer (0.25 volume of

original culture) (Appendix VI), then it was centrifuged at 5000 rpm for two min. The pellet

was re-suspended in 200 µl of ice-cold alkaline-lysis solution-I (Appendix VI) by vigorous

vortexing. The 400 µl of freshly prepared alkaline-lysis solution-II (Appendix VI) was

added to each tube and the contents were mixed by inverting the tubes for 4 to 5 times

and kept in ice for about 5 min. To this suspension, 300 µl of alkaline-lysis solution-III

(Appendix VI) was added and again mixed thoroughly by gently inverting the tubes 4-5

times. The tubes were stored on ice for 5 min and centrifuged at 13,000 for 8 min at 40

°C.

The supernatant was transferred to fresh tubes and equal volume of phenol:

chloroform: Isoamyl alcohol (25:24:1) was added to precipitate proteins and mixed well.

The tube was centrifuged at 13,000 rpm for 10 min at 4 °C. The aqueous layer was

transferred to a fresh tube and two volumes of isopropanol were added. The contents

were mixed and allowed to stand for 2 minutes at room temperature. The solution was

later centrifuged at 13,000 rpm for 5 min. The supernatant was discarded and pellet was

washed with 70 per cent ethanol and spun for 1 min at 13,000 rpm to recover the plasmid.

The supernatant was discarded, pellet was dried completely and dispensed into 30 µl of

T10E1 (pH 8.0) containing 3 µl of RNase A (10 mg/ml). The solution was kept at 50 °C for

15 min and then stored at -20 °C. The confirmation of the presence of cloned fragment

was done by PCR amplification.

3.12.5 Sequencing of clones and sequence analysis

The independent clones were sequenced using M13 primers at Sci-Genom Pvt.

Ltd., Kochi. The obtained sequences (forward and reverse) were multiple aligned and

contig was formed. Such contigs were processed to remove vector backbone by vector

backbone trim algorithm available with BioEdit software (Hall, 2011). Such processed

contigs were subjected for blast analysis against pigeonpea draft genome sequence

database by establishing local blast search using BioEdit software. The T-DNA flanking

genomic region was recovered based on local blast results.

72

3.13 The plant growth parameters

The selected plant growth parameters were observed in developed transgenic

plants and compared with the respective parental genotypes used during transformation

processes (ICPL 87119 and BSMR 736). The observations for different plant growth

parameters were taken in greenhouse conditions.

1. Stem colour: The stem colour of developed transgenic plants was observed and

compared with the typical characteristic green stem colour of ICPL 87119 and BSMR

736 non-transgenic plants.

2. Flower colour: The transgenic plants were observed for flower colour and compared

with that of ICPL 87119 (Yellow with pink streaks) or BSMR 736 (Yellow) non-

transgenic parental genotypes.

3. Growth habit: The growth habit of transgenic plants was analysed and categorised into

spread, semi spread and compact types and compared with that of ICPL 87119 (semi

spread) or BSMR 736 (semi spread) non-transgenic plants.

4. Leaf colour and shape: The observation of leaf colour and shape were taken for

identified transgenic plants and compared with that of ICPL 87119 (light green and

tapering) or BSMR 736 (dark green tapering) non-transgenic parental genotypes.

5. Branching pattern: The branching pattern in developed transgenic plants was observed

and categorised into more, medium and few (ICPL 87119 and BSMR 736; medium).

6. Fresh pod colour: The transgenic plants were observed for fresh pod colour (light

green and green with purple streaks) and compared with that of ICPL 87119 and

BSMR 736 non-transgenic plants.

7. Seed colour: The seed colour development was noticed in transgenic plants and

parental ICPL 87119, BSMR 736 non-transgenic parental genotypes.

8. Plant height: The trait, plant height, in case of transgenic plants was observed and

compared with that of non-transgenic control plants (ICPL 87119 and BSMR 736).

73

3.14 Statistical analysis

Required number of replications (technical and biological) was maintained for all

experiments and standard deviation was calculated. The experimental design used for

statistical analysis was completely randomized block design and means were evaluated

at five per cent level of significance using Duncan’s multiple range tests and Tuke’s test

(MSTAT-C program). For statistical analysis of per cent data, arcsine transformation was

performed for per cent data before subjecting it for any statistical test. Statistical

independent and unpaired ‘t’-test was performed to compare the performance of two

groups in different experimental treatments. The correlation analysis was performed using

SPSS statistical program for insect mortality, Cry protein and cry gene transcript levels

from different transgenic lines. The chi-square analysis was done to test the transgene

segregation pattern using MS-Excel program.

74

4. EXPERIMENTAL RESULTS

The present investigation was carried out to improvise the pigeonpea

transformation protocol using in vitro and in planta transformation methods. The

transformation protocol with good transformation efficiency was employed to develop

transgenic pigeonpea expressing different cry genes viz., cry1Ac, cry2Aa, cry1F and

cry1Acm separately. The transgenic lines were characterised for their bio-efficacy

against H. armigera. The investigation also encompassed the molecular characterisation

of selected transgenic lines of pigeonpea. The experimental results are presented below.

4.1 Effect of cytokinins and their concentration regimes on multiple shoot induction

and plantlet regeneration in pigeonpea

The surface sterilized seeds, cultured on MS basal and MS medium

supplemented with different cytokinins and their concentration regimes indicated 50-60

per cent germination after 7 days in both the genotypes viz., ICPL 87119 and BSMR

736. The different concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) of three cytokinins

viz., BAP, TDZ and zeatin were assessed to study their effect on shoot bud induction in

pigeonpea.

4.1.1 Multiple shoot bud induction in response to BAP concentration regimes

The number of cotyledonary node with cotyledon explants responded to different

concentration regimes of BAP for shoot bud induction ranged from 0.0 to 36.0 in case of

ICPL 87119 and from 0.0 to 36.5 in case of BSMR 736 (Table 1). The maximum number

of explants responded for shoot bud induction was noticed at 2.0 mg/L BAP

concentration level, whereas it was list at 10 mg/L BAP, in both genotypes. The number

of shoot bud induced per explant ranged from 0 to 52.6 for ICPL 87119 and from 0 to

53.7 for BSMR 736 (Table 2). The maximum number of shoot buds induced was

recorded in MS medium supplemented with 2.0 mg/L BAP. For embryo disc with half

cotyledon explants, the explants response ranged from 0 to 26.5 (ICPL 87119) and 0 to

27.5 (BSMR 736) in pigeonpea genotypes (Table 3). The number of explants responded

to BAP fortification was at its maximum when medium was fortified with 2.0 mg/L BAP.

Interestingly, there was a concomitant decrease in explants response with increase in

BAP concentration. Similar kind of explants response to multiple shoot bud induction was

75

Table 1: Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from cotyledonary node with cotyledons explant of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture (50 explants)

Growth hormone

concentrations

Number of explants responded (mean ±SD)

Grand mean

Benzylamino purine Thidiazuron Zeatin

ICPL 87119

BSMR 736

ICPL 87119

BSMR 736

ICPL 87119

BSMR 736

0 mg/l 0.0±0.0 c 0.0±0.0d 0.0±0.0e 0.0±0.0d 0.0±0.0c 0.0±0.0c 0.0±0.0d

1 mg/l 23.5±2.1b 21.0±1.4b 22.5±3.5c 23.5±2.1bc 29.5±2.1a 30.5±3.5a 25.1±3.9b

2 mg/l 36.0±1.4a 36.5±2.1a 23.5±2.1c 23.5±3.5bc 33.0±1.4a 32.0±1.4a 30.8±5.8a

3 mg/l 32.0±2.8a 32.5±2.1a 29.5±2.1ab 29.0±1.4ab 22.5±3.5b 22.0±1.4b 27.9±4.6a

4 mg/l 26.0±2.8b 24.5±2.4b 33.5±3.5a 33.5±2.1a 21.5±3.5b 21.5±2.1b 26.8±5.5b

6 mg/l 24.0±1.4 b 24.5±0.7b 25.5±2.1bc 24.5±3.5bc 21.5±2.1b 20.5±1.2b 23.4±1.9b

8 mg/l 22.5±2.1 b 21.5±1.4b 23.5±2.2c 22.5±3.5bc 19.0±2.8b 19.0±1.8b 21.3±1.9bc

10 mg/l 21.0±1.4 b 16.0±2.8c 17.5±3.5d 18.5±2.9c 18.5±2.1b 17.5±3.5b 18.2±1.7c

Grand mean 23.1±10.6a 22.1±11.1a 21.9±10.1a 21.9±9.9a 20.7±9.7a 20.4±9.8a 24.8±9.7

Note: The means followed with same letters are within student ‘t’ range at α=0.05.

76

Table 2: Number of shoot bud induced in response to different concentration regimes of cytokinins (benzylamino purine, thidiazuron and zeatin) from cotyledonary node with cotyledons and embryo discs with half cotyledons explants.

Growth hormone conc-

entrations

Number of shoot bud induced per explant (mean ±SD)

Cotyledonary node with cotyledons

Grand mean

Embryo discs with half cotyledon

Grand mean

Benzylamino purine Thidiazuron Zeatin Benzylamino purine Thidiazuron Zeatin

ICPL 87119 BSMR 736 ICPL 87119

BSMR 736 ICPL 87119

BSMR 736

ICPL 87119

BSMR 736

ICPL 87119

BSMR 736 ICPL 87119

BSMR 736

0 mg/l 0.0±0.0f 0.0±0.0

f 0.0±0.0

e 0.0±0.0

e 0.0±0.0

e 0.0±0.0

e 0.0±0.0

e 0.0±0.0

e 0.0±0.0

e 0.0±0.0

d 0.0±0.0

d 0.0±0.0

d 0.0±0.0

d 0.0±0.0

d

1 mg/l 29.9±5.6c 30.2±6.2

c 22.8±4.6

d 26.0±3.9

d 35.9±4.9

b 35.3±6.0

b 30.2±5.1

c 2.8±0.6

cd 3.1±0.6

bc 2.5±0.5

c 2.8±0.6

c 4.2±1.0

a 4.2±0.8

a 3.2±0.7

b

2 mg/l 52.6±5.9a 53.7±4.9

a 30.5±3.9

bc 32.2±3.3

bc 40.4±4.5

a 40.9±5.8

a 41.7±9.8

a 4.8±0.8

a 4.4±0.5

a 3.1±0.7

c 3.3±0.7

bc 3.5±0.5

b 3.6±0.5

b 3.7±0.6

a

3 mg/l 40.4±5.8b 41.2±4.9

b 31.9±4.3

b 34.9±2.6

b 30.8±3.0

c 29.3±3.9

c 34.7±5.0

b 3.5±0.5

b 3.2±0.6

b 4.0±0.9

b 3.9±1.1

ab 2.8±0.8

c 2.9±0.7

c 3.3±0.5

b

4 mg/l 32.9±5.2c 33.8±4.2

c 41.1±5.7

a 46.1±5.3

a 27.3±4.4

c 25.3±3.8

d 34.4±7.9

b 3.0±.0.7

bc 2.7±0.7

cd 4.7±1.0

a 4.5±0.8

a 2.7±0.7

c 2.6±0.5

c 3.3±0.9

b

6 mg/l 23.9±4.2d 25.9±1.7

d 31.4±2.9

bc 33.4±3.0

b 22.4±3.3

d 23.5±3.3

d 26.7±4.5

c 2.5±0.5

cd 2.6±0.5

d 2.7±0.7

c 2.9±0.6

c 2.5±0.5

c 2.4±0.5

c 2.6±0.2

c

8 mg/l 19.7±4.3de

21.2±3.7e 27.6±3.7

c 29.6±4.5

c 20.0±3.8

d 21.7±3.3

d 23.3±4.2

d 2.4±0.5

d 2.6±0.5

d 2.7±0.7

c 2.7±0.5

c 2.4±0.5

c 2.5±0.5

c 2.5±0.1

c

10 mg/l 18.7±2.8e 20.1±3.4

e 22.8±2.7

d 25.8±3.1

d 21.0±3.0

d 22.0±2.5

d 21.7±2.4

d 2.3±0.5

d 2.5±0.5

d 2.5±0.5

c 2.6±0.5

c 2.6±0.5

c 2.4±0.5

c 2.4±0.1

c

Grand mean 27.2±15.7a 28.2±15.9

a 26.0±12.0

a 28.5±13.1

a 24.7±12.3

a 24.7±12.0

a 26.5±13.0 2.6±1.4

a 2.6±1.2

a 2.7±1.4

a 2.8±1.3

a 2.5±1.2

a 2.5±1.2

a 3.1±1.2

Note: The means followed with same letters are within student ‘t’ range at α =0.05.

77

Table 3: Effect of benzylamino purine, thidiazuron and zeatin on direct multiple shoot induction from embryo discs with half cotyledon explants of pigeonpea genotypes, ICPL 87119 and BSMR 736, after 12 days of culture (50 explants)

Growth hormone concentrations

Number of explants responded (mean ±SD)

Grand mean Benzylamino purine Thidiazuron Zeatin

ICPL 87119 BSMR 736 ICPL 87119 BSMR 736 ICPL 87119 BSMR 736

0 mg/l 0.0±0.0e 0.0±0.0e 0.0±0.0c 0.0±0.0c 0.0±0.0c 0.0±0.0d 0.0±0.0d

1 mg/l 19.0±1.4bc 18.5±3.5bc 16.5±2.1b 18.0±2.8b 21.0±3.5a 21.0±1.4ab 19.0±1.8b

2 mg/l 26.5±2.1a 27.5±2.1a 19.0±1.4b 19.0±1.4b 21.5±4.9a 22.0±2.8a 22.5±3.6a

3 mg/l 23.0±1.4ab 23.0±2.8ab 22.0±2.8a 23.5±2.1a 16.0±2.8b 17.5±3.5abc 20.8±3.2ab

4 mg/l 16.5±2.1cd 18.0±1.4bc 22.0±5.6a 25.0±1.4a 16.5±2.1b 17.0±1.4bc 19.1±3.5b

6 mg/l 13.5±2.1d 15.0±1.4cd 17.5±3.5b 16.5±2.1b 15.0±4.1b 16.0±1.4c 15.5±1.3c

8 mg/l 12.0±1.4d 12.5±0.7d 16.5±2.1b 15.5±0.7b 16.0±1.4b 15.5±1.2c 14.6±1.9c

10 mg/l 12.0±2.8d 11.0±1.4d 17.5±0.7b 16.0±2.4b 16.5±2.1b 16.0±1.4c 14.8±2.6c

Grand mean 15.3±8.1a 15.6±8.3a 16.3±6.9a 16.6±7.6a 15.3±6.7a 15.6±6.8a 18.1±7.1


78

noticed in both pigeonpea genotypes. The number of shoot bud induced from embryo

discs with half cotyledon explants was ranged from 0 to 4.8 in ICPL 87119, whereas it

was from 0 to 4.4 in BSMR 736 (Table 2). In MS medium supplemented with 2.0 mg/L

BAP recorded highest number shoot buds (4.8; ICPL 87119) when embryo discs with

half cotyledon was used as explant. Interestingly, there was a concomitant decrease in

resonance of explants for shoot bud induction and number of shoot bud induced with

further increase in BAP levels in growth medium (Fig. 1; 2).

The behavioral response of two genotypes to same BAP level was non-significant

(p > 0.72) as revealed by ‘t’-test statistical analysis. At the same time, there was a

significant difference between cotyledonary node with cotyledons and embryo discs with

half cotyledon explants for their response to same BAP concentration regime (p < 0.05).

Similarly, there was no significant difference for number of shoot bud induced per

explants in response to same BAP level between two genotypes (p > 0.82). Whereas,

the effect of same BAP level on shoot bud induction from cotyledonary node with

cotyledons and embryo discs with half cotyledon was found be significant (p < 0.001).

4.1.2 Multiple shoot bud induction in response to TDZ concentration regimes

The number of cotyledonary node with cotyledon explants responded to different

TDZ concentration regimes ranged from 0 to 33.5 in case of both genotypes (Table 1).

Further, in both genotypes, maximum number of explants responded for shoot bud

induction was noticed at 4.0 mg/L TDZ concentration level (Plate 1). It was observed that

the number of shoot bud induced per explant ranged from 0 to 41.1 (ICPL 87119) and

from 0 to 46.1 (BSMR 736) in respective genotypes (Table 2). The embryo disc with half

cotyledon explants response to increasing TDZ concentration regimes ranged from 0 to

22.0 in ICPL 87119 and 0 to 25.0 in BSMR 736. The maximum number of explants

responding was recorded in BSMR 736 at 4.0 mg/L TDZ concentration regime (Table 3).

The number of shoot bud induced from embryo discs with half cotyledon explant was

ranged from 0 to 4.7 (ICPL 87119) and from 0 to 4.5 (BSMR 736) (Table 2). At TDZ

concentration regime of 4.0 mg/L, the highest number

shoot buds (4.7) was recorded in ICPL 87119 when embryo discs with half cotyledon

was used as explant. Similarly, it was noticed that any further increase in TDZ

concentration levels imparts reduction in multiple shoot bud induction in both explants

types of pigeonpea (Fig. 1 and 2).

79

80

81

Plate 1: Direct multiple shoot bud induction and plantlet regeneration from cotyledonary node with

cotyledons, embryo dises with half cotyledon explants of pigeonpea A. Multiple shoot buds

induced from cotyledonary node with cotyledons explants cultured on MS +2mg/L BAP after 10

days of inoculation. B.shoot growth after first round of subculture on MS + 4 mg/L TDZ. C.

Development of sugary white callus around induced shoot buds when explants were cultured on

MS +10 mg/L.TDZ.D. Multiple shoot buds induced from embryo discs with half cotyledon explants

cultured on MS + 1 mg/L zeatin after 15 days of inoculation.E. Shoots with profuse rooting on MS

+0.5 mg/LIBA. F. Multiple shoot buds induced (up to 52 shoot buds) from cotyledonary node with

cotyledons explants cultured on MS + 2mg/LBAP after 14days of inoculation.

82

There was no significant difference between the responses of genotypes to

different levels of TDZ concentrations (p > 0.43). On other hand, the effect of TDZ

concentration regime on explants response was found significant (p < 0.001). The

statistical analysis revealed that there was no significant difference between genotypes

for number of shoot bud induced per explant in response to same TDZ level (p > 0.83).

At the same time, the two explants types showed significant difference for their response

to multiple shoot bud induction at same TDZ concentration regimes in both genotypes (p

< 0.02). The analysis of means revealed that the treatment 4.0 mg/L of TDZ was most

effective for induction of multiple shoot buds per explants from both explants and

genotypes.

4.1.3 Multiple shoot bud induction in response to zeatin concentration regimes

The augmentation of MS with different zeatin concentration regimes resulted in

the multiple shoot bud induction in cotyledonary node with cotyledon explants, which

ranged from 0 to 33.0 in ICPL 87119 and from 0 to 32.0 in BSMR 736 (Table 1). In both

genotypes, the maximum number of explants responded for shoot bud induction was

recorded at 2.0 mg/L zeatin concentration regime. Further, the maximum number of

shoot bud induced per explant was recorded at 2.0 mg/L zeatin concentration level,

which was 41.1 for ICPL 87119 and 46.1 for BSMR 736 (Table 2). The maximum

number of explants responding was recorded at 2.0 mg/L zeatin concentration regime,

which was 21.5 in ICPL 87119 and 22.0 in BSMR 736 (Table 3). The number of shoot

bud induced from embryo discs with half cotyledon explant was ranged from 0 to 4.2 in

both genotypes (Table 2). From embryo discs with half cotyledon explants, at 1.0 mg/L

zeatin concentration regime the highest number shoot buds was recorded. There was a

concomitant decrease in resonance of explants for shoot bud induction and number of

shoot bud induced with further increase in zeatin levels in growth medium

(Fig. 1 and 2).

The ‘t’-test statistical analysis indicated that the response of two genotypes at

same level of zeatin was non-significant (p > 0.84). At the same time, there was a

significant difference between the two explants response to same level of zeatin

concentration regimes for multiple shoot bud induction (p < 0.03). It was observed that

there was no significant difference between two genotypes response for number of shoot

83

bud induced per explants at same zeatin concentration regime (p > 0.96). Whereas, the

two explants (cotyledonary node with cotyledons and embryo discs with half cotyledon)

noticed differ significantly for their response to shoot bud induction at same zeatin

concentration regime in both genotypes (p < 0.001). The analysis of means showed that

the two treatments, 1.0 mg/L and 2.0 mg/L zeatin, were on par for number of explants

responded to zeatin fortification. On other hand, the treatment with 2.0 mg/L zeatin level

was superior over other treatments in cotyledonary node with cotyledons explants and

the treatment with 1.0 mg/L zeatin level was superior in embryo discs with half cotyledon

explants for number of shoot bud induced per explant.

4.1.4 Rooting and establishment of plantlets

Elongated and well developed shoots (~ 3 cm long) were excised from shoot

clumps on basal MS medium and cultured on MS medium augmented with increasing

IBA concentration regimes. The frequency of rooting varied with different IBA

concentration regimes ranging from 20 to 80 per cent in both genotypes (Table 4). The

highest root induction was noticed in MS media with 0.5 mg/L IBA. The observed number

of roots per shoot ranged from 1.4 to 4.8 per shoot. The maximum number of roots

induced per shoot was recorded in MS fortified with 0.5 mg/L IBA, in both genotypes.

The induced roots were thick, white in color with less fine root hair developed on it (Plate

1). The analysis of means revealed that the response to root induction and number of

roots induced was maximum in MS supplemented with 0.5 mg/L IBA for both genotypes

(Table 4) (Fig. 3).

4.2 Effect of different treatments on in planta transformation in pigeonpea

The improvisation of in planta transformation protocol to increased transformation

efficiency was performed by using different treatment such as,

A. tumefaciens infection alone, A. tumefaciens infection using A. tumefaciens culture

with tobacco extract added in overnight grown culture and A. tumefaciens infection using

air evacuation to increase the proximity between A. tumefaciens and embryonic axis. For

each treatment, a set of 200 explants in four batches of 50 explants each were used for

transformation.

84

Table 4: Effect of indole butyric acid (IBA) concentration regimes on root induction and number of root induced per shoot in pigeonpea genotypes, ICPL 87119 and BSMR 736, after 10 days of in vitro culture

Growth hormone concentrations

Per cent rooting

(mean ±SD) Grand mean

Number of roots per shoot (mean ±SD) Grand

mean ICPL 87119 BSMR 736 ICPL 87119 BSMR 736

MS 25.0±7.0c 20.0±8.0c 22.5±3.5c 1.4±0.5e 1.5±0.4e 1.4±0.1e

½ MS 25.0±8.0c 15.0±8.0c 20.0±7.1c 1.8±0.4e 1.8±0.4e 1.8±0.2e

MS + 0.25 mg/l IBA 50.0±6.0b 55.0±5.0b 52.5±3.5b 2.6±0.5d 2.7±0.5d 2.6±0.1d

MS + 0.50 mg/l IBA 75.0±7.0a 80.0±4.0a 77.5±3.5a 4.6±0.8a 4.8±0.7a 4.7±0.1a

MS + 0.75 mg/l IBA 55.0±5.0ab 60.0±6.0b 57.5±3.5ab 4.0±0.7b 3.7±0.5b 3.8±0.2b

MS + 1.0 mg/l IBA 50.0±7.0b 55.0±5.0b 52.5±3.5b 3.4±0.6c 3.4±0.4bc 3.4±0.2bc

MS + 1.5 mg/l IBA 45.0±5.0bc 45.0±7.0b 45.0±2.0b 3.4±0.7c 3.3±0.7bc 3.4±0.3bc

MS + 2.0 mg/l IBA 50.0±4.0b 45.0±6.0b 47.5±3.5b 3.3±0.6c 3.1±0.5cd 3.2±0.4c

½ MS + 0.50 mg/l IBA 50.0±6.0b 50.0±7.0b 50.0±6.5b 3.3±0.7c 3.2±0.4c 3.2±0.4c

Grand mean 47.22±15.2a 47.22±19.8a 47.22±17.1a 3.1±1.1a 3.1±0.9a 3.0±0.9


85

86

The use of A. tumefaciens infection alone to injured embryonic axis attached to

single cotyledon resulted in observation of 160 explants responding after the co-

cultivation period (Table 5). Out of responded 160 explants, a total of 86 explants could

survive and developed into seedlings, also called as primary transformants (T0

generation). The seeds from these set of 86 transformants were harvested and plant to

row progeny test employing gene specific PCR assay resulted in the identification of six

putative transgenic plants in T1 generation. In case of embryonic axis attached to single

cotyledon infected with A. tumefaciens culture with tobacco extract, out of infected 200

explants, 170 noticed responding well (Table 6). Among 170 well responded explants,

153 could survive and developed into seedlings. The plant to progeny screening of these

primary transformants resulted in identification of a set of 13 putative transformants in T1

generation as revealed by cry gene specific PCR assay. On other hand, A. tumefaciens

infection using air evacuation to increase the proximity between A. tumefaciens and

embryonic axis resulted in the observation of comparatively lesser number of explants

responding (133 explants) to this treatment (Table 7). The seeds were harvested from

121 survived primary transformants and plant to row progeny screening of them

identified a set 24 putative transformants in T1 generation, based on PCR analysis.

The statistical analysis revealed 80.00 per cent explant response, 53.75 per cent

explant survival and 3.0 per cent transformation efficiency in case of A. tumefaciens

infection alone (Table 8) (Fig. 4). Whereas, in case of A. tumefaciens infection using A.

tumefaciens culture along with tobacco extract recorded 85.00 per cent explant

response, 90.00 per cent explant survival and 6.5 per cent transformation efficiency.

Similarly, 66.50 per cent explant response, 90.98 per cent explant survival and 12.0 per

cent increased transformation efficiency was recorded for the treatment involving air

evacuation to increase the proximity between A. tumefaciens and embryonic axis.

87

Table 5: The effect of targeting embryonic axis attached to single cotyledon for successful Agrobacterium tumefaciens infection and successful transformation.

Sl. No.

Number of explants

infected/ batch

Number of explants

responded

Number of

explants survived

Plant to row screening in next

generation (T1)

Number of PCR +ve plants

identified in T1

1 50 36 20 20 0

2 50 39 18 18 3

3 50 40 27 27 1

4 50 45 21 21 2

Total 200 160 86 86 6

Table 6: The effect of targeting embryonic axis attached to single cotyledon with tobacco extract added in overnight grown Agrobacterium culture on plant transformation.

Sl. No.

Number of explants

infected / batch

Number of explants responde

d

Number of explants survived


generation (T1)


identified in T1

1 50 45 40 40 2

2 50 43 38 38 3

3 50 42 38 38 4

4 50 40 37 37 4

Total 200 170 153 153 13

Table 7: The effect of targeting embryonic axis attached to single cotyledon with air evacuation to increase the proximity between Agrobacterium tumefaciens and embryonic axis on transformation

Sl. No.

Number of explants

infected/ batch

Number of explants responde

d

Number of explants survived


generation (T1)


identified in T1

1 50 37 35 35 6

2 50 33 31 31 7

3 50 31 30 30 5

4 50 32 25 25 6

Total 200 133 121 121 24

88

Table 8: Effect of different treatments used in in planta transformation of pigeonpea on explant survival, explants responded and transformation efficacy presented in per cent.

Sl. No.

Treatments Explant

responded (%)

Explant survived

(%)

Transformation efficiency (%)

1 Agrobacterium infection alone

80.00a 53.75b 3.0c

2 Tobacco extract was added in overnight grown Agrobacterium culture

85.00a 90.00a 6.5b

3 Air evacuation to increase the proximity between Agrobacterium and embryonic axis

66.50b 90.98a 12.0a

LSD value (α=0.05) 0.1518 0.1554 0.1558


89

90

4.3 Generation of transgenic pigeonpea conferring expression of cry1Ac gene

Transgenic plants expressing cry1Ac gene were developed using both improvised

in vitro and in planta transformation methods. The in planta method, modified in present

study, was found to be the most effective and less laborious when compared with in vitro

transformation and plantlet regeneration method. The transgenic pigeonpea plants were

developed and forwarded to advanced generations (up to T3 generation). The analyses

of developed transgenic lines was done by performing their bioefficacy analysis against

H. armigera, quantitative Cry1Ac protein analysis using ELISA assay and cry1Ac gene

segregation analysis using PCR assay. Furthermore, the molecular analyses of selected

good transgenic lines were assessed by RT-PCR analysis of cry1Ac transcripts,

Southern and northern blot, and juncture region analysis using TAIL-PCR assay.

4.3.1 Development of transgenic pigeonpea carrying cry1Ac gene

A total of 1400 explants were infected with A. tumefaciens strain carrying construct

with cry1Ac gene and nptII as marker gene using in planta transformation protocol (Fig.

5). A set of 741 primary transformants were established, of which 728 could grow well till

plant maturity (Table 9) (Plate 2). The T1 seeds were harvested form well-established

primary transformants after reaching to maturity stage and T1 generation was raised. The

T1 plant progeny screening using cry1Ac gene specific PCR assay identified a set of

eighty eight putative transgenic plants for integration of cry1Ac gene (Plate 3). The

identified putative transformants were grown till maturity and T2 seeds were harvested. In

the embryonic axis infection followed by in vitro screen effort, a set of 2800 explants

were infected and co-cultivated. Upon kanamycin selection and rooting, a set of ten

plants could be established (Table 10a). The plantlets surviving on Kan selection

medium were confirmed for presence of cry1Ac gene using gene specific PCR assay

(Plate 3). The in vitro generated transformants (T0 generation) were hardened and grown

in transgenic containment facility, T1 seeds were harvested. The T1 seeds were sown in

plant to row progeny manner and T1 plants carrying cry1Ac gene were identified based in

PCR assay (Table 10b).

91

Table 9: Summary of transformation carried out using cry1Ac gene in pigeonpea

Sl. No. Number of

explants co-cultivated/set

Number of explants

responded

Number of primary

transformants (T0) established in greenhouse

Number of primary transformants

tested in plant-to-row progeny for

identifying putative transformants

Number of putative

transformants identified (T1)

1 50 20 10 10 00

2 50 22 08 08 00

3 50 45 27 27 05

4 50 40 21 21 03

5 50 16 12 12 00

6 50 00 00 00 00

7 50 45 40 40 03

8 50 46 37 37 04

9 50 42 30 30 03

10 50 45 29 29 03

11 50 30 32 32 00

12 50 42 40 40 00

13 50 45 40 40 21

14 50 30 00 00 00

15 50 43 38 38 09

16 50 16 10 10 00

17 50 17 10 10 00

18 50 42 38 38 13

19 50 40 37 37 16

20 50 25 00 00 00

21 50 45 40 27 08

22 50 41 35 35 00

23 50 46 42 42 00

24 50 44 40 40 00

25 50 38 30 30 00

26 50 36 32 32 00

27 50 34 31 31 00

28 50 35 32 32 00

Total 1400 970 741 728 88

92

93

94

Table 10a: Summary of transformation work being carried out using cry1Ac gene following in vitro kanamycin selection method in pigeonpea

No. of sets

Number of explants co-

cultivated/set

Number of explants survived and established on Kan selection media

Number of plantlets rooted and established

in greenhouse

1 100 0 0

2 100 0 0

3 100 0 0

4 100 4 2

5 100 3 2

6 100 0 0

7 100 0 0

8 100 0 0

9 100 0 0

10 100 0 0

11 100 0 0

12 100 0 0

13 100 2 2

14 100 0 0

15 100 0 0

16 100 0 0

17 100 0 0

18 100 4 1

19 100 1 1

20 100 0 0

21 100 0 0

22 100 0 0

23 100 0 0

24 100 0 0

25 100 2 2

26 100 0 0

27 100 0 0

28 100 0 0

Total 2800 16 10

95

Table 10b: Identification of T1 plants progenies of 10 putative transformants carrying cry1Ac gene. Testing for the presence of gene was done through gene specific PCR assay

Transgenic plants

Number of plants

screened

Number of PCR positive plants

Number of PCR negative plants

AC1 09 03 06

AC2 07 04 03

AC3 07 06 01

AC4 21 14 07

AC5 09 06 03

AC6 09 04 05

AC7 08 05 03

AC8 16 05 11

AC9 10 05 05

AC10 12 05 07

96

4.3.2 The cry1Ac gene segregation analysis in T2 generation progenies

The obtained T2 seeds were sown in plant to row progeny manner to get T2

generation plants. From each parental T1 generation plants up to 40 seeds were sown

and obtained T2 plants were subjected for gene specific PCR assay (Plate 3). The gene

segregation pattern was assayed using chi-square test. Among developed eighty eight,

for forty eight transgenic plant progenies of T1 generations plants recorded chi-square

calculated value less than table chi-square value (3.84) (Table 11). For rest forty

transgenic plants the chi-square value was observed to be more than the table value,

clearly indicating that the cry1Ac gene segregation in those transgenic plant progenies

did not follow 3:1 gene segregation pattern in T2 generation.

4.3.3 Characterization of developed transgenic lines in T2 generation

Bioefficacy of the transgenic plants expressing cry1Ac against first instar

Helicoverpa larvae revealed significant variability in larval mortality in all targeted tissue

types viz., leaf, flower and pod. The larval mortality in case of leaf tissues ranged from

25.0% to 70.0% (Table 12a). Whereas, it was 22.5% to 52.5% in case of flower tissues

and 17.5% to 47.5% in case of pod tissue. It was noticed that the majority of putative

transformants were having insect mortality ranging from 40.0% to 60.0%, with the

highest mortality recorded in Ac140-13 (70.0%) and Ac272-18 (70.0%) transgenic line

(Fig. 6). It was noticed that the larval feeding was highest in transgenic lines Ac279-8

(30.0%) and Ac30-12 (25.0%). Few transgenic plants like, Ac16-7, Ac30-12, Ac46-11

showed consistency in the insect mortality levels across different tissue types, though

mortality was low. The larval feeding pattern on different tissue types of transgenic and

non-transgenic control plants is shown in Plate 4. The statistical analysis of variance

(ANOVA), indicated that the transgenic lines differ significantly (p < 0.001) from each

other for larval mortality in all tissue types. The ‘t’-test analysis of larval mortality in

different tissue types indicated significant difference between mortality levels in leaf and

flower (p ≤ 0.001), and in leaf and pod tissues (p ≤ 0.001). Interestingly, the larval

mortality levels were comparable between flower and pod tissue (p ≥ 0.41) types as

indicated by ‘t’-test analysis. The un-paired ‘t’-test analysis of bioefficacy levels noticed in

different transgenic plants with two different genomic backgrounds (ICPL87119 and

BSMR736) indicated that the effect of Cry1Ac through thransgenic plant tissues of

97

Table 11: The transgene segregation pattern in eighty eight transformants carrying cry1Ac in T2 generation revealed by gene specific PCR assay

Plant ID Number of plants tested using gene

specific PCR

Number of plants positive using gene

specific PCR

Number of plants negative using gene

specific PCR

Expected number of

positive plants

Expected number of negative plants

Calculated chi-square

Gene segregation in

3:1 ration

Ac5-2 24 14 10 18.00 6.00 3.56 Yes Ac8-19 22 14 08 16.50 5.50 1.52 Yes Ac8-15 23 13 10 17.25 5.75 4.19 No Ac12-11 21 12 09 15.75 5.25 3.57 Yes Ac12-15 24 13 11 18.00 6.00 5.56 No Ac16-1 29 17 12 21.75 7.25 2.50 Yes Ac16-7 25 14 11 18.75 6.25 4.81 No Ac16-11 21 11 10 15.75 5.25 5.73 No Ac18-12 26 15 11 19.50 6.50 4.15 No Ac20-2 21 13 08 15.75 5.25 1.92 Yes Ac20-3 29 21 08 21.75 7.25 0.10 Yes Ac21-4 23 14 09 17.25 5.75 2.45 Yes Ac25-1 20 10 10 15.00 5.00 6.67 No

Ac29-1 30 19 11 22.50 7.50 2.18 Yes Ac29-3 30 23 07 22.50 7.50 0.02 Yes Ac30-12 24 14 10 18.00 6.00 3.56 Yes Ac31-1 24 18 06 18.00 6.00 0.00 Yes Ac31-2 32 19 13 24.00 8.00 4.17 No Ac37-8 20 12 08 15.00 5.00 2.40 Yes Ac39-2 22 12 10 16.50 5.50 4.91 No Ac41-7 18 11 07 13.50 4.50 1.85 Yes Ac46-11 24 13 11 18.00 6.00 5.56 No Ac48-3 25 15 10 18.75 6.25 3.00 Yes

(Table chi-square = 3.84) Cont…

98


specific PCR


specific PCR

Number of plants negative using

gene specific PCR

Expected number of

positive plants



Gene segregation in

3:1 ration

Ac49-5 20 11 09 15.00 5.00 4.27 No

Ac50-1 32 20 12 24.00 8.00 2.66 Yes

Ac50-3 22 12 10 16.50 5.50 4.91 No

Ac51-1 22 13 09 16.50 5.50 2.97 Yes

Ac53-23 21 11 10 15.75 5.25 5.73 No

Ac55-16 19 11 08 14.25 4.75 2.96 Yes

Ac56-3 23 13 10 17.25 5.75 4.19 No

Ac57-10 24 15 09 18.00 6.00 2.00 Yes

Ac58-23 20 12 08 15.00 5.00 2.40 Yes

Ac61-4 20 11 09 15.00 5.00 4.27 No

Ac63-20 24 15 09 18.00 6.00 2.00 Yes

Ac66-12 25 13 12 18.75 6.25 7.05 No

Ac73-3 26 14 12 19.50 6.50 6.21 No

Ac75-3 20 14 06 15.00 5.00 0.27 Yes

Ac88-2 24 16 08 18.00 6.00 0.89 Yes

Ac110-10 25 16 09 18.75 6.25 1.61 Yes

Ac140-13 22 14 08 16.50 5.50 1.52 Yes

Ac150-6 24 13 11 18.00 6.00 5.56 No

Ac152-15 28 17 11 21.00 7.00 3.05 Yes

Ac161-4 20 14 06 15.00 5.00 0.27 Yes

Ac162-8 20 11 09 15.00 5.00 4.27 No

Ac167-4 16 09 07 12.00 4.00 3.00 Yes

Ac168-7 26 15 11 19.50 6.50 4.15 No

Ac170-2 24 10 14 18.00 6.00 14.22 No


99


specific PCR


specific PCR


gene specific PCR

Expected number of

positive plants



Gene segregation in

3:1 ration

Ac201-3 25 18 07 18.75 6.25 0.12 Yes

Ac202-3 24 15 09 18.00 6.00 2.00 Yes

Ac203-5 20 14 06 15.00 5.00 0.27 Yes

Ac204-2 22 13 09 16.50 5.50 2.97 Yes

Ac205-2 23 16 07 17.25 5.75 0.36 Yes

Ac206-6 24 13 11 18.00 6.00 5.56 No

Ac207-11 21 12 09 15.75 5.25 3.57 Yes

Ac208-19 19 10 09 14.25 4.75 5.07 No

Ac251-3 23 12 11 17.25 5.75 6.39 No

Ac252-3 21 11 10 15.75 5.25 5.73 No

Ac253-8 20 12 08 15.00 5.00 2.40 Yes

Ac254-12 21 11 10 15.75 5.25 5.73 No

Ac255-4 27 15 12 20.25 6.75 5.44 No

Ac256-9 26 14 12 19.50 6.50 6.21 No

Ac257-5 22 14 08 16.50 5.50 1.52 Yes

Ac258-12 24 13 11 18.00 6.00 5.56 No

Ac259-3 27 15 12 20.25 6.75 5.44 No

Ac260-9 24 14 10 18.00 6.00 3.56 Yes

Ac261-3 25 13 12 18.75 6.25 7.05 No

Ac262-16 24 15 09 18.00 6.00 2.00 Yes

Ac263-4 24 14 10 18.00 6.00 3.56 Yes

Ac264-15 26 16 10 19.50 6.50 2.51 Yes

Ac265-5 20 14 06 15.00 5.00 0.27 Yes

Ac266-13 23 12 11 17.25 5.75 6.39 No


100


specific PCR


specific PCR


gene specific PCR

Expected number of

positive plants



Gene segregation in

3:1 ration

Ac267-21 22 12 10 16.50 5.50 4.91 No

Ac268-18 24 14 10 18.00 6.00 3.56 Yes

Ac269-7 25 15 10 18.75 6.25 3.00 Yes

Ac270-6 24 12 12 18.00 6.00 8.00 No

Ac271-14 26 15 11 19.50 6.50 4.15 No

Ac272-18 24 16 08 18.00 6.00 0.89 Yes

Ac273-11 26 17 09 19.50 6.50 1.28 Yes

Ac274-3 21 12 09 15.75 5.25 3.57 Yes

Ac275-6 20 11 09 15.00 5.00 4.27 No

Ac276-2 24 13 11 18.00 6.00 5.56 No

Ac277-11 22 14 08 16.50 5.50 1.52 Yes

Ac278-9 23 15 08 17.25 5.75 1.17 Yes

Ac279-8 22 11 11 16.50 5.50 7.33 No

Ac280-3 24 14 10 18.00 6.00 3.56 Yes

Ac281-5 26 13 13 19.50 6.50 8.67 No

Ac282-1 24 13 11 18.00 6.00 5.56 No

Ac362-21 20 10 10 15.00 5.00 6.67 No

(Table chi-square = 3.84)

101

Table 12a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eighty eight putative transformants carrying cry1Ac gene in T2 generation

Plant ID Per cent corrected mortality


Leaf tissues Flower tissues Pod tissues Leaf tissues Flower tissues Pod tissues

Ac5-2 45.00abcd 32.50abc 37.50abc Ac48-3 50.00abcd 37.50abc 37.50abc

Ac8-19 60.00abc 47.50ab 42.50ab Ac49-5 35.00bcd 27.50bc 32.50abc

Ac8-15 45.00abcd 37.50abc 42.50ab Ac50-1 47.50abcd 37.00abc 35.00abc

Ac12-11 50.00abcd 42.50abc 42.50ab Ac50-3 50.00abcd 37.50abc 42.50ab

Ac12-15 40.00abcd 32.50abc 32.50abc Ac51-1 55.00abcd 37.50abc 42.50ab


Ac16-7 35.00bcd 27.50bc 32.50abc Ac55-16 50.00abcd 32.50abc 32.50abc

Ac16-11 50.00abcd 42.50abc 42.50ab Ac56-3 40.00abcd 27.50bc 27.50abc

Ac18-12 40.00abcd 32.50abc 32.50abc Ac57-10 60.00abc 42.50abc 42.50ab

Ac20-2 65.00ab 47.00ab 45.50a Ac58-23 60.00abc 42.50abc 37.50abc

Ac20-3 65.50ab 50.50ab 47.00a Ac61-4 40.00abcd 32.50abc 32.50abc

Ac21-4 55.00abcd 42.50abc 37.50abc Ac63-20 60.00abc 47.50ab 42.50ab

Ac25-1 35.00bcd 22.50c 22.50bc Ac66-12 30.00cd 22.50c 22.50bc


Ac29-3 47.50abcd 40.50abc 37.00abc Ac75-3 25.00d 22.50c 27.50abc

Ac30-12 25.00d 22.50c 22.50bc Ac88-2 55.00abcd 47.50ab 47.50a

Ac31-1 55.50abcd 42.50abc 40.00ab Ac110-10 45.00abcd 37.50abc 42.50ab

Ac31-2 52.50abcd 40.00abc 42.50ab Ac140-13 70.00a 52.50a 47.50a

Ac37-8 60.00abc 47.50ab 47.50a Ac150-6 50.00abcd 37.50abc 37.50abc


Ac41-7 55.00abcd 42.50abc 42.50ab Ac161-4 60.00abc 47.50ab 47.50a

Ac46-11 35.00bcd 32.50abc 32.50abc Ac162-8 45.00abcd 37.50abc 37.50abc

Note: The means followed with same letters are within student ‘t’ range at α=0.05. Cont…

102




Ac167-4 60.00abc 42.50abc 42.50ab Ac262-16 60.00abc 47.50ab 42.50ab

Ac168-7 40.00abcd 32.50abc 37.50abc Ac263-4 65.00ab 42.50abc 42.50ab

Ac170-2 30.00cd 22.50c 27.50abc Ac264-15 50.00abcd 37.50abc 32.50abc

Ac201-3 60.00abc 42.50abc 42.50ab Ac265-5 55.00abcd 42.50abc 42.50ab


Ac203-5 60.00abc 47.50ab 47.50a Ac267-21 35.00bcd 27.50bc 27.50abc

Ac204-2 60.00abc 47.50ab 47.50a Ac268-18 60.00abc 47.50ab 47.50a


Ac206-6 40.00abcd 27.50bc 27.50abc Ac270-6 45.00abcd 37.50abc 32.50abc

Ac207-11 60.00abc 42.50abc 42.50ab Ac271-14 50.00abcd 37.50abc 37.50abc

Ac208-19 30.00cd 22.50c 27.50abc Ac272-18 70.00a 47.50ab 47.50a

Ac251-3 45.00abcd 37.50abc 37.50abc Ac273-11 65.00ab 47.50ab 42.50ab


Ac253-8 55.00abcd 42.50abc 47.50a Ac275-6 45.00abcd 37.50abc 32.50abc


Ac255-4 30.00cd 27.50bc 27.50abc Ac277-11 55.00abcd 37.50abc 32.50abc

Ac256-9 45.00abcd 37.50abc 37.50abc Ac278-9 35.00bcd 22.50c 22.50bc

Ac257-5 60.00abc 47.50ab 47.50a Ac279-8 30.00cd 22.50c 17.50c


Ac259-3 30.00cd 27.50bc 27.50abc Ac281-5 40.00abcd 32.50abc 32.50abc

Ac260-9 60.00abc 42.50abc 42.50ab Ac282-1 40.00abcd 27.50bc 27.50abc


SD (±) 0.055 0.039 0.038

CD 0.002 0.001 0.001

CV (%) 6.04 4.70 4.35


103

104

105

pigeonpea was independent of genotypes used with respect to their bioassay against H.

armigera (p > 0.1) (Table 12b).

There was a significant variation in Cry1Ac protein levels of different transgenic

lines irrespective of tissue type viz., leaf, flower and pod as revealed through ELISA

assay (Plate 5). The Cry protein level ranged from 0.305 to 0.847 µg/g of fresh leaf

tissue (Table 13a). Whereas, it was 0.221 to 0.671 µg/g of fresh flower tissues and 0.250

to 0.685 µg/g of fresh pod tissue. Interestingly, it was noticed that the majority of putative

transformants were accumulating the Cry1Ac protein from 0.6 to 0.8 µg/g of fresh tissue,

wherein the highest Cry1Ac proteins was detected in Ac140-13 (0.847 µg/g in leaf)

transgenic line (Fig. 6). Similarly, in case of flower and pod tissues, the majority of

transgenic plants were accumulating Cry1Ac protein from 0.3 to 0.5 µg/g of fresh tissue.

The transgenic line Ac140-13 (0.671 µg/g) showed highest accumulation of Cry protein

in flower and Ac201-3 (0.685 µg/g) in pod tissues, as indicated by ELISA assay. On

other hand, the lower level of Cry protein was recorded in transgenic line Ac73-3 (0.250

µg/g) for leaf tissues and in Ac75-3 (0.305 µg/g in flower and 0.221 µg/g in pod) for

flower and pod tissues. The ‘t’-test analysis of Cry protein accumulation in different tissue

types indicated significant difference in leaf and flower (p ≤ 0.001) and in lead and pod

tissues (p ≤ 0.001), and the Cry protein accumulation was non-significant for flower and

pod tissue (p > 0.44) types. The positive correlation was noticed between insect morality

and Cry1Ac protein accumulation (r2=0.8303) (Fig. 7). Similarly, the frequency

distribution analysis of transgenic plants following 3:1 transgene segregation pattern and

other than 3:1 segregation pattern revealed that majority of high Cry1Ac protein

accumulating transgenic plants were following 3:1 transgene segregation pattern (Fig. 7).

4.3.4 Characterization of in vitro generated transgenic plants in T1 generation

The bioefficacy analysis of in vitro developed transgenic plants showed the larval

mortality ranged from 40.00% to 55.50% in case of leaf tissues. Similarly, it was 27.50%

to 42.50% in case of flower and pod tissues (Table 12c). The highest larval mortality

recorded in case of transgenic plant AC4 in leaf and flower tissues, whereas in case pod

tissues, it was maximum in AC2, AC5 and AC7. On other hand, least larval mortality was

noticed in transgenic line AC10 for all tissue types. In leaf tissues, the Cry1Ac protein

level ranged from 0.344 to 0.716 µg/g of fresh tissue weight. Similarly, the Cry protein

accumulation in flower tissues was noticed from 0.273 to 0.604 µg/g and from 0.291 to

106

Table 12b: Comparison of H. armigera mortality in transgenic lines of ICPL87119 and

BSMR736 (by unpaired ‘t’-test at α=0.05)

Genotypes ICPL87119 BSMR736

Mean 47.84 53.19

Variance 115.62 73.14

Observations 80 8

Hypothesized Mean Difference 0

Df 9

t Stat -1.64

P(T<=t) two-tail 0.13

t Critical two-tail 2.26

Table 12c: Per cent corrected mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving in vitro generated putative transformants carrying cry1Ac gene in T1 generation


Leaf tissues Flower tissues Pod tissues

AC1 45.00c 37.50b 32.50c

AC2 50.00b 42.50a 42.50a

AC3 40.00d 32.50c 32.50c

AC4 55.50a 42.50a 40.00ab

AC5 52.50ab 40.00ab 42.50a

AC6 45.00c 32.50c 37.50b

AC7 55.00a 42.50a 42.50a

AC8 45.00c 37.50b 37.50b

AC9 50.00b 32.50c 32.50c

AC10 40.00d 27.50d 27.50d

SD (±) 0.043 0.039 0.051

CD 0.002 0.001 0.003

CV (%) 5.12 4.69 5.26


107

Plate 5: Estimation of Cry 1Ac protein in different test tissues of developed

transgenic pigeonpea plants using ELISA assay. TR: Technical

Replications; NC: Negative control; NTC: Non-Transgenic Control

108

Table 13a: The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2

generation as revealed by ELISA assay (α=0.05)

Plant ID Cry1Ac protein level (µg/g FW)



Ac5-2 0.592 0.460 0.474 Ac48-3 0.637 0.542 0.550

Ac8-19 0.721 0.599 0.554 Ac49-5 0.332 0.267 0.301

Ac8-15 0.640 0.513 0.522 Ac50-1 0.345 0.287 0.291

Ac12-11 0.656 0.534 0.557 Ac50-3 0.612 0.528 0.538

Ac12-15 0.344 0.273 0.291 Ac51-1 0.708 0.536 0.572

Ac16-1 0.398 0.339 0.342 Ac53-23 0.627 0.508 0.523

Ac16-7 0.332 0.299 0.316 Ac55-16 0.626 0.450 0.464

Ac16-11 0.558 0.489 0.504 Ac56-3 0.423 0.383 0.398

Ac18-12 0.410 0.321 0.356 Ac57-10 0.690 0.584 0.594

Ac20-2 0.769 0.702 0.710 Ac58-23 0.780 0.636 0.586

Ac20-3 0.756 0.646 0.669 Ac61-4 0.378 0.300 0.318

Ac21-4 0.691 0.594 0.545 Ac63-20 0.719 0.638 0.638

Ac25-1 0.423 0.284 0.304 Ac66-12 0.310 0.292 0.301

Ac29-1 0.605 0.567 0.561 Ac73-3 0.404 0.363 0.250

Ac29-3 0.405 0.325 0.342 Ac75-3 0.305 0.221 0.266

Ac30-12 0.315 0.267 0.289 Ac88-2 0.682 0.584 0.572

Ac31-1 0.454 0.378 0.399 Ac110-10 0.529 0.454 0.489

Ac31-2 0.587 0.502 0.519 Ac140-13 0.847 0.671 0.599

Ac37-8 0.721 0.561 0.562 Ac150-6 0.557 0.461 0.486

Ac39-2 0.632 0.505 0.522 Ac152-15 0.640 0.575 0.574

Ac41-7 0.716 0.604 0.601 Ac161-4 0.700 0.626 0.645

Ac46-11 0.459 0.397 0.400 Ac162-8 0.636 0.505 0.520

Cont…

109




Ac167-4 0.712 0.616 0.612 Ac262-16 0.744 0.611 0.587

Ac168-7 0.426 0.319 0.339 Ac263-4 0.806 0.584 0.604

Ac170-2 0.340 0.262 0.299 Ac264-15 0.560 0.490 0.452

Ac201-3 0.725 0.615 0.685 Ac265-5 0.685 0.500 0.500

Ac202-3 0.629 0.530 0.470 Ac266-13 0.605 0.473 0.355

Ac203-5 0.760 0.604 0.602 Ac267-21 0.539 0.438 0.442

Ac204-2 0.688 0.603 0.610 Ac268-18 0.799 0.619 0.619

Ac205-2 0.683 0.517 0.532 Ac269-7 0.673 0.486 0.493

Ac206-6 0.447 0.322 0.344 Ac270-6 0.567 0.439 0.365

Ac207-11 0.768 0.616 0.637 Ac271-14 0.620 0.421 0.434

Ac208-19 0.354 0.278 0.290 Ac272-18 0.807 0.632 0.642

Ac251-3 0.627 0.504 0.528 Ac273-11 0.835 0.536 0.504

Ac252-3 0.515 0.414 0.439 Ac274-3 0.543 0.405 0.417

Ac253-8 0.648 0.545 0.558 Ac275-6 0.587 0.426 0.397

Ac254-12 0.547 0.434 0.447 Ac276-2 0.406 0.360 0.362

Ac255-4 0.342 0.265 0.274 Ac277-11 0.708 0.447 0.410

Ac256-9 0.527 0.467 0.471 Ac278-9 0.457 0.311 0.338

Ac257-5 0.749 0.618 0.615 Ac279-8 0.337 0.259 0.223

Ac258-12 0.698 0.598 0.612 Ac280-3 0.701 0.467 0.488

Ac259-3 0.325 0.250 0.261 Ac281-5 0.453 0.403 0.403

Ac260-9 0.701 0.497 0.512 Ac282-1 0.467 0.358 0.363

Ac261-3 0.552 0.411 0.410 Ac362-21 0.406 0.325 0.334

SD (±) 0.016 0.019 0.018

CD 0.006 0.010 0.012

CV (%) 3.35 4.13 4.06

110

111

0.601 µg/g of fresh pod tissue (Table 13b). The higher level of Cry1Ac proteins was

detected in AC4 transgenic line irrespective of tissue type. The statistical analysis

revealed that the transgenic plants differ significantly for both insect mortality levels and

Cry1Ac protein accumulation (p < 0.01). There was a strong positive correlation noticed

between the different tissue types, and insect mortality and Cry1Ac protein in transgenic

lines (Table 13c).

4.3.5 The cry1Ac gene segregation analysis in T3 generation progenies

In initial efforts of plant transformation a set of eight transgenic plants viz., Ac16-1,

Ac20-2, Ac20-3, Ac29-1, Ac29-3, Ac31-1, Ac31-2 and Ac50-1 carrying cry1Ac gene were

identified and forwarded to T3 generation. The T3 seeds were harvested from T2

generation plants and were sown in plant to row progeny manner to get T3 generation

plants. From each parental T2 generation plant up to 40 seeds were sown and obtained

plants were subjected for gene specific PCR assay (Plate 3). Using chi-square test, the

cry1Ac gene segregation pattern was assayed. Among eight transgenic lines, in five

transgenic lines we identified homozygosity. The homozygosity in T2 parental plants viz.,

20-2-7, 20-3-2, 29-1-10, 31-2-9, 31-2-12, 31-1-2 and 31-1-3 belonging to five transgenic

lines were confirmed using cry1Ac gene specific PCR assay (Plate 3) (Table 14). Many

of other plant progenies was found to heterozygous as revealed by chi-square analysis

(table chi-square value is 3.84).

4.3.6 Characterization of eight transgenic lines carrying cry1Ac gene in T3 generation

The insect bioassay of the transgenic pigeonpea plants recorded significant

variability in larval mortality for eight transgenic lines. It was noticed that the larval

mortality was ranged from 41.25 to 61.25 per cent in case of leaf tissues whereas; it was

32.5 to 47.5 per cent in case of flower tissues and 35.0 to 52.5 per cent in case of pod

(Table 15a). The highest larval mortality recorded in case of transgenic line Ac20-2 for all

tissue types (Plate 4). On other hand, least larval mortality was noticed in transgenic line

Ac16-1. The analysis of variance indicated that the transgenic lines differ significantly (p

< 0.001) for larval mortality in all tissue types. The ‘t’-test analysis of larval mortality in

112

Table 13b: The Cry1Ac protein level detected in in vitro generated transgenic pigeonpea plants of T1 generation as revealed by ELISA assay in leaf, flower and pod

tissues (α=0.05).



AC1 0.454 0.378 0.399

AC2 0.587 0.502 0.519

AC3 0.423 0.383 0.398

AC4 0.716 0.604 0.601

AC5 0.637 0.542 0.550

AC6 0.540 0.413 0.422

AC7 0.656 0.534 0.557

AC8 0.527 0.408 0.423

AC9 0.526 0.450 0.464

AC10 0.344 0.273 0.291

SD (±) 0.011 0.016 0.021

CD 0.012 0.011 0.014

CV (%) 4.12 4.89 5.04

113

Table 13c: The correlation analysis of insect mortality levels and estimated Cry1Ac

protein in leaf, flower and pod tissues of T2 generation plants (αααα=0.01)

Insect mortality

in leaf tissues

Insect mortality in

flower tissues

Insect mortality

in pod tissues

Cry1Ac protein in leaf

tissues

Cry1Ac protein in

flower tissues

Cry1Ac protein in

pod tissues

Insect mortality in leaf tissues

1 0.936** 0.861** 0.911** 0.893** 0.867**

Insect mortality in flower tissues

1 0.921** 0.846** 0.875** 0.834**

Insect mortality in pod tissues

1 0.798** 0.839** 0.855**

Cry1Ac protein in leaf tissues

1 0.946** 0.915**

Cry1Ac protein in flower tissues

1 0.970**

Cry1Ac protein in pod tissues

1

**Correlation is significant at the 0.01 level (two tailed).

114

Table 14: Transgene segregation pattern in eight transgenic lines carrying cry1Ac gene in

T3 generation

Plant ID

Total number of

plant progenies

tested


Number of PCR

negative plants

Expected number

of positive plants

Expected number

of negative plants


Estimated plant

zygosity

20-2-1 15 14 01 11.25 03.75 02.69 Hetero 20-2-2 16 12 04 12.00 04.00 00.00 Hetero 20-2-3 14 07 07 10.50 03.50 04.67 Hetero. 20-2-4 16 11 05 12.00 04.00 00.33 Hetero. 20-2-6 16 12 04 12.00 04.00 00.00 Hetero. 20-2-7 15 15 00 11.25 03.75 05.00 Homo.

29-3-2 05 05 00 03.75 01.25 01.67 Homo. 29-3-3 19 10 09 14.25 04.75 05.07 Hetero. 29-3-6 10 08 02 07.50 02.50 00.13 Hetero. 29-3-8 08 05 03 06.00 02.00 00.67 Hetero.

29-3-10 09 05 04 06.75 02.25 01.81 Hetero. 29-3-11 15 08 09 11.25 03.75 08.29 Hetero.

20-3-1 14 09 05 10.50 03.50 00.86 Hetero. 20-3-2 11 11 00 08.25 02.75 03.67 Homo. 20-3-4 13 12 01 09.75 03.25 02.08 Hetero. 20-3-5 14 07 07 10.50 03.50 04.67 Hetero. 20-3-6 11 04 07 08.25 02.75 08.76 Hetero. 20-3-9 14 08 06 10.50 03.50 02.38 Hetero.

29-1-6 12 08 04 09.00 03.00 00.44 Hetero. 29-1-7 11 05 06 08.25 02.75 05.12 Hetero. 29-1-8 13 07 06 09.75 03.25 03.10 Hetero. 29-1-9 15 14 01 11.25 03.75 02.69 Hetero.

29-1-10 10 10 00 07.50 02.50 03.33 Homo. 29-1-15 11 10 01 08.25 02.75 01.48 Hetero.

16-1-1 24 16 08 18.00 06.00 00.89 Hetero. 16-1-2 23 19 04 17.25 05.75 00.71 Hetero. 16-1-3 10 10 00 07.50 02.50 03.33 Homo. 16-1-4 11 05 06 08.25 02.75 05.12 Hetero. 16-1-5 07 07 00 05.25 01.75 02.33 Hetero. 16-1-6 10 09 01 07.50 02.50 01.20 Hetero.

31-2-6 16 11 06 12.00 04.00 01.08 Hetero. 31-2-7 15 14 01 11.25 03.75 02.69 Hetero. 31-2-9 16 16 00 12.00 04.00 05.33 Homo.

31-2-10 21 12 09 15.75 05.25 03.57 Hetero. 31-2-11 23 15 08 17.25 05.75 01.17 Hetero. 31-2-12 16 16 00 12.00 04.00 05.33 Homo.

31-1-1 13 12 01 09.75 03.25 02.08 Hetero. 31-1-2 11 11 00 08.25 02.75 03.67 Homo. 31-1-3 10 10 00 07.50 02.50 03.33 Homo. 31-1-5 18 10 08 13.50 04.50 03.63 Hetero. 31-1-6 11 07 04 08.25 02.75 00.76 Hetero. 31-1-7 20 10 10 15.00 05.00 06.67 Hetero.

Contd…

115

Plant ID

Total number of

plant progenies

tested


Number of PCR

negative plants

Expected number

of positive plants

Expected number

of negative plants


Estimated plant

zygosity

50-1-1 18 13 05 13.50 04.50 00.07 Hetero.

50-1-2 20 12 08 15.00 05.00 02.40 Hetero.

50-1-3 14 05 09 10.50 03.50 11.52 Hetero.

50-1-4 17 10 07 12.75 04.25 02.37 Hetero.

50-1-5 21 14 07 15.75 05.25 00.78 Hetero.

50-1-6 17 12 05 12.75 04.25 00.18 Hetero.

Where; Hetero.: Heterozygous; Homo.: Homozygous

116

Table 15a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eight putative transformants carrying cry1Ac gene in T3 generation.

Event I D

Corrected per cent larval mortality


Ac29-3 42.50d 35.00d 37.50d

Ac31-2 47.50c 37.50c 40.00c

Ac29-1 51.25b 42.50d 45.00b

Ac16-1 41.25e 32.50e 35.00e

Ac20-2 61.25a 47.50a 52.50a

Ac20-3 53.75a 45.00a 47.50a

Ac31-1 50.00b 37.50b 37.50d

Ac50-1 45.00d 35.00d 37.50d

SD (±) 0.078 0.033 0.030

CD 0.019 0.010 0.017

CV (%) 4.91 5.00 6.24


Table 15b: The ‘t’-test analysis of Helicoverpa armigera mortality levels from T2 and T3

generation of eight cry1Ac transgenic lines (α=0.05)


T2

generation T3

generation T2

generation T3

generation T2

generation T3

generation

Mean 53.50 49.06 42.13 39.06 40.25 41.56

Variance 68.07 42.75 21.98 28.46 20.57 37.39

Observations 8 8 8 8 8 8

Pooled Variance 55.41 25.22 28.98

Hypothesized Mean Difference

0 0 0

Df 14 14 14

t Stat 1.19 1.22 -0.49

P(T<=t) two-tail 0.25 0.24 0.63

t Critical two-tail 2.14 2.14 2.14

117

different tissue types indicated significant difference between mortality levels transgenic

lines in leaf and flower (p ≤ 0.003), and in leaf and pod tissues (p ≤ 0.02). The larval

mortality between two tissue types viz., flower and pod were on par with each other with

p ≥ 0.19, by ‘t’-test analysis. Further, ‘t’-test analysis revealed that there was no

significant difference between the mortality levels observed in T2 and T3 generation in all

tissue types of eight transgenic lines (Table 15b).

It was interesting to notice the significant variation in Cry1Ac protein levels of eight

transgenic lines irrespective of tissue types as revealed through ELISA assay. In leaf

tissues, the Cry1Ac protein level ranged from 0.322 to 0.736 µg/g of fresh tissue weight

(Table 16). Similarly, the Cry protein accumulation in flower tissues was noticed from

0.254 to 0.646 µg/g and from 0.269 to 0.691 µg/g of fresh pod tissue. The higher level of

Cry1Ac protein was detected in Ac20-2 transgenic line irrespective of tissue type. On

other hand, the lower level of Cry1Ac protein was recorded in transgenic line Ac50-1.

The statistical analysis indicated that the transgenic lines were differencing significantly

for Cry protein accumulation with p < 0.001 in case of leaf, flower and pod tissue types.

Interestingly, the ‘t’-test analysis of Cry protein accumulation in different tissue types

indicated that there was no significant difference in leaf and flower

(p > 0.19), flower and pod tissues (p > 0.38) and between leaf and pod tissue types (p

>0.29). The correlation analysis between insect morality and Cry1Ac protein

accumulation revealed positive correlation between them with r2=0.8262 (Fig. 8).

4.3.7 The cry1Ac transcript analysis using qRT-PCR assay

Absolute real time analysis of cry1Ac transcript levels in different tissue types of

eight transgenic lines indicated significant variation in cry1Ac transcript levels as

revealed by standard graph (Plate 6). The cry1Ac transcript level was ranged from 24.6

to 165.1 ng/µl in fresh leaf tissue (Table 17a). Similarly, the cry1Ac transcript level in

flower was ranged from 15.6 to 149.5 ng/µl and in pod from 18.3 to 152.4 ng/µl.

Interestingly, the highest transcript level was detected in all tissue types of Ac20-2

transgenic lines. On other hand, the lower level of cry1Ac transcripts was recorded in

case of transgenic line Ac50-1. The ANOVA indicated the observation of significant

difference between transgene transcript levels of eight transgenic lines (p < 0.01). The

‘t’-test analysis of transcript levels between tissue types revealed non-significant

difference in the transcripts levels of cry1Ac gene in different tissue types within

118

Table 16: The Cry1Ac protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay

Event ID

Cry1Ac protein level (µg/g FW)

Leaf tissue Flower tissue Pod tissue

Ac29-3 0.375d 0.304e 0.324e

Ac31-2 0.526bc 0.469cd 0.498cd

Ac29-1 0.566b 0.512bc 0.529bc

Ac16-1 0.340d 0.298e 0.312e

Ac20-2 0.736a 0.646a 0.691a

Ac20-3 0.709a 0.617ab 0.634ab

Ac31-1 0.437cd 0.361de 0.388de

Ac50-1 0.322d 0.254e 0.269e

SD (±) 0.030 0.028 0.008

CD 0.021 0.061 0.050

CV (%) 5.99 5.53 2.93


119

120

121

Table 17a: The cry1Ac transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

Event ID cry1Ac transcript level in leaf tissue

(ng/µl)

cry1Ac transcript level in flower tissue

(ng/µl)

cry1Ac transcript level in pod tissue

(ng/µl)

Ac29-3 056.3f 049.6f 052.1f

Ac31-2 102.5d 098.5d 102.1d

Ac29-1 120.6c 105.2c 110.4c

Ac16-1 045.2g 023.9g 025.8g

Ac20-2 165.1a 149.5a 152.4a

Ac20-3 156.9b 145.3b 149.3b

Ac31-1 075.6e 057.4e 061.0e

Ac50-1 024.6h 015.6h 018.3h

SD (±) 3.358 3.005 2.916

CD 0.943 0.973 0.941

CV (%) 3.59 3.72 3.47


122

developed transgenic plants (p ≥ 0.31). The correlation analysis between Cry1Ac protein

accumulation and transgene transcript levels showed positive correlation (r2=0.988) (Fig.

8). The correlation analysis of larval mortality, Cry1Ac protein and cry1Ac transcript

revealed strong positive in leaf, flower and pod tissues for these traits of T3 generation

transgenic lines (Table 17b).

4.3.8 Southern blot analysis

The Southern blotting was performed to study the integration pattern of T-DNA in

to plant genome, for which from three selected transgenic plants genomic DNA was

digested. It was performed with HindIII as the enzyme that cuts in the T-DNA region and

probed with 600 bp cry1Ac PCR product. An observation of strong signal and the

difference in the hybridization pattern in the three selected transgenic plants viz., Ac29-1,

Ac20-2 and Ac20-3, revealed the single copy integration in these plants (Plate 7).

Whereas, there was no hybridization signal noticed with the DNA of non-transformed

plants. Interestingly, in case of Ac20-2 and Ac20-3 the hybridization signals revealed

similar kind of banding pattern in both transgenic lines.

4.3.9 Northern blot analysis

The presence of cry1Ac transcript in transgenic pigeonpea plants was also

confirmed with northern blotting using 600 bp cry1Ac PCR product as a probe (Plate 8).

The hybridization signals were noticed in all selected transgenic plant samples viz.,

Ac31-2, Ac29-1, Ac31-1, Ac29-3, Ac20-2 and Ac20-3. The observation of strong signals

revealed the production of transcripts in respective transgenic plants.

4.3.10 Juncture region analysis of T-DNA integration

The site of insertion of T-DNA/cry1Ac in the pigeonpea genome was identified by

recovering the genomic sequence flanking the left border (LB) of T-DNA by TAIL-PCR in

transgenic plants. Primary TAIL-PCR with 35S revers and arbitrary primer AD-3

produced a smear in PCR product. The multiple bands were noticed in secondary TAIL-

PCR when amplified product was electrophoresed on one per cent agarose gel.

However, there was a reduction in number of multiple bands produced with development

of one to more thick bands in the tertiary TAIL-PCR (Plate 9). The bright bands with

more than 1.5 kb amplicon size were eluted and cloned into pTZ57R/T and sequenced.

123

Table 17b: The correlation analysis of insect mortality levels, estimated Cry1Ac protein and cry1Ac transcript in leaf, flower and pod

tissues of T3 generation plants (αααα=0.01).

Insect mortality

in leaf tissues


Insect mortality

in pod tissues

Cry1Ac protein in

leaf tissues

Cry1Ac protein in

flower tissues

Cry1Ac protein in

pod tissues

cry1Ac transcript

level in leaf tissue

cry1Ac transcript

level in flower tissue

cry1Ac transcript

level in pod tissue


1 0.957** 0.935** 0.909** 0.890** 0.904** 0.887** 0.868** 0.867**


1 0.982** 0.965** 0.952** 0.953** 0.950** 0.941** 0.941**


1 0.950** 0.941** 0.944** 0.930** 0.925** 0.923**

Cry1Ac protein in leaf tissues

1 0.996** 0.996** 0.994** 0.991** 0.990**

Cry1Ac protein in flower tissues

1 0.998** 0.995** 0.991** 0.990**

Cry1Ac protein in pod tissues

1 0.995** 0.990** 0.989**

cry1Ac transcript level in leaf tissue

1 0.993** 0.993**

cry1Ac transcript level in flower tissue

1 1.000**

cry1Ac transcript level in pod tissue

1

**Correlation is significant at the 0.01 level (two tailed)

124

125

Sequence of all the three lines viz., Ac20-2, Ac20-3 and Ac29-1 were analysed by using

BioEdit bioinformatics algorithm.

The obtained sequences (forward and reverse) were assembled and contigues

were formed. Prior to that the contigues were assembled with available T-DNA left

border sequences and T-DNA backbone was removed. The local blast was set for these

processed contigue sequences and it was noticed that flaking genomic region recovered

from Ac20-2 showed 99 per cent homology (268 bp of 270 bp) with Scaffold130851 of

pigeonpea genome (Table 18). It was interesting to notice that flaking genomic region

recovered from Ac20-3 also showed 100 per cent homology (311 bp of 311 bp) with

Scaffold130851 of pigeonpea genome. On other hand, in case of Ac29-1, the recovered

flaking genomic region indicated 100 per cent homology (85 bp of 85 bp) with

Scaffold137204 of pigeonpea draft genome.

4.4 Generation of transgenic pigeonpea conferring expression of cry2Aa gene

The in planta transformation method was employed for development of transgenic

plants carrying cry2Aa gene. The developed pigeonpea transformants were forwarded to

advanced generations (up to T3 generation). The characterization of transgenic lines

carrying cry2Aa gene was performed by their bioefficacy analysis against H. armigera

larvae and quantitative Cry2Aa protein analysis using ELISA. The transgene segregation

pattern was analysed using cry2Aa gene specific PCR assay. The selected transgenic

lines were subjected for molecular characterization using qRT-PCR for cry2Aa transcript

level, Southern and northern blot, and juncture region analysis using TAIL-PCR assay.

4.4.1 Development of transgenic pigeonpea carrying cry2Aa gene

A set of 600 explants were subjected for A. tumefaciens infection using

A. tumefaciens strain carrying cry2Aa gene construct with nptII as marker gene (Fig. 9).

A total of 348 primary transformants were established in transgenic containment facility

(Plate 10). All the primary transformants were allowed to grow till plant maturity and T1

126

Table 18: Juncture region analysis of cry1Ac cassette in AC20-2, AC20-3 and AC29-1 transgenic lines as revealed by TAIL-PCR analysis

Transgenic lines

Pigeonpea contig Position (bp) Query coverage E value

Ac20-2 Scaffold130851 37339-37340 99% (268/270 bp) e-143

Ac20-3 Scaffold130851 37339-37340 100% (311/311 bp) e-175

Ac29-1 Scaffold137204 398597-398598 100% (85/85bp) 1e-040

127

seeds were collected. The plant to row progeny screening in T1 generation, using cry2Aa

gene specific PCR assay identified a set of sixty five putative transformants carrying

cry2Aa gene (Table 19) (Plate 11). The identified putative transformants were grown till

maturity and T2 seeds were harvested.

4.4.2 The cry2Aa gene segregation analysis in T2 generation progenies

The collected T2 seeds were sown in plant to row progeny manner and T2

generation progenies were raised (Plate 10). The chi-square analysis of observed

number of plants with and without cry2Aa gene indicated that among sixty five transgenic

lines, sixteen were following 3:1 (positive: negative) ration for transgene segregation

(Table 20) (Plate 11). Whereas, in case of rest forty nine transgenic lines the chi-square

value was observed to be more than the table value (3.84), clearly indicating that the

cry2Aa gene segregation in those transgenic plant progenies did not follow 3:1

segregation pattern for transgene in T2 generation.


The insect bioassay in case of cry2Aa transgenic plants showed significant

variability in larval mortality in different tissues viz., leaf, flower and pod. It was noticed

that the larval mortality in case of leaf tissues ranged from 5.25% to 65.75% (Table 21).

On other hand, larval mortality was between 5.25% to 40.5% in case of flower tissues

and 10.25% to 50.50% in case of pod tissue. It was interesting to notice that the majority

of transformants were having insect mortality between 30.0% - 40.0% (Fig. 10). The

highest larval mortality was recorded in 21A12-24 (65.75%) transgenic line for leaf

tissues, in 21A3-4 and 21A6-12 transgenic line (40.50%) for flower and in 1A2-1

(50.50%) for pod tissues. It was noticed that the larval feeding was highest in transgenic

line 21A5-11, which was comparable with non-transgenic plant. The larval feeding

pattern on different tissue types of transgenic and non-transgenic control plants is shown

in Plate 12. The transgenic lines, 21A4-21, 21A12-26, 1A2-30, 1A2-42 were found

consistent in their performance across different tissue types. The statistical analysis of

variance/ANOVA indicated that there was significant difference in observed insect

mortality levels of different transgenic lines. The ‘t’-test analysis of larval mortality in

different tissue types indicated significant difference between mortality levels in leaf and

flower, and leaf and pod, and between flower and pod tissues types (p < 0.01).

128

129

Table 19: Summary of transformation carried out using cry2Aa gene in pigeonpea

Sl. No.

Number of explants co-

cultivated/set

Number of explants

responded

Number of primary

transformants (T0)

established in greenhouse

Number of primary

transformants tested in plant-to-row progeny for

identifying putative

transformants

Number of putative


1 50 38 30 30 6

2 50 35 20 20 3

3 50 31 30 30 0

4 50 35 32 32 7

5 50 33 31 31 0

6 50 39 25 25 5

7 50 36 29 29 12

8 50 35 33 33 6

9 50 37 35 35 0

10 50 34 27 27 12

11 50 34 31 31 11

12 50 32 25 25 3

Total 600 419 348 348 65

130

131

Table 20: The transgene segregation pattern in sixty five transformants carrying cry2Aa in T2 generation revealed by gene specific PCR assay

Plant ID

Number of plants tested using gene specific

PCR

Number of plants positive using gene specific

PCR

Number of plants negative using gene specific

PCR

Expected number of

positive plants

Expected number of negative

plants


Gene segregation in

3:1 ration

1A2-1 36 24 12 27.00 09.00 01.33 Yes 21A2-2 32 22 10 24.00 08.00 00.67 Yes 21A2-3 35 17 18 26.25 08.75 13.04 No 21A3-4 40 25 15 30.00 10.00 03.33 Yes 21A3-5 38 18 20 28.50 09.50 15.47 No 21A6-6 34 20 14 25.50 08.50 04.75 No 21A6-7 37 20 17 27.75 09.25 08.66 No 21A6-8 34 15 19 25.50 08.50 17.29 No 21A6-9 29 20 09 21.75 07.25 00.56 Yes 21A5-10 30 21 09 22.50 07.50 00.40 Yes 21A5-11 38 20 18 28.50 09.50 10.14 No 21A6-12 40 25 15 30.00 10.00 03.33 Yes 21A6-13 40 17 23 30.00 10.00 22.53 No 21A5-14 34 23 11 25.50 08.50 00.98 Yes 21A5-15 39 20 19 29.25 09.75 11.70 No 21A5-16 36 22 14 27.00 09.00 03.70 Yes 21A5-17 40 16 24 30.00 10.00 26.13 No 21A5-18 33 19 14 24.75 08.25 05.34 No 21A5-19 36 22 14 27.00 09.00 03.70 Yes 21A4-20 35 17 18 26.25 08.75 13.04 No 21A4-21 40 24 16 30.00 10.00 04.80 No 21A12-24 33 21 12 24.75 08.25 02.27 Yes 21A12-25 40 28 12 30.00 10.00 00.53 Yes


132

Plant ID


PCR


PCR


PCR

Expected number of

positive plants


plants


Gene segregation in

3:1 ration

21A12-26 37 20 17 27.75 09.25 08.66 No 21A12-27 36 20 16 27.00 09.00 07.26 No 1A22-28 34 16 18 25.50 08.50 14.16 No 1A22-29 39 18 21 29.25 09.75 17.31 No 1A2-30 40 17 23 30.00 10.00 22.53 No 21A6-31 38 20 18 28.50 09.50 10.14 No 21A6-32 36 19 17 27.00 09.00 09.48 No 21A6-33 37 16 21 27.75 09.25 19.90 No

2A22-39 33 16 17 24.75 08.25 12.37 No

1A2-40 39 19 20 29.25 09.75 14.37 No 1A2-41 40 24 16 30.00 10.00 04.80 No 1A2-42 36 21 15 27.00 09.00 05.33 No 21A6-43 37 20 17 27.75 09.25 08.66 No

21A4-50 35 19 16 26.25 08.75 08.01 No 21A4-51 37 19 18 27.75 09.25 11.04 No 21A4-52 38 18 20 28.50 09.50 15.47 No 21A5-53 39 20 19 29.25 09.75 11.70 No 21A5-57 39 22 17 29.25 09.75 07.19 No 21A5-58 34 11 23 25.50 08.50 32.98 No 21A5-59 34 24 10 25.50 08.50 00.35 Yes

21A5-60 33 21 12 24.75 08.25 02.27 Yes 21A4-61 36 20 16 27.00 09.00 07.26 No 21A4-62 40 28 12 30.00 10.00 00.53 Yes 21A4-63 40 20 20 30.00 10.00 13.33 No


133

Plant ID


PCR


PCR


PCR

Expected number of positive plants


plants


Gene segregation in

3:1 ration

21A4-64 38 19 19 28.50 09.50 12.67 No

21A4-65 40 22 18 30.00 10.00 08.53 No

21A4-66 31 14 17 23.25 07.75 14.72 No

21A4-67 34 06 28 25.50 08.50 59.65 No

21A6-68 26 19 07 19.50 06.50 00.05 Yes

21A6-69 40 21 19 30.00 10.00 10.80 No

21A6-70 27 04 23 20.25 06.75 52.16 No

21A6-71 33 09 24 24.75 08.25 40.09 No

21A4-72 39 20 19 29.25 09.75 11.70 No

21A4-73 36 22 14 27.00 09.00 03.70 Yes

21A4-74 40 17 23 30.00 10.00 22.53 No

21A4-75 40 21 19 30.00 10.00 10.80 No

21A4-76 36 13 23 27.00 09.00 29.04 No

21A4-77 39 20 19 29.25 09.75 11.70 No

21A4-78 34 09 25 25.50 08.50 42.71 No

21A12-79 40 21 19 30.00 10.00 10.80 No

21A12-80 38 21 17 28.50 09.50 07.89 No

21A12-81 35 18 17 26.25 08.75 10.37 No


134

Table 21: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving sixty five putative transformants carrying cry2Aa gene in T2 generation




1A2-1 55.25bc 35.50ab 50.50a 21A12-25 60.25ab 30.25bc 45.25b

21A2-2 60.50ab 35.25ab 45.50b 21A12-26 30.75de 25.75cd 30.75e

21A2-3 25.50ef 20.25de 25.50f 21A12-27 20.25fg 10.25fgh 20.25g

21A3-4 60.25ab 40.50a 50.25a 1A22-28 25.50ef 10.75fg 20.75g

21A3-5 25.25ef 20.50de 25.75f 1A22-29 30.50de 15.25ef 25.25f

21A6-6 35.50d 25.50cd 30.25e 1A2-30 25.25ef 20.50de 25.50f

21A6-7 25.50ef 15.25ef 25.75f 21A6-31 35.25d 20.50de 30.50e

21A6-8 30.50de 30.75bc 35.25d 21A6-32 25.50ef 20.25de 25.25f

21A6-9 65.25a 35.25ab 50.50a 21A6-33 30.50de 15.25ef 25.25f

21A5-10 55.75bc 30.75bc 40.50c 2A22-39 25.50ef 15.50ef 25.50f

21A5-11 05.25h 05.25i 10.25i 1A2-40 35.25d 10.50fg 20.50g

21A6-12 60.75ab 40.50a 50.25a 1A2-41 25.75ef 10.25fgh 20.25g

21A6-13 25.25ef 15.50ef 20.50g 1A2-42 30.25de 25.25cd 30.25e

21A5-14 55.50bc 30.25bc 40.50c 21A6-43 25.75ef 15.50ef 30.50e

21A5-15 15.50g 05.25hi 10.25i 21A4-50 30.25de 20.50de 35.50d

21A5-16 55.25bc 20.50de 35.25d 21A4-51 30.50de 15.50ef 25.50f

21A5-17 30.25de 15.50ef 30.50e 21A4-52 35.50d 10.25egh 20.50g

21A5-18 20.50fg 15.25ef 20.50g 21A5-53 30.25de 15.75ef 25.50f

21A5-19 60.50ab 30.25bc 40.50c 21A5-57 30.25de 15.25ef 30.50e

21A4-20 25.50ef 20.50de 30.50e 21A5-58 25.50ef 10.75fg 15.25h

21A4-21 30.25de 25.50cd 30.50e 21A5-59 55.50bc 30.25bc 45.75b

21A12-24 65.75a 25.50cd 40.50c 21A5-60 50.50c 35.50ab 45.25b

Note: The means followed with same letters are within student ‘t’ range at α=0.05. Cont…

135




21A4-61 30.25de 15.50ef 25.75f 21A4-72 35.50d 15.25ef 25.50f

21A4-62 55.75bc 35.25ab 45.25b 21A4-73 60.25ab 30.25bc 40.50c

21A4-63 35.25d 20.25de 30.50e 21A4-74 05.75h 05.50ghi 15.25h

21A4-64 35.75d 15.50ef 25.50f 21A4-75 35.25d 10.50fg 20.75g

21A4-65 25.25ef 10.25fgh 20.25g 21A4-76 30.75de 15.25ef 25.25f

21A4-66 30.50de 15.75ghi 20.25g 21A4-77 25.25ef 15.25ef 20.50g

21A4-67 20.50fg 10.25fgh 20.50g 21A4-78 35.50d 20.50de 30.50e

21A6-68 65.25a 35.75ab 45.50b 21A12-79 35.50d 20.50de 25.25f

21A6-69 35.25d 15.25ef 25.25f 21A12-80 35.25d 10.25fgh 20.25g

21A6-70 35.50d 15.50ef 30.25e 21A12-81 30.25de 15.50ef 25.50f

21A6-71 25.50ef 15.50ef 25.50f

SD (±) 0.021 0.026 0.012

CD 0.005 0.007 0.003

CV (%) 3.43 4.74 2.11


136

137

138

The significant variation in Cry2Aa protein levels of different transgenic lines

irrespective of tissue types was revealed through ELISA assay (Plate 13). It was

observed that the Cry2Aa protein level in leaf tissue ranged from 0.013 to 3.231 µg/g of

fresh leaf tissue (Table 22a). Whereas, it was 0.010 to 2.854 µg/g in case of fresh flower

tissues and 0.012 to 2.811 µg/g in case of fresh pod tissue. Further, it was noticed that

the majority of putative transformants were accumulating the Cry2Aa protein from 0.0 to

0.5 µg/g of fresh tissue (Fig. 10). The highest Cry2Aa protein accumulation was recorded

in 21A12-24 transgenic line in both leaf and flower tissues, whereas in case of pod tissue

it was more in 21A4-73. On other hand, the lower level of Cry2Aa protein was recorded

in transgenic line 21A5-11 for all tissues types. The analysis of variance indicated

significant difference between Cry2Aa protein levels of different transgenic lines (p <

0.01). Interestingly, the ‘t’-test analysis of Cry2Aa protein accumulation in different tissue

types indicated non-significant difference between Cry protein levels among them (p >

0.45). There was a positive correlation between insect morality and Cry2Aa protein

accumulation with r2=0.8171 (Fig. 11). Similarly, the frequency distribution analysis of

transgenic plants following 3:1 transgene segregation pattern and other than 3:1

segregation pattern showed that majority of high Cry2Aa protein accumulating transgenic

plants were following 3:1 transgene segregation pattern (Fig. 11). The correlation

analysis of insect mortality and Cry2Aa protein levels in different tissues types of

developed transgenic plants indicated the positive correlation among them at α =0.01

(Table 22b).

4.4.4 The cry2Aa gene segregation analysis in T3 generation progenies

Based on the observation of transgene segregation analysis, bioefficacy test and

Cry2Aa protein accumulation in T2 generation progenies, a set of fifteen transgenic lines

were selected and advanced in to T3 generation. The transgenic lines advanced to T3

generation were 21A2-2, 21A3-4, 21A6-9, 21A5-10, 21A6-12, 21A5-14, 21A5-16, 21A5-

19, 21A12-24, 21A12-25, 21A5-59, 21A5-60, 21A4-62, 21A6-68 and 21A4-73, wherein

transgene reported 3:1 segregation pattern, comparatively high larval mortality and

Cry2Aa protein accumulation. The T3 seeds collected from T2 plants were sown in plant

139

140

Table 22a: The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2

generation as revealed by ELISA assay (α=0.05)

Plant ID Cry2Aa protein level (µg/g FW)



1A2-1 1.320 0.969 1.001 21A12-25 2.387 1.859 1.958

21A2-2 2.704 1.981 2.014 21A12-26 0.315 0.224 0.234

21A2-3 0.180 0.121 0.145 21A12-27 0.140 0.096 0.091

21A3-4 2.708 2.341 2.258 1A22-28 0.023 0.025 0.024

21A3-5 0.557 0.485 0.491 1A22-29 0.258 0.159 0.210

21A6-6 0.446 0.315 0.325 1A2-30 0.038 0.029 0.031

21A6-7 0.777 0.419 0.108 21A6-31 0.338 0.289 0.275

21A6-8 0.835 0.589 0.612 21A6-32 0.098 0.089 0.075

21A6-9 2.913 2.124 2.302 21A6-33 0.173 0.124 1.350

21A5-10 2.550 2.152 2.189 2A22-39 0.159 0.128 0.124

21A5-11 0.013 0.010 0.012 1A2-40 0.214 0.189 0.191

21A6-12 2.003 1.485 1.541 1A2-41 0.104 0.095 0.099

21A6-13 0.048 0.051 0.045 1A2-42 0.192 0.124 0.141

21A5-14 1.950 1.428 1.521 21A6-43 0.140 0.115 0.121

21A5-15 0.085 0.059 0.069 21A4-50 0.088 0.074 0.079

21A5-16 1.833 1.305 1.285 21A4-51 0.133 0.110 0.101

21A5-17 0.491 0.298 0.324 21A4-52 0.476 0.296 0.301

21A5-18 0.124 0.099 0.102 21A5-53 0.136 0.110 0.128

21A5-19 2.204 1.897 1.793 21A5-57 0.186 0.128 0.134

21A4-20 0.138 0.990 0.105 21A5-58 0.124 0.112 0.108

21A4-21 0.305 0.199 0.201 21A5-59 2.076 1.856 1.812

21A12-24 3.231 2.854 2.795 21A5-60 1.782 1.412 1.329

Cont…

141




21A4-61 0.157 0.124 0.119 21A4-72 0.062 0.051 0.058

21A4-62 2.161 1.854 1.799 21A4-73 3.080 2.749 2.811

21A4-63 0.126 0.110 0.109 21A4-74 0.090 0.074 0.081

21A4-64 0.189 0.125 0.154 21A4-75 0.087 0.078 0.064

21A4-65 0.131 0.110 0.124 21A4-76 0.042 0.041 0.038

21A4-66 0.121 0.102 0.113 21A4-77 0.120 0.110 0.118

21A4-67 0.143 0.121 0.119 21A4-78 0.077 0.061 0.068

21A6-68 3.112 2.458 2.621 21A12-79 0.170 0.149 0.157

21A6-69 0.198 0.154 0.161 21A12-80 0.133 0.124 0.119

21A6-70 0.191 0.148 0.156 21A12-81 0.103 0.098 0.100

21A6-71 0.095 0.068 0.071

SD (±) 0.016 0.016 0.017

CD 0.002 0.001 0.001

CV (%) 2.04 2.63 2.52

142

143

Table 22b: The correlation analysis of insect mortality levels and estimated Cry2Aa protein in leaf, flower and pod tissues of T2

generation plants (αααα=0.01)




Cry2Aa protein in leaf

tissues

Cry2Aa protein in flower tissues

Cry2Aa protein in pod

tissues


1 0.821** 0.890** 0.904** 0.884** 0.886**


1 0.945** 0.795** 0.781** 0.767**


1 0.834** 0.820** 0.806**

Cry2Aa protein in leaf tissues

1 0.985** 0.977**


1 0.971**

Cry2Aa protein in pod tissues

1

**Correlation is significant at the 0.01 level (two trials).

144

to row progeny manner. The gene specific PCR assay identified T3 plant progenies

carrying cry2Aa gene. The chi-square test analysis of observed number of plants with

and without transgene indicated that majority of plants were heterozygous in nature for

cry2Aa locus. Among fifteen transgenic lines, in four transgenic lines homozygous nature

of cry2Aa locus was detected. The T2 parental plants with homozygous nature were

21A2-2-1, 21A3-4-7, 21A5-16-1 and 21A4-62-6 (Plate 11) (Table 23).

4.4.5 Characterization of fifteen transgenic lines carrying cry2Aa gene in T3 generation

The bioefficacy analysis of T3 generation transgenic lines revealed consistent

nature of their efficacy against neonate larvae of Helicoverpa armigera. The bioassay

results reported that the larval mortality in case of leaf tissue, which ranged from 43.33 to

68.33 per cent. The larval mortality ranges in case of flower tissue were 23.5 to 48.5 per

cent and in case of pod it ranged from 28.5 to 53.25 per cent (Table 24a). The highest

larval mortality recorded in case of transgenic line 21A6-68 for all tissue types. On other

hand, least larval mortality was noticed in transgenic line 21A5-16. The analysis of

variance showed that the transgenic lines differ significantly (p < 0.01) for larval mortality

in all tissue types. The ‘t’-test analysis of larval mortality levels in different tissue types

indicated significant difference between mortality levels in leaf and flower (p ≤ 0.001) and

in leaf and pod tissues (p ≤ 0.001). It was interesting to notice that the larval mortality

levels between flower and pod were on par with each other with p ≥ 0.06. The ‘t’-test

analysis of mortality levels in T2 and T3 generations for developed transgenic plants

carrying cry2Aa gene reported non-significant difference in insect mortalities between

two generations for all tissue types (p ≥ 0.08) (Table 24b).

The similar pattern of consistent Cry2Aa protein accumulation was noticed in

selected fifteen transgenic lines in T3 generation. The Cry2Aa protein levels in leaf

tissues was recorded and ranged from 0.370 to 1.417 µg/g of fresh tissue weight (Table

25). The Cry2Aa protein accumulation in flower tissues was noticed ranged from 0.312 to

1.021 µg/g and in pod it ranged from 0.309 to 0.989 µg/g. The higher level of Cry2Aa

proteins was recorded in 21A6-68 transgenic line irrespective of tissue type. On other

hand, the Cry2Aa accumulation was lowest in transgenic line 21A4-62. The statistical

analysis indicated that the transgenic lines were differencing significantly for Cry2Aa

145

Table 23: Transgene segregation pattern in fifteen transgenic lines carrying cry2Aa gene in T3 generation

Plant ID

Total number of progenies

tested


Number of PCR

negative plants

Expected number of

positive plants



Estimated plant

zygosity

A2-2-1 16 16 00 11.25 3.75 05.00 Homo.

A2-2-5 16 06 10 12.00 4.00 12.00 Hetero.

A2-2-7 15 10 05 11.25 3.75 00.56 Hetero.

A2-2-8 15 11 04 11.25 3.75 00.02 Hetero.

A2-2-9 19 14 05 14.25 4.75 00.02 Hetero.

A2-2-12 12 09 03 09.00 3.00 00.00 Hetero.

A3-4-2 16 15 01 12.00 4.00 03.00 Hetero.

A3-4-4 12 08 04 09.00 3.00 00.44 Hetero.

A3-4-5 19 13 06 14.25 4.75 00.44 Hetero.

A3-4-7 17 17 00 12.75 4.25 05.67 Homo.

A3-4-8 17 10 07 12.75 4.25 02.37 Hetero.

A3-4-9 15 11 04 11.25 3.75 00.02 Hetero.

A6-9-3 17 11 06 12.75 4.25 00.96 Hetero.

A6-9-4 21 14 07 15.75 5.25 00.78 Hetero.

A6-9-6 19 11 08 14.25 4.75 02.96 Hetero.

A6-9-9 20 08 12 15.00 5.00 13.07 Hetero.

A6-9-10 10 02 08 07.50 2.50 16.13 Hetero.

A6-9-12 13 08 05 09.75 3.25 01.26 Hetero.

A5-10-1 11 07 04 08.25 2.75 00.76 Hetero.

A5-10-4 13 08 05 09.75 3.25 01.26 Hetero.

A5-10-5 14 06 08 10.50 3.50 07.71 Hetero.

A5-10-8 12 07 05 09.00 3.00 01.78 Hetero.

A5-10-13 10 06 04 07.50 2.50 01.20 Hetero.

A5-10-14 14 08 06 10.50 3.50 02.38 Hetero.

A6-12-1 15 11 04 11.25 3.75 00.02 Hetero.

A6-12-2 14 08 06 10.50 3.50 02.38 Hetero.

A6-12-3 15 09 06 11.25 3.75 01.80 Hetero.

A6-12-4 15 10 05 11.25 3.75 00.56 Hetero.

A6-12-8 16 07 09 12.00 4.00 08.33 Hetero.

A6-12-9 14 09 05 10.50 3.50 00.86 Hetero.

A5-14-1 18 14 04 13.50 4.50 00.07 Hetero.

A5-14-2 15 12 03 11.25 3.75 00.20 Hetero.

A5-14-4 17 12 05 12.75 4.25 00.18 Hetero.

A5-14-5 14 10 04 10.50 3.50 00.10 Hetero.

A5-14-6 16 12 04 12.00 4.00 00.00 Hetero.

A5-14-10 14 08 06 10.50 3.50 02.38 Hetero.

Contd…

146

Plant ID

Total number

of progenies

tested


Number of PCR

negative plants

Expected number

of positive plants

Expected number

of negative plants


Estimated plant

zygosity

A5-16-1 15 15 00 11.25 3.75 05.00 Homo.

A5-16-2 18 16 02 13.50 4.50 01.85 Hetero.

A5-16-3 12 08 04 09.00 3.00 00.44 Hetero.

A5-16-4 14 10 04 10.50 3.50 00.10 Hetero.

A5-16-11 16 11 05 12.00 4.00 00.33 Hetero.

A5-16-12 14 08 06 10.50 3.50 02.38 Hetero.

A5-19-4 12 08 04 09.00 3.00 00.44 Hetero.

A5-19-5 14 08 06 10.50 3.50 02.38 Hetero.

A5-19-7 10 06 04 07.50 2.50 01.20 Hetero.

A5-19-8 14 09 05 10.50 3.50 00.86 Hetero.

A5-19-9 13 10 03 09.75 3.25 00.03 Hetero.

A5-19-10 14 08 06 10.50 3.50 02.38 Hetero.

A12-24-1 14 07 07 10.50 3.50 04.67 Hetero.

A12-24-2 15 08 07 11.25 3.75 03.76 Hetero.

A12-24-3 13 06 07 09.75 3.25 05.77 Hetero.

A12-24-4 14 09 05 10.50 3.50 00.86 Hetero.

A12-24-6 15 10 05 11.25 3.75 00.56 Hetero.

A12-24-8 10 06 04 07.50 2.50 01.20 Hetero.

A12-25-3 16 10 06 12.00 4.00 01.33 Hetero.

A12-25-4 15 10 05 11.25 3.75 00.56 Hetero.

A12-25-6 20 13 07 15.00 5.00 01.07 Hetero.

A12-25-7 15 10 05 11.25 3.75 00.56 Hetero.

A12-25-8 14 08 06 10.50 3.50 02.38 Hetero.

A12-25-10 12 08 04 09.00 3.00 00.44 Hetero.

A5-59-7 12 08 04 09.00 3.00 00.44 Hetero.

A5-59-5 12 09 03 09.00 3.00 00.00 Hetero.

A5-59-1 14 09 05 10.50 3.50 00.86 Hetero.

A5-59-6 13 08 05 09.75 3.25 01.26 Hetero.

A5-59-3 12 05 07 09.00 3.00 07.11 Hetero.

A5-59-4 10 05 05 07.50 2.50 03.33 Hetero.

A5-60-2 12 08 04 09.00 3.00 00.44 Hetero.

A5-60-3 17 11 06 12.75 4.25 00.96 Hetero.

A5-60-9 11 09 02 08.25 2.75 00.27 Hetero.

A5-60-10 13 08 05 09.75 3.25 01.26 Hetero.

A5-60-11 06 04 02 04.50 1.50 00.22 Hetero.

A5-60-1 10 07 03 07.50 2.50 00.13 Hetero.

A4-62-10 09 05 04 06.75 2.25 01.81 Hetero.

A4-62-3 12 11 01 09.00 3.00 01.78 Hetero.

A4-62-2 14 08 06 10.50 3.50 02.38 Hetero.

A4-62-7 12 08 04 09.00 3.00 00.44 Hetero.

A4-62-6 11 11 00 08.25 2.75 03.67 Homo.

A4-62-8 09 06 03 06.75 2.25 00.33 Hetero.

Cont…

147

Plant ID

Total number

of progenies

tested


Number of PCR

negative plants

Expected number

of positive plants

Expected number

of negative plants


Estimated plant

zygosity

A6-68-4 12 08 04 09.00 3.00 00.44 Hetero.

A6-68-1 14 09 05 10.50 3.50 00.86 Hetero.

A6-68-10 12 08 04 09.00 3.00 00.44 Hetero.

A6-68-4 15 11 04 11.25 3.75 00.02 Hetero.

A6-68-11 14 10 04 10.50 3.50 00.10 Hetero.

A6-68-2 13 09 04 09.75 3.25 00.23 Hetero.

A4-73-7 15 10 05 11.25 3.75 00.56 Hetero.

A4-73-3 10 08 02 07.50 2.50 00.13 Hetero.

A4-73-5 12 08 04 09.00 3.00 00.44 Hetero.

A4-73-4 12 07 05 09.00 3.00 01.78 Hetero.

A4-73-1 11 07 04 08.25 2.75 00.76 Hetero.

A4-73-2 14 08 06 10.50 3.50 02.38 Hetero.

Where; Hetero.: Heterozygous; Homo.: Homozygous

148

Table 24a: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fifteen putative transformants carrying cry2Aa gene in T3 generation

Event ID Corrected per cent larval mortality


21A2-2 58.33abc 43.50ab 50.50a

21A3-4 58.33abc 43.00ab 49.00a

21A6-9 63.33ab 43.00ab 45.00ab

21A5-10 48.33cd 38.00abc 43.00abc

21A6-12 53.33bcd 33.50bcd 36.50bcd

21A5-14 48.33cd 28.00de 32.00d

21A5-16 48.33cd 23.50e 28.50d

21A5-19 58.33abc 48.50a 52.50a

21A12-24 68.33a 43.25ab 47.25a

21A12-25 58.33abc 38.50abc 44.50ab

21A5-59 48.33cd 30.50cde 34.50cd

21A5-60 43.33d 28.00de 32.00d

21A4-62 43.33d 28.25de 33.25cd

21A6-68 68.33a 48.25a 53.25a

21A4-73 53.33bcd 28.50de 34.50cd

SD (±) 0.024 0.063 0.063

CD 0.004 0.012 0.012

CV (%) 3.22 6.75 5.84


149

Table 24b: The ‘t’-test analysis of mortality levels from T2 and T3 generation of fifteen

cry2Aa transgenic lines (α=0.05)


T2

generation T3

generation T2

generation T3

generation T2

generation T3

generation

Mean 57.15 54.66 31.75 36.42 42.73 41.08

Variance 52.12 65.95 30.33 68.42 28.08 70.77

Observations 15 15 15 15 15 15

Pooled Variance 59.04 49.37 49.43

Hypothesized Mean Difference 0 0 0

Df 28 28 28

t Stat 0.89 -1.82 0.64

P(T<=t) two-tail 0.38 0.08 0.53

t Critical two-tail 2.05 2.05 2.05

150

Table 25: The Cry2Aa protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T3 generation as revealed by ELISA assay

Event ID Cry2Aa protein level (µg/g FW)

Leaf tissue Flower tissue Pod tissue

21A2-2 1.046d 0.910b 0.915c

21A3-4 0.975e 0.825c 0.812d

21A6-9 1.170b 0.914b 0.931b

21A5-10 0.667i 0.589h 0.605i

21A6-12 0.829g 0.712f 0.728g

21A5-14 0.609j 0.548i 0.543j

21A5-16 0.485k 0.401j 0.398k

21A5-19 0.892f 0.759d 0.782e

21A12-24 1.127c 0.912b 0.939b

21A12-25 0.889f 0.729e 0.742f

21A5-59 0.457l 0.356k 0.371l

21A5-60 0.424m 0.387j 0.392k

21A4-62 0.370n 0.312l 0.309m

21A6-68 1.417a 1.021a 0.989a

21A4-73 0.798h 0.685g 0.691h

SD (±) 0.017 0.019 0.018

CD 0.005 0.005 0.004

CV (%) 2.19 2.78 2.65


151

protein accumulation with p < 0.001 in case of leaf, flower and pod tissue types.

Interestingly, the ‘t’-test analysis of Cry protein accumulation in different tissue types

indicated that there was significant difference in Cry2Aa protein levels in leaf and flower

(p < 0.08), whereas it was on par between flower and pod tissues (p > 0.07) and

between leaf and pod tissue types (p >0.09). The correlation analysis between insect

morality and Cry2Aa protein accumulation revealed positive correlation between them

with r2=0.8434 (Fig. 12).

4.4.6 The cry2Aa transcript analysis using qRT-PCR assay

The observation of absolute real time analysis of cry2Aa transcripts in different

tissue types of selected fifteen transgenic lines indicated significant variation in cry2Aa

transcript levels (Plate 14). It was noticed that the cry2Aa transcript level ranged from

48.5 to 134.5 ng/µl in fresh leaf tissue of transgenic plants (Table 26a). Similar

observation was noticed in flower tissues where the cry2Aa transcript level ranged from

41.2 to 110.2 ng/µl and in pod from 42.8 to 100.5 ng/µl. The highest transcript level was

recorded in 21A6-68 for leaf and flower tissues and in 21A2-2 for pod. The lower level of

cry2Aa transcripts was recorded in case of transgenic line 21A4-62. The ANOVA

statistical analysis indicated the observation of significant difference between transgene

transcript levels of transgenic lines (p < 0.01). The ‘t’-test analysis of transcript levels

between tissue types revealed non-significant difference in the transcripts levels of

cry2Aa gene in different tissue types within developed transgenic plants (p ≥ 0.11). The

correlation analysis between Cry2Aa protein accumulation and transgene transcript

levels revealed presence of positive correlation (r2=0.9469)

(Fig. 12). The correlation analysis of larval mortality, Cry2Aa protein and cry2Aa

transcript reported positive in leaf, flower and pod tissues for these traits of T3 generation



The genomic DNA from four selected transgenic plants was digested using HindIII

restriction endonuclease and Southern blotting was performed to analyse the T-DNA

integration pattern in to plant genome. The blotted digested genomic DNA was probed

with 676 bp labelled cry2Aa PCR product. A single but weak hybridization signals were

noticed for two samples belonging to 21A2-2 and 21A3-4 transgenic lines. The Southern

152

153

154

Table 26a: The cry2Aa transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

Event ID cry2Aa transcript level in leaf tissue

(ng/µl)

cry2Aa transcript level in flower tissue

(ng/µl)

cry2Aa transcript level in pod tissue

(ng/µl)

21A2-2 124.2c 105.1b 100.5a

21A3-4 100.4e 095.6d 094.6b

21A6-9 115.2d 099.8c 095.4b

21A5-10 084.6h 081.5g 078.9d

21A6-12 091.2g 085.4ef 081.6c

21A5-14 078.1i 071.5h 068.9e

21A5-16 054.9j 050.6j 048.6g

21A5-19 098.4e 084.5f 078.2d

21A12-24 128.1b 104.3b 094.6b

21A12-25 095.2f 087.3e 082.4c

21A5-59 054.8j 049.5j 047.2g

21A5-60 052.6k 045.7k 042.9h

21A4-62 048.5l 041.2l 042.8h

21A6-68 134.5a 110.2a 099.8a

21A4-73 086.2h 061.5i 062.9f

SD (±) 2.421 2.734 2.639

CD 0.779 0.774 0.823

CV (%) 2.73 3.49 3.54


155

Table 26b: The correlation analysis of insect mortality levels, estimated Cry2Aa protein and cry2Aa transcript in leaf, flower and pod

tissues of T3 generation plants (αααα=0.01)

Insect mortality

in leaf tissues


Insect mortality

in pod tissues

Cry2Aa protein in

leaf tissues

Cry2Aa protein in

flower tissues

Cry2Aa protein in

pod tissues

cry2Aa transcript


cry2Aa transcript


cry2Aa transcript

level in pod tissue


1 0.829** 0.811** 0.960** 0.934** 0.935** 0.936** 0.891** 0.863**


1 0.988** 0.848** 0.852** 0.856** 0.857** 0.873** 0.855**


1 0.838** 0.848** 0.848** 0.854** 0.866** 0.853**

Cry2Aa protein in leaf tissues

1 0.984** 0.976** 0.973** 0.939** 0.925**


1 0.998** 0.986** 0.962** 0.959**

Cry2Aa protein in pod tissues

1 0.986** 0.962** 0.958**

cry2Aa transcript level in leaf tissue

1 0.970** 0.958**

cry2Aa transcript level in flower tissue

1 0.994**

cry2Aa transcript level in pod tissue

1


156

hybridization pattern was different for both samples. On other hand, there were strong

hybridization signals noticed in other two other samples viz., 21A6-9 and 21A6-68.

Interestingly, the hybridization pattern was same in both the samples, indicated the

possibility of same position of T-DNA integration in plant genome. An observation of

single hybridization signal in all tested transgenic plants indicated the single copy

integration of transgene (Plate 15).


The transcription of cry2Aa gene was further confirmed using northern blot

analysis using 676 bp labelled cry2Aa PCR product as a probe (Plate 16). The

hybridization signals were noticed in all selected transgenic plant samples viz., 21A2-2,

21A3-4, 21A6-9, 21A12-24 and 21A6-68. The observation of strong signals revealed the

transcription of cry2Aa gene in transgenic lines.

4.4.9 Juncture region analysis of T-DNA integration

The site of insertion of T-DNA carrying cry2Aa gene in the pigeonpea genome

was identified by recovering the genomic sequence flanking the left border (LB) of

T-DNA. The smear was noticed in the primary TAIL-PCR product obtained from 35S

revers and arbitrary primer AD-2. The secondary TAIL-PCR reported development of

multiple bands when diluted primary product was used as template. However, there was

a reduction in number of multiple bands produced with development of single thick bands

in the tertiary TAIL-PCR (Plate 17). The bright bands with more than one kb amplicon

size were eluted and cloned into pTZ57R/T and sequenced. Sequence of all the five

lines viz., 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68 were analysed by using

BioEdit bioinformatics algorithm.

The sequences were analysed and detected vector backbone sequences were

trimmed. Such processed forward and revers sequences were assembled and contigues

were formed. The local blast was set using BioEdit tool for processed contigue

sequences and it was observed that flaking genomic region recovered from 21A2-2

showed 100 per cent homology for 89 nucleotide bases of 144 bp with Scaffold137204 of

pigeonpea genome (Table 27). Similarly, the flaking genomic region recovered from

21A3-4 showed 100 per cent homology for 58 bp out of 186 bp and genomic region

157

158

Table 27: Juncture region analysis of cry2Aa cassette in selected transgenic lines as revealed by TAIL PCR analysis

Transgenic lines Pigeonpea contig Position (bp) Query coverage E value

21A5-14 Scaffold137204 398597-398598 89/144 (100%) 4e-043

21A6-7 Scaffold134438 5542-5543 58/186 (100%) 2e-024

21A5-60 Scaffold127179 23885-23886 89/150 (100%) 4e-043

21A3-4 Scaffold127179 23885-23886 85/195 (100%) 8e-036

21A5-10 Scaffold134438 5481-5482 119/243 (99%) 2e-058

recovered from 21A12-24 showed 99 per cent homology for 119 bp out of 243 bp with

Scaffold134438. On other hand, in case of 21A6-9 and 21A6-68, the recovered flaking

159

genomic region indicated 100 per cent homology (89 bp of 150 bp and 85 bp of 195 bp

respectively) with Scaffold127179 of pigeonpea draft genome.

4.5 Generation of transgenic pigeonpea conferring expression of cry1F gene

The transgenic pigeonpea expressing cry1F was developed using improvised in

planta transformation method (Fig. 13). The developed transgenic pigeonpea plants

were characterized for their bioefficacy against H. armigera larvae and quantitative

Cry1F protein analysis using ELISA. The transgene segregation pattern was assessed

using cry1F gene specific PCR assay. The developed transgenic lines were subjected

for cry1F transcript analysis using qRT-PCR, Southern and northern blot analysis.

4.5.1 Development of transgenic pigeonpea carrying cry1F gene

A total of 350 explants were infected with Agrobacterium tumefaciens strain

carrying cry1F gene carrying construct and set of 177 primary transformants were

established, which was considered as T0 generation (Table 28). The T1 seeds were

harvested form well-established primary transformants and T1 generation was raised.

The T1 progeny plant screening using cry1F gene specific PCR assay identified a set of

fourteen putative transgenic plants for integration of cry1F gene (Plate 18). The identified

putative transformants were grown till maturity and T2 seeds were harvested.

4.5.2 The cry1F gene segregation analysis in T2 generation progenies

The obtained T2 seeds were sown in plant to row progeny manner to get T2

generation plants. From each parental T1 generation plant up to 40 seeds were sown

and obtained plants were subjected for gene specific PCR assay. The gene segregation

pattern was assayed using chi-square test. Among developed fourteen, for seven

transgenic plants the chi-square calculated value was recorded less than table chi-

square value (3.84) (Table 29) (Plate 19). The observation of chi-square analysis results

160

Table 28: Summary of transformation carried out using cry1F gene in pigeonpea

Sl. No Number of

explants co-cultivated/set

Number of explants

responded

Number of primary

transformants (T0)


Number of primary transformants tested

in plant-to-row progeny for

identifying putative transformants

Number of putative


1 50 38 25 25 4

2 50 34 20 20 0

3 50 32 26 26 0

4 50 37 30 30 0

5 50 35 28 28 6

6 50 31 25 25 0

7 50 30 23 23 4

Total 350 237 177 177 14

161

162

Table 29: The transgene segregation pattern in fourteen transformants carrying cry1F in T2 generation revealed by gene specific PCR assay

Plant ID


PCR


PCR


PCR




Gene segregation in

3:1 ration

1F-22 28 17 11 21.00 7.00 03.05 Yes

1F-32 25 15 10 18.75 6.25 03.00 Yes

1F-38 24 16 08 18.00 6.00 00.89 Yes

1F-19 28 15 13 21.00 7.00 06.86 No

1F-10 24 15 09 18.00 6.00 02.00 Yes

1F-20 27 16 11 20.25 6.75 03.57 Yes

1F-25 27 14 13 20.25 6.75 07.72 No

1F-21 28 12 16 21.00 7.00 15.43 No

1F-37 20 12 08 15.00 5.00 02.40 Yes

1F-9 28 15 13 21.00 7.00 06.86 No

1F-36 25 12 13 18.75 6.25 09.72 No

1F-35 25 12 13 18.75 6.25 09.72 No

1F-33 35 22 13 26.25 8.75 02.75 Yes

1F-45 24 12 12 18.00 6.00 08.00 No


163

revealed the 3:1 (positive: negative) cry1F gene segregation pattern in transgenic plant

progenies of 1F-22, 1F-32, 1F-38, 1F-10, 1F-20, 1F-33 and 1F-37 in T2 generation. For

rest seven transgenic plants the chi-square value was noticed more than the table value,

clearly indicating that the cry1F gene segregation in those transgenic plant progenies

was not following 3:1 gene segregation pattern in T2 generation.


The bioefficacy of the plants against first instar H. armigera larvae revealed

significant variability in larval mortality and damage in all targeted tissue types viz., leaf,

flower and pod. The larval mortality in case of leaf tissues ranged from 22.5 to 62.5 per

cent (Table 30). It was 20.0 to 57.5 per cent in case of flower tissues and 10.0 to 47.5

per cent in case of pod. It also was noticed that the majority of putative transformants

were having insect mortality from 40.0 to 60.0 per cent in both leaf and flower tissue, with

the highest mortality recorded in 1F-22 transgenic line for leaf and in 1F-33 for flower

(Fig. 14). On other hand, in case of pod, the majority of putative transformants showed

insect mortality from 30.0 to 50.0 per cent with the highest mortality recorded in 1F-22

and 1F-33. The larval feeding was highest in transgenic line 1F-25. The larval feeding

pattern on different tissue types of transgenic and non-transgenic control plants is shown

in Plate 20. The statistical analysis, analysis of variance (ANOVA), indicated that the

transgenic lines differ significantly (p < 0.001) from each other for larval mortality in all

tissue types and for leaf damage. The ‘t’-test analysis of larval mortality in different tissue

types indicated significant difference between mortality levels in leaf and flower (p ≤

0.01), and in flower and pod tissues (p ≤ 0.01). Interestingly, the larval mortality levels

were comparable between leaf and pod tissue (p ≥ 0.9) types as indicated by ‘t’-test

analysis.

There was a significant variation in Cry1F protein levels of different transgenic

lines irrespective of tissue types viz., leaf, flower and pod as revealed through ELISA

assay (Plate 21). The Cry protein level ranged from 0.170 to 1.032 µg/g of fresh leaf

tissue (Table 31). Whereas, it was 0.113 to 0.870 µg/g of fresh flower tissues and 0.117

to 0.782 µg/g of fresh pod tissue. Interestingly, it was noticed that the majority of putative

transformants were accumulating the Cry1F protein from 0.4 to 0.6 µg/g of fresh leaf

tissue, wherein the highest Cry1F proteins was detected in 1F-22 transgenic line (Fig.

12). Similarly, in case of flower and pod tissues, the majority of transgenic plants were

164

Table 30: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving fourteen putative transformants carrying cry1F gene in T2 generation.



1F-22 62.5a 55.0a 47.5a

1F-32 57.5ab 50.0ab 42.5abc

1F-38 60.0a 47.5ab 42.5abc

1F-19 47.5c 40.0bc 37.5bc

1F-10 50.0bc 50.0ab 45.0ab

1F-20 47.5c 42.5b 37.5bc

1F-25 22.5e 20.0d 10.0d

1F-21 47.5c 35.0c 40.0abc

1F-37 57.5ab 55.0a 40.0abc

1F-9 30.0d 25.0d 12.5d

1F-36 47.5c 47.5ab 40.0abc

1F-35 50.0bc 47.5ab 37.5bc

1F-33 60.0a 57.5a 47.5a

1F-45 47.5c 47.5ab 35.0c

SD (±) 0.057 0.060 0.058

CD 0.043 0.047 0.044

CV (%) 5.15 5.42 5.25


165

166

167

168

Table 31: The Cry1F protein level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay

Plant ID Cry1F protein level (µg/g FW)


1F-22 1.032a 0.870a 0.782a

1F-32 0.834b 0.557b 0.523b

1F-38 0.683d 0.511c 0.499c

1F-19 0.489g 0.327h 0.301g

1F-10 0.570e 0.412f 0.412d

1F-20 0.552e 0.462d 0.425d

1F-25 0.170j 0.113k 0.117i

1F-21 0.457h 0.301i 0.309g

1F-37 0.556e 0.433e 0.422d

1F-9 0.286i 0.194j 0.181h

1F-36 0.522f 0.394g 0.342f

1F-35 0.470h 0.386g 0.383e

1F-33 0.714c 0.547b 0.513bc

1F-45 0.502g 0.430e 0.410d

SD (±) 0.025 0.027 0.026

CD 0.007 0.006 0.007

CV (%) 4.60 4.47 4.69


169

accumulating Cry1F protein from 0.3 to 0.6 µg/g of fresh tissue. The transgenic line 1F-

22 showed highest accumulation of Cry protein in both flower and pod tissues as well, as

indicated by ELISA assay. On other hand, the lower level of Cry protein was recorded in

transgenic line 1F-25 and 1F-9. The statistical analysis of variance clearly indicated the

significant difference in Cry1F protein levels among developed transgenic lines (p <

0.001). The ‘t’-test analysis of Cry protein accumulation in different tissue types indicated

significant difference in leaf and flower (p ≤ 0.01), in flower and pod tissues (p < 0.03)

and between leaf and pod tissue (p ≤ 0.03) types as indicated by ‘t’-test analysis. There

was a positive correlation between insect morality and Cry1F protein accumulation with

r2=0.781 (Fig. 15). Further, the frequency distribution analysis of transgenic plants

following 3:1 transgene segregation pattern and other than 3:1 segregation pattern

showed that majority of high Cry1F protein accumulating transgenic plants were

following 3:1 transgene segregation pattern (Fig. 15).

4.5.4 The cry1F transcript analysis using qRT-PCR assay

Absolute real time analysis of cry1F transcript levels in different tissue types

indicated significant variation in cry1F transcript levels in developed transgenic lines as

revealed by standard graph (Plate 22). The cry1F transcript level ranged from 47.6 ng/µl

to 105.3 ng/µl in fresh leaf tissue (Table 32). Similarly, the cry1F transcript level in flower

was ranged from 45.2 ng/µl to 101.0 ng/µl and in pod from 48.5 ng/µl to 103.2 ng/µl.

Interestingly, the highest transcript level was detected in all tissue types of 1F-25 and 1F-

9 transgenic line. On other hand, the lower level of cry1F transcripts was recorded in

transgenic line 1F-35. The statistical analysis of variance clearly indicated the significant

difference in cry1F transcript levels among developed transgenic lines (p < 0.001). The

‘t’-test analysis revealed non-significant difference in the transcripts levels of cry1F gene

in different tissue types within developed transgenic plants (p ≥ 0.94). There was a

positive correlation between Cry1F protein accumulation and cry1F transcript levels

(r2=0.0431) (Fig. 15). The correlation analysis of larval mortality, Cry1F protein and cry1F

transcript reported positive in leaf, flower and pod tissues for these traits of T2 generation


170

171

172

Table 32a: The cry1F transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

Event ID cry1F transcript level in leaf tissue (ng/µl)

cry1F transcript level in flower tissue (ng/µl)

cry1F transcript level in pod tissue (ng/µl)

1F-22 105.3c 101.0c 103.2c

1F-32 080.5d 081.2d 079.5d

1F-38 075.1e 073.4e 072.5e

1F-19 060.2h 061.9h 061.9g

1F-10 071.6f 070.8f 070.4e

1F-20 070.5fg 068.1g 072.1e

1F-25 140.2a 137.6a 142.3a

1F-21 059.4h 060.2h 060.1g

1F-37 068.7g 066.9g 068.1f

1F-9 128.2b 130.8b 129.3b

1F-36 054.3i 053.6i 052.0h

1F-35 047.6j 045.2k 048.5i

1F-33 078.5d 079.1d 078.0d

1F-45 049.2j 050.1j 048.6i

SD (±) 1.414 1.212 1.313

CD 0.905 0.889 0.843

CV (%) 1.82 1.52 1.69


173

Table 32b: The correlation analysis of insect mortality levels, estimated Cry1F protein and cry1F transcript in leaf, flower and pod

tissues of T2 generation plants (αααα=0.05; 0.01)

Insect mortality

in leaf tissues


Insect mortality

in pod tissues

Cry1F protein in

leaf tissues

Cry1F protein in

flower tissues

Cry1F protein in

pod tissues

cry1F transcript


cry1F transcript


cry1F transcript

level in pod tissue


1 0.625* 0.750** 0.827** 0.780** 0.817** 0.796** 0.791** 0.771**


1 0.497 0.556 0.647* 0.647* 0.539 0.511 0.524


1 0.747** 0.664* 0.687* 0.811** 0.821** 0.788**

Cry1F protein in leaf tissues

1 0.954** 0.952** 0.924** 0.921** 0.906**

Cry1F protein in flower tissues

1 0.992** 0.876** 0.851** 0.858**

Cry1F protein in pod tissues

1 0.882** 0.858** 0.864**

cry1F transcript level in leaf tissue

1 0.995** 0.996**

cry1F transcript level in flower tissue

1 0.992**

cry1F transcript level in pod tissue

1

*Correlation is significant at the 0.05 level (two tailed). **Correlation is significant at the 0.01 level (two tailed).

174


The Southern blotting was performed to study the integration pattern of T-DNA in

to plant genome, for which from six selected transgenic plants the genomic DNA was

digested (Plate 23). It was performed with EcoRI as the enzyme that cuts in the T-DNA

region and probed with 600 bp cry1F PCR product. An observation of strong signal and

the difference in the hybridization pattern in the four selected transgenic plants viz., 1F-

22, 1F-32, 1F-38 and 1F-33, revealed the single copy integration in these plants.

Whereas, there was no hybridization signal noticed with the DNA of non-transformed

plants. On other hand, in two plant samples viz., 1F-25 and 1F-9, hybridization signals

were noticed in two places indicating integration of two T-DNA copies in plant genome.


The presence of cry1F transcript in transgenic pigeonpea plants was also

confirmed with northern blotting using 600 bp cry1F PCR product as a probe (Plate 24).

The hybridization signals were noticed in all selected transgenic plant samples viz., 1F-

25, 1F-32, 1F-33, 1F-9, 1F-22 and 1F-38. The observation of strong signals revealed the

production of transcripts in respective transgenic plants.

4.6 Generation of transgenic pigeonpea conferring expression of cry1Acm gene

The in planta transformation method was employed and transgenic pigeonpea

expressing cry1Acm was developed. The characterization of developed transgenic

plants was done by performing insect bioassay against H. armigera larvae and

quantitative Cry1Acm protein analysis using ELISA. The segregation pattern of cry1Acm

was assessed using gene specific PCR assay. Further, the developed transgenic lines

were also subjected for cry1Acm transcript analysis using qRT-PCR, Southern and

northern blot analysis.

4.6.1 Development of transgenic pigeonpea carrying cry1Acm gene

The A. tumefaciens infection was made to a total of 250 explants using

Agrobacterium tumefaciens strain carrying cry1Acm gene carrying construct .The

transformation efforts resulted in the development of total of 120 primary transformants,

which were established in green house facility (Table 33). The T1 seeds were collected

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form well-established primary transformants and T1 generation was raised. The T1

progeny plant screening identified a set of eleven putative transgenic plants carrying

cry1Acm gene as revealed by gene specific (cry1Acm) PCR assay (Plate 25). The

identified putative transformants were grown till maturity and T2 seeds were harvested.

4.6.2 The cry1Acm gene segregation analysis in T2 generation progenies

The collected T2 seeds were sown in plant to row progeny manner and T2

generation progenies were raised. The chi-square analysis of observed number of plants

with and without cry1Acm gene indicated that among eleven transgenic lines, seven

were following 3:1 (positive: negative) ration of transgene segregation (Table 34) (Plate

26). Whereas, in case of rest four transgenic lines the chi-square value was observed to

be more than the table value (3.84), clearly indicating that the cry1Acm gene segregation

in those transgenic plant progenies did not following 3:1 segregation pattern for

transgene in T2 generation.


The insect bioassay against first instar Helicoverpa armigera larvae reported

significant variability in larval mortality targeted tissue types. It was noticed that, the

larval mortality in case of leaf tissues ranged from 40.0% to 62.5% (Table 35). Similarly,

in case of flower bioassay it ranged 37.5% to 57.5% and 35.0% to 52.5% in case of pod.

The majority of putative transformants were having insect mortality from 50.0% to 62.5%

in leaf bioassay, with the highest mortality recorded in M135-4 transgenic line (Fig. 17).

The larval feeding was highest in transgenic line M33-12. The larval feeding pattern on

different tissue types of transgenic and non-transgenic control plants is shown in Plate

27. The statistical analysis (ANOVA) indicated that the transgenic lines differ significantly

(p < 0.001) from each other for larval mortality in all tissue types. The ‘t’-test analysis of

larval mortality in different tissue types indicated non-significant difference between

mortality levels in leaf, flower and pod tissues with p > 0.15.

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Table 33: Summary of transformation carried out using cry1Acm gene in pigeonpea

Sl. No Number of explants

co-cultivated/set

Number of explants

responded

Number of primary transformants (T0)


Number of primary transformants tested in plant-to-row progeny for identifying

putative transformants

Number of putative transformants identified (T1)

1 50 45 20 20 2

2 50 46 27 27 2

3 50 42 20 20 3

4 50 45 29 29 3

5 50 46 24 24 1

Total 250 224 120 120 11

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Table 34: The transgene segregation pattern in eleven transformants carrying cry1Acm in T2 generation revealed by gene specific PCR assay

Plant ID


PCR


PCR


PCR




Gene segregation in

3:1 ration

M2 24 16 08 18.00 6.00 0.89 Yes

M3 24 13 11 18.00 6.00 5.56 No

M5 22 14 08 16.50 5.50 1.52 Yes

M7 23 12 11 17.25 5.75 6.39 No

M10 22 15 07 16.50 5.50 0.55 Yes

M12 21 13 08 15.75 5.25 1.92 Yes

M16 24 15 09 18.00 6.00 2.00 Yes

M33 25 14 11 18.75 6.25 4.81 No

M55 24 14 10 18.00 6.00 3.56 Yes

M133 25 13 12 18.75 6.25 7.05 No

M135 22 13 09 16.50 5.50 2.97 Yes


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Table 35: Per cent corrected cumulative mortality of neonate Helicoverpa armigera larvae observed over 5 days bioassay involving eleven putative transformants carrying cry1Acm gene in T2 generation



M2-14 60.0ab 50.0abc 50.0ab

M3-4 42.5cd 40.0bc 35.0c

M5-1 47.5cd 50.0abc 52.5a

M7-5 45.0cd 42.5bc 40.0bc

M10-3 62.5a 57.5a 52.5a

M12-10 50.0cd 50.0abc 47.5ab

M16-1 60.0ab 52.5ab 50.0ab

M33-12 45.0cd 37.5c 40.0bc

M55-6 52.5bc 50.0abc 47.5ab

M133-10 40.0d 42.5bc 42.5abc

M135-4 62.5a 57.5a 52.5a

SD (±) 0.060 0.061 0.066

CD 0.054 0.066 0.056

CV (%) 5.85 6.40 5.74


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182

183

Analysis of Cry protein in different tissue types indicated significant variation in

Cry1Acm protein levels of different transgenic lines irrespective of tissue types (Plate

28). The Cry protein level in leaf tissue ranged from 0.322 to 0.907 µg/g of fresh leaf

tissue; 0.202 to 0.503 µg/g of fresh flower tissue and 0.186 to 0.405 µg/g of fresh pod

tissue (Table 36). The highest Cry1Acm protein was detected in M135-4 transgenic line,

as indicated by ELISA assay (Fig. 17). On other hand, the lower level of Cry protein was

recorded in transgenic line M133-10. The statistical analysis of variance clearly indicated

the significant difference in Cry1Acm protein levels among developed transgenic lines (p

< 0.001). The ‘t’-test analysis of Cry protein accumulation in different tissue types

indicated non-significant difference for Cry1Acm protein accumulation in flower and pod

tissues (p > 0.08). Whereas, there it was significant between leaf and flower; and leaf

and pod tissues (p < 0.001) as indicated by ‘t’-test analysis. There was a positive

correlation between insect morality and Cry1Acm protein accumulation with r2=0.6542

(Fig. 18). Further, the frequency distribution analysis of transgenic plants following 3:1

transgene segregation pattern and other than 3:1 segregation pattern showed that

majority of high Cry1Acm protein accumulating transgenic plants were following 3:1

transgene segregation pattern (Fig. 18).

4.6.4 The cry1Acm transcript analysis using qRT-PCR assay

The absolute real time analysis of cry1Acm transcripts varied among different

transgenic lines but was constitutive between different tissue types as indicated by

standard graph (Plate 29). The cry1Acm transcript level was ranged from 40.15 to 105.5

ng/µl in fresh leaf tissue; in flower it ranged from 39.80 to 103.0 ng/µl and in pod from

39.83 to 102.9 ng/µl (Table 37a). Interestingly, the highest transcript level was detected

in all tissue types of M135-4 transgenic line. On other hand, the lower level of cry1Acm

transcripts was recorded in transgenic line M3-4. The statistical analysis of variance

showed the significant difference in cry1Acm transcript levels among developed

transgenic lines (p < 0.001).The ‘t’-test analysis revealed non-significant difference in the

transcripts levels of cry1Acm gene in different tissue types within developed transgenic

plants (p ≥ 0.45). Further, positive correlation was noticed between Cry1Acm protein

accumulation and cry1Acm transcript levels with r2=0.4886 (Fig. 15). The correlation

analysis of larval mortality, Cry1Acm protein and cry1Acm transcript showed positive in

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Table 36: The Cry1Acm protein level detected in leaf, flower and pod tissues developed transgenic pigeonpea plants of T2 generation as revealed by ELISA assay

Plant ID Cry1Acm protein level (µg/g FW)


M2-14 0.519e 0.394d 0.323e

M3-4 0.328gh 0.230h 0.204f

M5-1 0.519e 0.363e 0.335d

M7-5 0.359f 0.282f 0.204f

M10-3 0.542de 0.392d 0.322e

M12-10 0.568d 0.426c 0.363c

M16-1 0.734b 0.482b 0.392b

M33-12 0.352fg 0.253g 0.186g

M55-6 0.621c 0.427c 0.384b

M133-10 0.322h 0.202i 0.188g

M135-4 0.907a 0.503a 0.405a

SD (±) 0.023 0.014 0.023

CD 0.011 0.013 0.014

CV (%) 4.48 4.07 2.03


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187

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Table 37a: The cry1Acm transcript level detected in leaf, flower and pod tissues of developed transgenic pigeonpea plants as revealed by real time PCR assay

Event ID cry1Acm transcript level in leaf tissue

(ng/µl)

cry1Acm transcript level in flower tissue (ng/µl)

cry1Acm transcript level in pod tissue

(ng/µl)

M2-14 72.10d 71.60e 70.82e

M3-4 40.15g 39.80i 39.83h

M5-1 75.75c 73.75d 75.00d

M7-5 59.50e 60.00g 60.00f

M10-3 85.40b 87.10b 84.20c

M12-10 86.65b 85.00c 87.00b

M16-1 87.45b 87.00b 85.90bc

M33-12 50.00f 51.75h 51.20g

M55-6 87.65b 86.05bc 86.05bc

M133-10 60.00e 62.30f 60.60f

M135-4 105.5a 103.0a 102.9a

SD (±) 3.599 3.342 2.185

CD 0.923 0.677 0.923

CV (%) 4.88 4.55 2.99


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leaf, flower and pod tissues for these traits of T2 generation transgenic lines (Table 37b).


The genomic DNA from four selected transgenic plants was digested using

BamHI restriction endonuclease and Southern blotting was performed to analyse the T-

DNA integration pattern in to plant genome. The blotted digested genomic DNA was

probed with 452 bp labelled cry1Acm PCR product. A single but weak hybridization

signals were noticed for all samples viz., M2, M10, M16 and M135 transgenic lines. The

Southern hybridization pattern was different for all samples, indicated different position of

T-DNA insertion in plant genome. Further, an observation of single hybridization signal in

all tested transgenic plants indicated the single copy integration of transgene (Plate 30).


The presence of cry1Acm transcript in transgenic pigeonpea plants was also

confirmed with northern blotting using 452 bp cry1Acm PCR product as a probe (Plate

31). The hybridization signals were noticed in all selected transgenic plant samples viz.,

M135, M55, M2, M5 and M7. The observation of strong signals revealed the production

of transcripts in respective transgenic plants.

4.7 The plant growth parameters comparison between parental genotypes and

transgenic lines developed

The different plant growth parameters such as, stem colour, flower colour, growth

habit, leaf colour and shape, branching pattern, fresh pod colour, seed colour, and plant

height were phenotypically observed in transgenic and non-transgenic control plants. It

was interesting to notice the change in flower colour development, slight pinkish yellow,

in few transgenic lines such as Ac63-20, Ac61-4, with BSMR 736 genomic background.

Whereas, it was observed that typical yellow flower colour developed in BSMR 736 non-

transgenic plants (Plate 32). Similarly, it was also noticed that another plant parameter

altered in transgenic plants i.e. fresh pod colour. The typical fresh pod colour developed

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Table 37b: The correlation analysis of insect mortality levels, estimated Cry1Acm protein and cry1Acm transcript in leaf, flower and

pod tissues of T2 generation plants (αααα=0.01).

Insect mortality

in leaf tissues


Insect mortality

in pod tissues

Cry1Acm protein in

leaf tissues

Cry1Acm protein in

flower tissues

Cry1Acm protein in

pod tissues

cry1Acm transcript


cry1Acm transcript


cry1Acm transcript

level in pod tissue


1 0.878** 0.789** 0.809** 0.857** 0.781** 0.781** 0.789** 0.766**


1 0.912** 0.843** 0.871** 0.874** 0.919** 0.921** 0.909**


1 0.762** 0.815** 0.838** 0.874** 0.877** 0.870**

Cry1Acm protein in leaf tissues

1 0.946** 0.914** 0.921** 0.906** 0.910**

Cry1Acm protein in flower tissues

1 0.971** 0.926** 0.910** 0.918**

Cry1Acm protein in pod tissues

1 0.922** 0.901** 0.913**

cry1Acm transcript level in leaf tissue

1 0.997** 0.999**

cry1Acm transcript level in flower tissue

1 0.997**

cry1Acm transcript level in pod tissue

1


191

192

193

in case of ICPL 87119 was green with purple strips, whereas in case of few transgenic

plants (1F-22) with ICPL 87119 as genomic background recorded dark purple colour

development in freshly developing pods. Further the other important plant growth

parameter, wherein the changes were noticed was plant height. Apart from theses, there

were no phenotypically visible changes noticed in other parameters of transgenic and

non-transgenic pigeonpea plants. The per cent recovery of parental phenotypes for

selected traits in transgenic plants carrying different cry genes is presented in Table 38

(Fig. 19). It was noticed that the non-parental phenotypes were observed in transgenic

plants expressing cry1Ac and cry1F gene.

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Table 38: The per cent deviation observed between transgenic plants and parental genotypes for selected morphological traits

Plant growth parameters Transformants

carrying cry1Ac

Transformants carrying cry2Aa

Transformants carrying cry1F

Transformants carrying cry1Acm

Stem colour 0.00 0.00 0.00 0.00

Flower colour 4.55 0.00 7.14 0.00

Growth habit 2.27 0.00 0.00 0.00

Leaf colour and shape 0.00 0.00 0.00 0.00

Branching pattern 2.27 0.00 0.00 0.00

Fresh pod colour 3.41 0.00 7.14 0.00

Seed colour 0.00 0.00 0.00 0.00

Plant height 2.27 0.00 0.00 0.00

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5. DISCUSSION

Pigeonpea belonging to family Fabaceae is cultivated in more than 25 countries of

the world including Indian subcontinent, Africa and Central America. It ranks sixth among

grain legumes in production and grown over 4.7 million hectares of agriculture land in

world wide with the production of around 3.69 million tons annually

(http://faostat.fao.org). India, alone contributes more than 80 per cent of total world

pigeonpea production (Parde et al., 2012). Pigeonpea production and productivity are

constrained by several biotic and abiotic stresses, whose levels of resistance in world

germplasm accessions are low to moderate. Attempts to develop pigeonpea cultivars

resistant to biotic stresses, such as resistance to legume pod borer (Helicoverpa

armigera) and fusarium wilt by conventional breeding methods have shown limited

success due to narrow genetic variability among the germplasm accessions. Breeding

incompatibility problems associated with wild species warrant the exploration of

alternative approaches. Due to high level of resistance to different insecticides in H.

armigera, pigeonpea cultivation also has been difficult. Such problem has been resorted

by Bt transgenic technology adopted in cotton and other crops world wide. Genetic

engineering technology plays a significant role as an additional tool for the introduction of

agronomically useful traits in a high yielding background.

There are very few reports on in vitro regeneration of pigeonpea through

organogenesis from unorganised callus (Kumar et al., 1983). Many independent studies

have reported the multiple shoot production and plantlet regeneration through

organogenesis from different explants viz., cotyledons, embryonic axes, cotyledonary

node from mature seeds and seedling petioles (Franklin et al., 1998; Srinivasan et al.,

2004). The attempts have been made to initiate in vitro culture from different tissue

sources in pigeonpea (Geetha et al., 1998). However, the in vitro regeneration protocols

further need to be fine-tuned and improvised to achieve high frequency regeneration of

plantlets. In this contest, the enhancement in multiple shoot bud induction by use of

cytokinins in nutrient medium expected to be one of the potential approaches.

Cytokinins, as a hormone, are associated with cell division, modification of apical

dominance, shoot differentiation etc and incorporated in tissue culture media for cell

197

division and differentiation of adventitious shoots from callus and organs. The commonly

used cytokinins for plant tissue culture includes benzylamino purine (BAP), isopentenyl-

adenine (2-ip), furfurylamino purine (kinetin), thidiazuron (TDZ) and zeatin. The effect of

cytokinins such as, BAP, kinetin and their varied levels of concentrations on multiple

shoot induction has been studied in pigeonpea (Geetha et al., 1998). In another study,

frequency of multiple shoot bud induction has been titrated using different cytokinins viz.,

BAP, kinetin, TDZ in eleven Indian cultivars of pigeonpea using leaf as explant (Kashyap

et al., 2011).

Independent studies have reported that the type of explant, genotype and

concentrations of cytokinin usually influence the frequency of shoot bud regeneration

(Geetha et al., 1998; Kashyap et al., 2011). The present study aims at improvisation of

multiple shoot induction frequency and plantlet regeneration in pigeonpea using

cotyledonary node with cotyledons and embryo discs with half cotyledon explants. Two

pigeonpea genotypes viz., ICPL 87119 (Asha) and BSMR 736 were tested for their

response to different cytokinins such as, BAP, TDZ and Zeatin, most commonly used

cytokinins for organogenesis. The study identified the best concentration level of

cytokinin in medium for highest number of shoot bud inductions. The medium fortified

with identified concentration levels of respective cytokinins can be used for crop

improvement programmes involving production of transgenic pigeonpea with improved

transformation efficiency by formation of more number of shoots per explant co-

cultivated.

5.1 Improvisation of multiple shoot induction and plantlet regeneration in pigeonpea

In present study, two genotypes viz., ICPL 87119 and BSMR 736 reported their

behavioral response to same BAP level, which was comparable in both genotypes. On

other hand it was interesting to notice that the two explants i.e. cotyledonary node with

cotyledons and embryo discs with half cotyledon reported different behavioral response

to same BAP concentration regime. As far as the number of shoot bud induced per

explants was concern both genotypes showed similar kind of response to same BAP

level. There was a significant difference in number of shoot bud induced from two

explants in response to same BAP fortification level. The analysis of means indicated

198

that the two treatments, 2.0 mg/L and 3.0 mg/L BAP, were on par for number of explants

responded to BAP fortification. On the other hand, the treatment with 2.0 mg/L BAP level

was superior over other treatments for number of shoot bud induced per explant.

Interestingly, there was a concomitant decrease in resonance of explants for shoot bud

induction and number of shoot bud induced with further increase in BAP levels in growth

medium. The similar kind of response by explants to increasing concentrations of

cytokinins has been documented by Geetha et al. (1998) in pigeonpea. The effect of two

cytokinins viz., BAP and Kinetin, and their concentration regimes have been studied for

shoot bud induction from cotyledonary node, epicotyl, hypocotyl, cotyledon and leaf

explants; reported that the 2.0 mg/L BAP level could potentially induced maximum

number of shoot buds from all explants (Geetha et al., 1998).

The results of present study reported parallel response of two pigeonpea

genotypes to different TDZ concentration levels. The effect of different TDZ

concentration regime on two explants response was significant. At the same time, both

genotypes responded well and equally for induction of multiple shoot bud in response to

same TDZ level. Further, there was a distinct response of two explants for multiple shoot

bud induction at same TDZ concentration regimes. The TDZ concentration regime 4.0

mg/L was the most effective for induction of multiple shoot buds per explants from both

explants and genotypes. Recently, Shekhar et al. (2012) have titrated different TDZ

concentration levels (1 to 6 mg/L) for shoot bud induction from mature zygotic embryo

explants of pigeonpea. They identified 4.0 mg/L TDZ as the most effective for multiple

shoot induction from mature zygotic embryo explants in pigeonpea (Shekhar et al.,

2012).

Similarly, the results of present study also revealed that any further increase in

TDZ concentration levels imparts reduction in multiple shoot bud induction in both

explants of pigeonpea. The same kind of explant response to TDZ levels in growth

medium has been reported in cotyledonary node of Cassia sophera and mature zygotic

embryo explants of pigeonpea (Shekhar et al., 2012; Parveen and Shahzad, 2010).

Furthermore, TDZ (1.0 mg/L) in combination to 2,4-D (1 mg/L) have been found effective

for shoot generation from cotyledon and hypocotyl explants of L. campestre as well

199

(Ivarson et al., 2013). Studies have reported that embryonic axis with cotyledons are the

best for producing maximum of 55.02 shoots per explant when cultured on MSB5

medium with 2 mg/L 2-isopentenyladenine (2-iP), 1 mg/L thidiazuron (TDZ) and 0.4 mg/L

kinetin (KIN) in chickpea (Cicer arietinum L.) (Pawar et al., 2012). Although, in present

study, the best TDZ concentration observed for more number of shoot induction seems

to be high (4.0 mg/L), in previous reports same TDZ concentration level has been

reported for more number of shoot induction from mature zygotic embryo in pigeonpea

(Shekhar et al., 2012). Further, any artefacts such as deformed shoots, when explants

were cultured on MS augmented with different TDZ concentration regimes, were not

observed in the present study.

The behavioral response of both genotypes and explants to zeatin fortification

was same as that of BAP and TDZ. Results indicated that ICPL 87119 and BSMR 736

response to zeatin augmentation was on par with each other. Whereas, two explants

(cotyledonary node with cotyledons and embryo discs with half cotyledon) responded

distinctly for the number of multiple shoot buds induced at same level of zeatin

concentration regimes. Further, results indicated absence of merit difference for number

of induced multiple shoot buds in two genotypes experiencing same zeatin concentration

regime: whereas, the two explants recorded merit difference for shoot bud induction at

same zeatin concentration regime in both genotypes. The mean analysis reported that

the two treatments, 1.0 mg/L and 2.0 mg/L zeatin, were on par and superior over other

treatments for number of shoot bud induction per explant. As that of BAP and TDZ, the

similar kind of response curve was also noticed for shoot bud induction at different zeatin

levels. The effect of change of zeatin concentration on shoot bud induction was similar to

that of effect of change in BAP or TDZ concentration levels. The observation of

development of white sugary callus might be the reason for reduction in multiple shoots,

when medium is supplemented with higher concentrations of cytokinins. Similar zeatin

levels (2.0 mg/L) in growth medium have been documented as most effective for shoot

regeneration from cotyledonary node in pigeonpea (Srivastava et al., 2013). In another

study, the zeatin (2.0 mg/L) in combination to 2,4-D (1.0 mg/L) has been reported for

shoot generation from cotyledon and hypocotyl explants of L. campestre (Ivarson et al.,

2013).

200

Present study reported induction of more number of shoot buds in cotyledonary

node with cotyledons explants when compared with that of embryo discs with half

cotyledon explants. The cotyledonary node with cotyledons explants has immense

potential for multiple shoot bud induction. It is interesting to notice such difference, and

might be it was due to removal of half cotyledon retaining embryo discs with other half

part of it. Such detachment of half cotyledon might have resulted in reduction in

cotyledonary node compactness, which might be essential for more number of shoot bud

induction. Although there are no previous reports indicating this phenomenon, based on

present experimental results it seems obvious. Many individual studies have reported the

response of different explants to multiple shoot bud induction in response to cytokinin

fortification (Geetha et al., 1998; Kashyap et al., 2011). As per previous literature or

information until this time the first report indicating such huge number of multiple shoot

bud induction from cotyledonary node with cotyledons explants in pigeonpea has been

emerged through present investigation.

Furthermore, results of present study clearly indicated that the MS with 0.5 mg/L

IBA was most suitable for healthy root induction with comparatively more number of

roots. Further increase in IBA levels in rooting medium did not affect the root induction

significantly. Many studies have documented the effectiveness of lower concentrations of

IBA on root induction in pigeonpea (Geetha et al., 1998; Guruprasad et al., 2011).

Pigeonpea suffers from many biotic and abiotic stresses, and the level of

resistance in world germplasm accessions is low to moderate. Hence, there is a great

need and opportunity to use tissue culture based methods to improve pigeonpea crop

resistance to different stresses. The results of present study reported the efficient

multiple shoot bud induction and plantlet regeneration method for pigeonpea. As

pigeonpea genotypes used in present study, ICPL 87119 and BSMR 736, are moderate

to resistant to fusarium wilt and sterility mosaic disease, in vitro plantlet regeneration

methods in such genotypes can be exploded for genetic engineering programs to

develop multiple stress tolerance in pigeonpea.

201

5.2 Improvisation of in planta transformation protocol in pigeonpea

The development of transgenics in pigeonpea has remains dogged due to poor in

vitro regeneration and low transformation frequency. Although numerous efforts have

been made for the development of efficient protocols of Agrobacterium tumefaciens and

microprojectile bombardment-based genetic transformation of pigeonpea, the in vitro

regeneration conditions so far described are available for only a few of the many

cultivars/genotypes of pigeonpea (Rao et al., 2008). It has also been reported that the

morphogenetic response of pigeonpea crop is known to be a genotype-specific

phenomenon (Mohan and Krishnamurthy, 1998).

An in planta transformation method have been successfully applied in wide range

of crop plants such as, mulberry, soybean, rice and cotton (Ping et al., 2003; Supartana

et al., 2005; Keshamma et al., 2008). This method of transformation also has been

documented in other crop plants like, pigeonpea, fieldbean, sunflower, groundnut and

safflower (Rao and Rohini, 1999a; 1999b; Rohini and Rao, 2000a; 2000b; 2001). In

resent efforts in planta transformation method has been successful in maize, rice and

wheat transformation (Mehrotra and Goyal, 2012). Further, very recently in planta

method has also been used for development of transgenic pigeonpea for expression of a

chimeric cry1AcF gene encoding Cry1Ac and Cry1F domains (Ramu et al., 2012). In

case of alfalfa, the marker free transgenic plants have been developed employing in

planta transformation method (Weeks et al., 2008).

In present study, for improvisation of in planta transformation protocol in

pigeonpea different treatment such as, A. tumefaciens infection alone, A. tumefaciens

infection using A. tumefaciens culture with tobacco extract added in overnight grown

culture and A. tumefaciens infection using air evacuation to increase the proximity

between A. tumefaciens and embryonic axis were employed. Results of present

investigation indicated merit differences between explants response to different in planta

transformation treatments with p < 0.03. The two treatments viz., A. tumefaciens

infection alone and A. tumefaciens infection using A. tumefaciens culture with tobacco

extract were comparable with each other with respect to explants response to

treatments. Whereas, there was a significant reduction in explants response to A.

tumefaciens infection using air evacuation treatment with 18.5 per cent.

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Similarly, the number of primary transformants developed during test treatments

was significant at p < 0.001. The two treatments viz., A. tumefaciens culture with tobacco

extract and A. tumefaciens infection using air evacuation were on par and superior for

development of more number of primary transformants when compared with treatment

involving A. tumefaciens infection alone. Further, study reported that the in planta

transformation treatment, A. tumefaciens infection using air evacuation, was most

effective in developing more number of transformants in pigeonpea with 12.0 per cent

transformation efficiency. Other two treatments were found to be statistically on par with

each in case of transformation efficiency.

Clough and Bent (1998) have reported two folds increase in transformation rate

when flower tissues were air evacuated with A. tumefaciens culture in place of flower dip

method in Arabidopsis thaliana. Dehestani et al. (2010) employed in planta

transformation method in Arabidopsis and showed that plants infected with A.

tumefaciens strain GV3850 only reported highest transformation frequency of 1.54 per

cent. The use of vacuum infiltration during A. tumefaciens infection has improved

transformation efficiency to 3.0 per cent through their study. Further, Habashi et al.

(2012) has studied the effect of vacuuming during Agroinoculation in two pear (Pyrus

communis L.) cultivars, Bartlett and Harrow Delight and showed significant increase in

transformation efficiency (10.63 per cent) when compared with the common

Agroinoculation method (4.06 per cent).

According to Rao et al. (2008) the use of in planta transformation method has

provided many advantageous like, ensured generation of pigeonpea transgenic plants

with considerable ease in a short time, applicable across different genotypes/cultivars of

the crop and offers immense potential as a supplemental or an alternative protocol for

generating transgenic plants of difficult-to-regenerate pigeonpea. Present study reported

identification of effective method involving air evacuation during A. tumefaciens infection

for pigeonpea transformation. Such high transformation efficiency might be due to the

removal of air from injured parts of actively developing embryo, which further might have

allowed the A. tumefaciens to reach and infect damaged tissues parts more efficiently.

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As the targeted explants involved developing embryo, the injured tissues may be healed

faster and grow normal due to actively dividing and growing cells after A. tumefaciens

infection and co-cultivation period. As the air evacuation during A. tumefaciens infection

improved in planta transformation efficiency to greater extent, can be employed for

genetic engineering programmes from crop improvement in pigeonpea.

5.3 Generation of transgenic pigeonpea carrying different cry genes for pod borer

resistance

The pigeonpea variety ICPL87119 have been derived from a cross between C11

and a breeding line, and is medium duration variety with resistance to Fusarium wilt and

pigeonpea sterility mosaic virus (Dharmaraj and Lohithaswa, 2004). The extra short-

duration varieties and the medium-duration genotypes have been found to be severely

damaged (90 per cent) by pod borer (Choudhary et al., 2013). Many independent studies

have reported that pigeonpea pod walls with more wax, total phenols, less reducing and

non-reducing sugars, total amino acids and high amount of soluble protein content have

been associated with plants resistance reaction in pigeonpea (Sharma et al., 2009;

MacFoy et al., 1983; Moudgal et al., 2008). The back cross progenies have been

developed and reported a range of resistance traits in the progeny lines for pod borer,

bruchid and pod fly resistance ranged from 6.85 to 22.84 per cent (Mallikarjuna et al.,

2011).

Substantial progress has been made towards development of large-scale

genomic resources in pigeonpea especially during the last decade, these efforts have

resulted in the development of large-scale molecular markers, construction of

comprehensive genetic maps, identification of various marker-trait associations and

initiation of molecular breeding in this crop (Raju et al., 2010; Mallikarjuna et al., 2011;

Dubey et al., 2011). Although inter as well as intra specific genetic maps in pigeonpea

have been developed, preliminary mapping efforts have resulted in mapping of Fusarium

wilt resistance and SMD only (Kotresh et al., 2006; Ganapathy et al., 2010). The

presence of genetic variability for resistance to insects particularly pod borer is not

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available in the primary gene pool of pigeonpea (Choudhary et al., 2013). Although the

advance back cross QTL based breeding is most suitable for introducing novel alleles

from wild relatives, the transfer of desirable alleles from wild relatives is not so simple

because of difficulty in efficient tracking for desired and non-desired alleles in breeding

lines (Tanksley and Nelson, 1996).

Hence, many plant transformation studies using genetic engineering have been

undertaken for development of transgenic in pigeonpea expressing different alien genes

such as, cowpea protease inhibitor gene (pCPI) (Lawrence and Koundal, 2001),

synthetic cryIE-C (Surekha et al., 2005), synthetic cry1Ab (Sharma et al., 2006), etc.

Recently, in planta transformation method has been successfully used to express

chimeric cry1AcF in pigeonpea (Ramu et al., 2012). It has been reported that Cry1Ac

belongs to 3D-Cry group and binds to V-ATPAse subunit A and actin of brush border in

case of M. sexta and H. virescens. Further the mode of action of Cry1Ac toxins involve

binding of the toxin with other components of the midgut cells (McNall and Adang, 2003;

Krishnamoorthy et al., 2007). Cry2Aa belongs to an unusual subset of crystalline

proteins and possessed broad insect species specificity against Lepidoptera and Diptera

(Liang and Dean, 1994). The Cry2Aa protoxins are significantly smaller (72 kDa) and are

processed primarily at the N-terminal end. Such activated toxin follows complex

sequential binding lines with different insect gut Cry-binding proteins and results in

membrane insertion and pore formation (Bravo et al., 2011).

The Cry1F protein mechanism of action involves the enzymatic cleavage of the

protoxin to form core toxin (Gao et al., 2006). Many studies have reported the activity of

Cry1F against lepidopteran species (Balog et al., 2011; Oppert et al., 2010). The Cry1F

toxin susceptible species also includes tobacco budworm, beet armyworm, soybean

looper, cotton bollworm, fall armyworm, lesser cornstalk borer, wax moth and European

corn borer (Adamczyk et al., 2008; Blanco et al., 2010; Buntin, 2008; Tindall et al., 2009).

The native cry1Ac gene sequence from B. thuringiensis have been in silico modified and

artificially synthesized to overcome codon bias and other undesirable regulatory coding

sequences for its improved expression in transgenic plants (Mohan, 2008). The study

resulted in the development of modified cry1Acm with increased GC content and

modifications in specific undesirable eukaryotic regulatory sequences (Mohan, 2008).

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In present study, eighty eight transgenic plants were developed carrying cry1Ac

gene using improvised in planta transformation protocol. The transgene segregation

analysis in T2 generation plants revealed 3:1 (positive : negative) cry1Ac gene

segregation pattern in forty eight transgenic lines. According to Mendel’s law of

inheritance, the characters govern by single gene segregate in 3:1 (dominant: recessive)

ratio in F2 generation (Bowler, 1989). The observation of 3:1 (positive : negative) ratio for

cry1Ac gene in T2 generation plants indicated the possibility of singly copy transgene

integration in pigeonpea genome. Although, in case of A. tumefaciens mediated

transformation the possibility of multiple copy insertion in very less to rare, the

observation of absence of 3:1 transgene segregation pattern in large number of

transgenic lines i.e. forty, was interesting. One of the possible reasons for observation of

such behaviour might be due to the presence of more than single copy transgene

insertion. Another possibility could be the number of plants tested to study transgene

segregation pattern might be less, as the 3:1 monogenic segregation ratio is population

phenomenon (Bowler, 1989). The in vitro transformation efforts resulted in generation of

ten transgenic plants. The transformation frequency observed in case of in vitro

transformation method was very less i.e. 0.36 per cent, whereas it was 6.29 per cent in

case of in planta plant transformation method for cry1Ac gene.

In case of cry2Aa, sixty five putative transformants were developed using

improvised in planta transformation protocol. The assessment of transgene segregation

pattern identified sixteen transgenic plants following 3:1 (positive : negative) cry2Aa gene

segregation pattern in T2 generation progenies. Such observation of 3:1 (positive :

negative) ratio for transgene segregation clearly indicated the possibility of singly copy

transgene integration in pigeonpea genome in those sixteen lines. Similar to that of

cry1Ac gene carrying transgenic plants, in case of cry2Aa transformants, the observation

of absence of 3:1 transgene segregation pattern in many transgenic lines i.e. forty nine,

was interesting. And it was probably either due to the presence of more than single copy

transgene insertion or the less number of plants tested to study transgene segregation

pattern. In order to achieve multiple generation per year, the transgenic plants were not

allowed to reach their full harvestable/ maturity stage and only initial pod yield was

harvested. Hence, due to availability of limited seed material, 30-40 seeds per transgenic

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line were sown and obtained next generation plants were used to study transgene

segregation analysis.

Further, fourteen transgenic plants were developed carrying cry1F gene using

improvised in planta transformation protocol. The transgene segregation analysis in T2

generation plants revealed 3:1 (positive : negative) cry1F gene segregation pattern in

seven transgenic lines. The observation of 3:1 (positive : negative) for cry1F gene in T2

generation plants indicated the possibility of singly copy transgene integration in

pigeonpea genome. The possible reasons for observation of absence of 3:1 transgene

segregation pattern in remaining seven transgenic lines was might be due to the

presence of more than single copy transgene insertion or could be due to the number of

plants tested to study transgene segregation pattern might be less.

In case of cry1Acm, eleven putative transformants were developed. The

assessment of transgene segregation pattern identified, among eleven, seven transgenic

plants were following 3:1 (positive : negative) cry1Acm gene segregation pattern in T2

generation progenies. Such observation of 3:1 (positive : negative) for transgene

segregation revealed the possibility of singly copy transgene integration in pigeonpea

genome. Similar to that of other cry gene carrying transgenic plants, in cry1Acm

transformants the observation of absence of 3:1 transgene segregation pattern in four

transgenic lines might due to the presence of more than single copy transgene insertion

or the less number of plants tested to study transgene segregation pattern.

Bhat and Srinivasan (2002) discussed genetic analyses of transgenic plants and

indicated that handling of primary transgenics to obtain progeny generation needs few

careful considerations, wherein the breeding behaviour of the plant species is the

deciding factor. It is important to take into account the performance of parent and

progeny. To obtain homozygosity researchers resort to selfing of the T0 plants. It results

in T1 progenies with the complex mixture of genotypes and their composition depends on

the genetic constitution of the T0 parents. In case of multiple copy integration of

transgene, determination of exact genotypic constitution of individual plants may be

difficult due to difficulties in distinguishing hemizygotes from homozygotes by Southern

analysis (Bhat and Srinivasan, 2002).

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5.4 Molecular characterization of pigeonpea transgenic lines expressing different cry

genes

Different transgene characterization methods viz., insect bioassay, protein

quantitation using ELISA, mRNA quantitation using qRT-PCR assay, Sothern, western

and northern blot, and TAIL-PCR assay for juncture region analysis have been employed

in many studies (Sharma et al., 2006; Ramu et al., 2012). Sharma et al. (2006) have

developed transgenic plants expressing synthetic cry1Ab in pigeonpea, which showed

varied range of expression of gene in different tissues of the whole plant, highest

expression in flowers (0.1 per cent of total soluble protein) and least in the leaves (0.025

per cent of total soluble protein). Gopalaswamy et al. (2007) evaluated the usefulness of

detached leaf assay to test the bioefficacy of transgenic pigeonpea and determined

levels of Cry1Ab or SBTI (soybean trypsin inbhibitor) proteins in the transgenic

pigeonpea plants as not sufficient to cause significant deterrent effects on leaf feeding,

larval survival and larval weight of H. armigera and found to be quite useful for evaluation

of transgenic pigeonpea plants for resistance to H. armigera (Gopalaswamy et al., 2007).

Recently, transgenic pigeonpea plants carrying chimeric cry1AcF have been developed

and showed that chimeric Cry1AcF levels in developed transgenic plants ranged 3-15

µg/g of fresh tissue weight with the insect mortality ranged from 0 to 100 per cent

indicating the potential effect of Cry1AcF against H. armigera (Ramu et al., 2012).

Bioassays can be used to test the functionality of the transgene product. However,

in primary transgenics, somaclonal and transgene effects are expected to be confounded

and hence bioassay analysis of transgenic plant progenies is recommended (Bhat and

Srinivasan, 2002). Considering the same, in present study, transgenic plants developed

were analysed through bioassay in T2 and T3 generations. The bioefficacy of developed

transgenic plants expressing cry1Ac in terms of larval mortality in three tissue viz., leaf,

flower and pod reported distinct response of transgenic plants toward insect resistance.

The larval mortality in case of leaf tissues ranged from 25.00 to 70.00 per cent, in case of

flower from 22.50 to 52.50 per cent and in case of pod from 17.50 to 52.50 per cent over

non-transgenic control. The expression of Cry1Ac protein resulted in reduction in leaf

tissues damage over non-transgenic plants. There was a concomitant decrease in leaf

damage with increase in larval mortality.

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Amoungst transgenic plants expressing cry2Aa gene, the larval mortality in case

of leaf tissues ranged from 5.25 to 68.33 per cent, between 5.25 to 48.5 per cent in case

of flower tissues and 10.25 to 53.25 per cent in case of pod tissue. When compared with

cry1Ac gene carrying transgenic plants, cry2Aa transformants showed wide range of

insect mortality starting from very low, 5.25 per cent, to almost comparable with the

highest mortality level as that of cry1Ac transgenic lines. In case of flower tissues cry1Ac

transgenic plants were more effective when compared with cry2Aa transformants.

Whereas, both the transgenic plants carrying cry1Ac and cry2Aa, separately, were

equally effective in leaf and pod bioassay. Similar to that of cry1Ac transgenic plants,

there was considerable reduction in leaf tissue damage in case of cry2Aa transgenic

plants as well. The expression of Cry2Aa protein reported significant reduction in leaf

tissues damage over non-transgenic plants.

Further, the transgenic plants carrying cry1F gene reported varied response in

case of larval mortality in tissue types. The larval mortality in case of leaf tissues ranged

from 22.5 to 62.5 per cent, in case of flower from 20.0 to 55.0 per cent and in case of

pod from 10.0 to 47.5 per cent over non-transgenic control. There was considerable

reduction in leaf tissue damage in case of transgenic plants. The larval mortality levels of

cry1F transgenic lines were comparable with both cry1Ac and cry2Aa transgenic lines in

case of flower bioassay. Whereas, the bioefficacy levels in leaf and pod tissues were

relatively low in case of cry1F transgenic plants when compared with other two cry gene

carrying transformants. There was a concomitant decrease in leaf damage with increase

in larval mortality probably due to reduced larval feeding on transgenic plant tissues

expressing cry1F gene.

The transgenic plants carrying cry1Acm gene also showed varied response in

case of larval mortality and leaf tissue damage in tissue types. The larval mortality in

case of leaf tissues ranged from 40.0 to 62.5 per cent, in case of flower from 37.5 to 57.5

per cent and in case of pod from 35.0 to 52.5 per cent over non-transgenic control. The

results indicated that there was considerable reduction in leaf tissue damage in case of

transgenic plants in response to Cry1Acm protein accumulation. When compared with

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other transgenic plants carrying different cry genes. The larval mortality levels of

cry1Acm transgenic lines were comparable with other transgenic lines. There was a

concomitant decrease in leaf damage with increase in larval mortality.

Surekha et al. (2005) has performed detached leaf feeding tests on transgenic

pigeonpea plants expressing cry1Ac for the pest S. litura. Study has reported significant

variation between transgenic samples and the control wild-type plants with highest

mortality of 80 per cent (Surekha et al., 2005). The effect of transgenic pigeonpea

carrying cry1Ab and SBTI genes on the growth and development of H. armigera have

been reported using detached leaf bioassay (Gopalaswamy et al., 2007). Ramu et al.

(2012) has indicated that the both the damage and the mortality in insects varied from 0

to 100 per cent among the putative transformants carrying cry1AcF. The effect of the

cry1AcF gene has been seen on the larva as there was a considerable difference in the

size of the larva that fed on the transgenics and wild type (Ramu et al., 2012). Selected

transgenic chickpea lines viz., BS2A, BS5A and BS6H have been tested in insect

bioassays using neonate H. armigera and compared to the non-transgenic cv ICCV

89314 and cv Semsen (Acharjee et al., 2010). Study reported significantly greater larval

mortality among the larvae consumed transgenic leaves, with highest mortality on leaves

of BS6H, up to100 per cent (Acharjee et al., 2010). Further the toxicity of T0 and T1

plants expressing Cry1Ac protein have been tested using either isolated or whole plants

in feeding assays with second or third instar neonate larvae of H. armigera (Sanyal et al.,

2005). There was inscrease in feeding of H. armigera larvae within two days of exposure

to transgenic chickpea expressing high levels of Bt toxins. The effective larval weight

reduction was 40.0 to 90.0 per cent with high mortality (Sanyal et al., 2005).

The ELISA has shown a significant advantage for protein analysis in transgenic

plants. A sandwich ELISA is the preferable immunoassay used for the detection of Bt

protein, where an analyte is sandwiched in between the two antibodies; a capture

antibody and the detector antibody (Kamle and Ali, 2013). ELISA has been successfully

used for the detection of protein encoded by cp4-epsps gene in a RR soybean (Rogan,

1999). In present study, the analysis of Cry1Ac protein in different tissues of developed

transgenic plants reported significant variation in Cry1Ac protein levels in leaf, flower and

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pod tissues of different transgenic lines. The ELISA results indicated that the majority of

putative transformants were having 0.4 to 0.6 µg/g of Cry1Ac protein in different tissue

types. There was a positive correlation between the Cry1Ac protein level and insect

mortality. With increase in Cry protein level, there was a concomitant increase in insect

mortality in transgenic plants. It was noticed that transgenic plants expressing higher

level of Cry1Ac protein showed significant reduction in tissue damage as well. The

similar kind of correlations has been reported in transgenic pigeonpea plants expressing

cry1AcF gene (Ramu et al., 2012).

There was a significant variation in Cry2Aa protein levels of different tissue types

as noticed in case of transgenic plants expressing cry2Aa gene. The majority of cry2Aa

transformants were having 0.0 to 0.5 µg/g of Cry2Aa protein in different tissue types.

There was a positive correlation between the Cry2Aa protein level and insect mortality

and with increase in Cry2Aa protein level, there was a concomitant increase in insect

mortality in transgenic plants. In few transgenic lines such as, 21A12-24, although there

was a very high accumulation of Cry2Aa protein, up to 3.231 µg/g, the insect mortality

levels were as comparable with transgenic plants expressing cry1Ac gene (0.847 µg/g of

Cry1Ac). This observation gives a probable indication of more effective activity of Cry1Ac

protein when compared to that of Cry2Aa against Helicoverpa armigera attack in

pigeonpea.

The analysis of Cry1F protein in different tissues of developed transgenic plants

reported significant variation in Cry1F protein levels in leaf, flower and pod tissues of

different transgenic lines. The ELISA results indicated that the majority of putative

transformants were having 0.4 to 0.6 µg/g of Cry1F protein in leaf, 0.3 to 0.6 µg/g of

Cry1F protein in both flower and pod tissue. With increase in Cry protein level, there was

a concomitant increase in insect mortality in transgenic plants. On other hand, transgenic

plants expressing higher level of Cry1F protein showed significant reduction in tissue

damage. The cry1F protein accumulation in developed transgenic plants were

comparable and slightly above (1.032 µg/g in leaf tissue; 0.870 µg/g in flower and 0.782

µg/g in pod) to that of cry1Ac protein accumulation (0.847 µg/g in leaf tissue; 0.671 µg/g

in flower and 0.685 µg/g in pod) in cry1Ac transgenic plants.

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Further, Cry1Acm protein analysis in different tissues of developed transgenic

plants indicated accumulation of Cry1Acm protein, 0.3 to 0.9 µg/g of Cry1F protein in

leaf, 0.2 to 0.5 µg/g of Cry1F protein in both flower and pod tissue. With increase in

Cry1Acm protein level, there was a concomitant increase in insect mortality in transgenic

plants. On other hand, transgenic plants expressing higher level of Cry1Acm protein

showed remarkable reduction in tissue damage when compared with non-transgenic

control plant tissue. The cry1Acm protein accumulation in developed transgenic plants

were comparable the cry1Ac protein accumulation in cry1Ac transgenic plants. Based on

comparative analysis of different transgenic events expressing cry1Ac, cry2Aa, cry1F

and cry1Acm genes independently, it was indicative that the transgenic events

expressing differential levels of cry genes were varying for their insecticidal activity

against H. armigera.

In previous studies, Sanyal et al. (2005) has estimated the Cry1Ac protein levels

in developed transgenic chickpea plants, which ranged from 14.5 to 23.5 ng/mg

extractable protein with high levels of toxicity against pod borer. Similarly, Mehrotra et al.

(2011) has reported that the Cry protein accumulation in cry1Ab and cry1Ac insecticidal

genes pyramided transgenic plants ranged between 15-20 ng/mg and showed high-level

of resistance against pod borer larvae of H. armigera.

RT-PCR and northern hybridization techniques are employed to assess the

expression of the introduced gene (Bhat and Srinivasan, 2002). Studies have reported

the use of real time PCR for quantification of targeted cDNA molecules (Kamle and Ali,

2013). The real time PCR has also been undertaken for validating and estimating the

number of copies of inserted genes into the host genome (Zhang et al., 2003). The

method has been reported for several genetically modified crops such as maize,

cassava, rapeseed, wheat, cotton and brinjal (Aguilera et al., 2008; Ballari et al., 2013;

Beltrán et al., 2009; Lee et al., 2006; Li et al., 2004; Wu et al., 2007). Beltrán et al. (2009)

developed real-time polymerase chain reaction-based methods for the primary scrutiny

of putative transgenic plants. They tested for the presence of transgenes, estimated copy

number and quantified messenger RNA (mRNA) levels of genes introduced through A.

tumefaciens.

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In present study, real-time PCR assay was employed to quantify the cry gene

transcript levels in transgenic plants. The real time transcript analysis of cry1Ac transcript

level reported that transgene transcripts ranged from 24.6 to 165.1 ng/µl in leaf, 15.6 to

149.5 ng/µl in flower and from 18.3 to 152.4 ng/µl in pod tissues of transgenic plants.

Interestingly, Ac20-2 transgenic lines indicated the highest cry1Ac transcript level in all

tissue types. Similarly, the real time transcript analysis of cry2Aa reported that transgene

transcripts ranged from 48.5 to 134.5 ng/µl in leaf, 41.2 to 110.2 ng/µl in flower and from

42.8 to 100.5 ng/µl in pod tissues of transgenic plants. In terms of transcription of cry

genes, cry1Ac transgenic plants were more effective than that of transformants

expressing cry2Aa gene. Considering the lower levels of Cry1Ac protein levels, although

the transcript levels of cry1Ac were high when compared with cry2Aa transgenic plants,

the possible reason for such behaviour may be the less stability of cry1Ac transcript in

plant cell. On other hand, cry2Aa transcripts might be more stable leading to more

accumulation of Cry2Aa protein in transgenic plant cell. It has been reported that the

high levels of expression of cry1Ac gene cannot be routinely achieved and this has been

attributed to the instability of the transcript (Rawat et al., 2011).

The real time transcript analysis of cry1F transcript level reported that transgene

transcripts ranged from 47.6 to 105.3 ng/µl in leaf, 45.2 to 101.0 ng/µl in flower and from

48.5 to 103.2 ng/µl in pod tissue of transgenic plants. Interestingly, 1F-25 and 1F-9

transgenic lines indicated the highest cry1F transcript level in all tissue types. On other

hand, the observed insect mortality and Cry1F protein was significantly less in 1F-25 and

1F-9 transgenic lines, contradicting the positive correlation of it with cry gene transcript

level. The transcript levels of cry1F gene in developed transgenic plants were

comparatively lower than that of cry1Ac and cry2Aa gene transcripts in their respective

transgenic plants. Further, in case of cry1Acm transgenic plants, M135-4 transgenic line

showed highest cry1Acm transcript accumulation in all tissue types. The transcript levels

of cry1Acm gene in developed transgenic plants were comparatively lower among all

other cry gene carrying transgenic plants. The northern blot analysis further confirmed

the presence of cry transcripts in selected transgenic pigeonpea plants and validated the

results obtained from qRT-PCR based transcript analysis.

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Southern analysis is an important component of transgene analysis to prove the

integration of the foreign gene into host genome (Bhat and Srinivasan, 2002). Southern

hybridization helps to assess the number of independent transgene insertions, which is

important to find transformants with single, unaltered transgene insertions and

considered ideal for analysis (Bhat and Srinivasan, 2002). Southern analysis of selected

three transgenic lines indicated presence of single cry1Ac carrying T-DNA copy

insertions in pigeonpea genome. The hybridization pattern was similar for two lines

Ac20-2 and Ac20-3, whereas Ac29-1 produced distinct band with higher molecular

weight. It was interesting to notice the same position of T-DNA insertion in pigeonpea

genome for transgenic line Ac20-2 and Ac20-3 as revealed through juncture region

analysis. The recovered genomic flanking region from these two lines identified

sequence homology with pigeonpea genome sequence Scaffold130851. In case of

transgenic line Ac29-1, the position of T-DNA integration was in the genomic region

represented in Scaffold137204. Although, the pigeonpea draft genome is available, as it

is not processed completely, we could not retrieve more information about identified

genomic region for its gene content and other genomic features. The identification of

same position of T-DNA insertion and similar Southern hybridization pattern gave the

possibility of Ac20-2 and Ac20-3 as same transgenic line.

For cry2Aa transgenic plants, southern analysis of selected four transgenic lines

indicated integration of single cry2Aa carrying T-DNA in pigeonpea genome. The

hybridization pattern was similar for two lines 21A6-9 and 21A6-68, whereas 21A2-2 and

21A3-4 produced distinct banding pattern with higher molecular weight. It was interesting

notice the same position of T-DNA insertion in pigeonpea genome in case of transgenic

line 21A6-9 and 21A6-68 as revealed through juncture region analysis. The recovered

genomic flanking region from 21A6-9 and 21A6-68 transgenic lines identified sequence

homology with Scaffold127179. Further, in case of transgenic line 21A2-2 the position of

T-DNA integration was in the genomic region represented in Scaffold137204. In the

same Scaffold, the recovered flanking genomic region from cry1Ac transgenic line Ac29-

1 also reported sequence homology.

Similarly, in case of transgenic line 21A3-4 the position of T-DNA integration was

in the genomic region represented in Scaffold134438. The identification of same position

of T-DNA insertion and similar Southern hybridization pattern observed in case of 21A6-

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9 and 21A6-68 given the possibility of these two transgenic lines as same transgenic

lines. The junction region analysis of 21A3-4 and 21A12-24 transgenic line reported

recovery of genomic region homologous to Scaffold134438. The Southern analysis of

21A12-24 expected to give interesting insights regarding the two transgenic lines 21A3-4

and 21A12-24 expressing cry2Aa gene.

The two transgenic lines carrying cry1Ac gene (Ac20-2 and Ac20-3) reported

same position of transgene insertion in pigeonpea genome. As these lines were

generated from same primary transformant i.e. Ac20 (T0 generation) might be the reason

for such observation. Similar kind of result was also recorded in case of two transgenic

lines carrying cry2Aa gene i.e. 21A6-9 and 21A6-68, having generated from same

primary transformant i.e. 21A6. Contradictory to above notion, although identified from

the different primary transformats viz., 21A3 and 21A12, the observation of same

position of insertion in case of 21A3-4 and 21A12-24 transgenic lines was interesting.

Southern analysis of 1F-25 and 1F-9 indicated presence of two T-DNA copy

insertions in pigeonpea genome, the probable reason for the reduced Cry protein and

insect mortality. The presence of two transgene copy might have resulted in post

transcriptional or post-translational silencing of the transgene expression. There are

many factors such as, DNA methylation, trans-inactivation, co-suppression may reduce

the gene expression (Li et al., 2002). The similar type transgene silencing phenomenon

has been reported in transgenic tobacco expressing uidA gene (Li et al., 2002). Study

reported the multiple copy insertion could result in the reduced to no GUS activity in

transgenic tobacco plants. The other transgenic plants, 1F-22, 1F-32, 1F-38 and 1F-33,

tested for Southern analysis indicated single copy T-DNA insertion in pigeonpea genome

and also found superior in insect mortality, cry1F gene expression in terms of both

protein and transcript level.

The genomic Southern blotting in case of cry1Acm gene carrying transformants

confirmed the integration of T-DNA in to plant genome. A weak hybridization signals

were recorded for all four samples viz., M2, M10, M16 and M135. The observation of

different Southern hybridization banding pattern clearly indicated different position of T-

DNA insertion in plant genome. Furthermore, an observation of single hybridization

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signal in all tested transgenic plants unambiguously reported the single copy integration

of transgene.

Surekha et al. (2005) have used Southern blotting analysis for confirmation of 18

pigeonpea transgenic samples. Based on Southern blotting they studied the presence of

transgene and number of the inserts, size of the inserts in different plants confirming the

independent origin of the transgenic lines (Surekha et al., 2005). The PCR amplicon of

cry1Ab fragment have been used as probe for the Southern hybridization analysis and

hybridization signals detected in eight of the 12 plants analyzed for the integration of Bt

gene in the genome of these plants. The blotting analysis revealed that four of six

transgenic plants possessed single-copy integrations (Sharma et al., 2006).

Rao et al. (2008) performed Southern blot and confirmed the integration of the

transgene in the pigeonpea genome. Further, in similar kind of study, Ramu et al. (2012)

studied the transgene integration pattern using genomic Southern analysis, where strong

signal and the difference in the hybridization pattern in the selected transgenic plants

revealed the single copy integration in these plants. The Southern analysis of the

genomic DNA from T1 progenies of selected transgenic chickpea plants have shown

single DNA fragments of size ranging from 4.16 to 6.57 kb hybridizing with 1.845 kb

radiolabelled cry1Ab/ c probe (Mehrotra et al., 2011). Acharjee et al. (2010) have

Southern blotted the genomic DNA from pooled progeny of nine independent transgenic

chickpea lines and showed the integration of cry2Aa gene in eight of the nine lines, with

integration of one, two or more copies of the transgene.

Further, in present study, the observation of transgenic plants for selected plant

growth parameters indicated the appearance of non-parental phenotypes for five traits

viz., flower colour, growth habit, branching pattern, fresh pod colour and plant height.

The change in these traits was noticed in transgenic plants carrying cry1Ac and cry1F

genes. The appearance of such non-parental types was either may be due to integration

of cry gene or the somatic changes that might have happed during the transformation

and regeneration process. Rawat et al. (2011) have reported the development of

abnormal phenotypes such as extreme retardation in the growth of the plant, reduced

flowering, abscission of the flowers following crossing and no setting of bolls in

transgenic cotton plant.

216

As in case of developing countries, conventional methods of pest control have

been least effective and the greatest improvement in yields due to use of genetically

modified insect resistance technology has occurred there. It has been reported that at

the aggregate level, the global farm income gain due to use of genetically modified insect

resistance technology in maize and cotton have been $6.71 billion and $5.3 billion

respectively during 2012 (Brookes and Barfoot, 2014). Cumulatively since from 1996, the

global farm income gain has been $32.3 billion for genetically modified insect resistant

maize and $36.3 billion for that of cotton. Furthermore, from the past 17 years, the

adoption of crop biotechnology (by 17.3 million farmers in 2012) has clearly

demonstrated the important economic benefits of genetic engineering technology.

Reports also showed that many of farmers, especially from developed countries, have

benefited due to lower costs of production (Brookes and Barfoot, 2014). Current statistics

and understanding of economic benefits from use of genetically modified insect resistant

cultivars clearly points at immense scope for use of genetic engineering technology in

crop improvement programme.

The gene pyramiding in transgenic plants expected to be potentially more viable

strategy for supressing insect evolution leading to resistance against single cry genes

(Cao et al., 2002). Although transgenic lines carrying different cry gene (cry1Ac, cry2Aa,

cry1F and cry1Acm) developed in present study showed insect resistance up to 60.0 to

70.0 per cent, they can be further potentially used for gene pyramiding of different cry

genes for development of broad spectrum and durable resistance. Based on the

experience of Bt transgenic cotton performance world wide and in India as well, pyramid

products expressing cry1Ac anlong with any other gene would be ideal for commercial

products amoung the lines developed in present study. It would ideal in terms of

resistance management also. The pigeonpea transgenic expressing cry1F, cry1Acm etc.

would also be ideal in terms of bio-activity against H. armigera, if second generation Bt

cottons are grown around. Some lines showed uniform expression (as per bioassay) in

different tissue types which is principally appreciable. Although, the insect mortalities are

low in such lines, this lot will make separate study material for enhancement.

217

Furthermore, as the transformation method followed in present study found efficient and

easy for pigeonpea transformation, can be explored to developed more number of

transgenic plants with new cry gene sources that can be characterised for identification

of superior transgenic line with complete resistance to H. armigera.

218

6. SUMMARY AND CONCLUSIONS

Pigeonpea is an important pulse crop, particularly in India. Pigeonpea

production is limited by wide range of insects in which pigeonpea pod borer,

Helicoverpa armigera is major. Although, conventional breeding approaches have

been attempted in pigeonpea crop improvement, development of genetically modified

crops for insect resistance would be an ideal solution in the interest of the pigeonpea

farmers and crop productivity. Many Cry proteins are well known for their insecticidal

activity, and expression of them could improve insect resistance in many crop plants

such as, cotton, maize and soybean. The improvisation in in vitro regeneration

methods through organogenesis is important for future transgenic programs in crops

like pigeonpea. The in planta transformation method have shown immense potential in

transgenic development in many crop plants. Development of transgenic pigeonpea

expressing insecticidal cry genes and understanding their bioefficacy against

pigeonpea pod borer attack is important. Present study involved improvisation of in

vitro and in planta transformation and plantlet regeneration methods, development and

characterization of transgenic pigeonpea carrying four cry genes viz., cry1Ac, cry2Aa,

cry1F and cry1Acm separately. The summary of present investigation is presented

here;

1. The increasing concentration regimes (1, 2, 3, 4, 6, 8 and 10 mg/L) of three

cytokinins viz., BAP, TDZ and zeatin were assessed to study their effect on shoot

bud induction in two pigeonpea genotypes, ICPL 87119 and BSMR 736.

2. The explants response to different concentration regimes of BAP for shoot bud

induction ranged from 0.0 to 72.0 per cent in case of ICPL 87119 and from 0.0 to

73.0 per cent in case of BSMR 736. The number of shoot bud induced per

explant ranged from 0 to 52.6 for ICPL 87119 and from 0 to 53.7 for BSMR 736.

The maximum number of explants responded and number of shoot induced

noticed at 2.0 mg/L BAP concentration regime.

3. In case of embryo disc with half cotyledon explants, the explants response

ranged from 0 to 53.0 per cent (ICPL 87119) and 0 to 55.0 per cent (BSMR 736)

in pigeonpea genotypes. The number of shoot bud induced ranged from 0 to 4.8

219

in ICPL 87119 and from 0 to 4.4 in BSMR 736. In MS medium supplemented

with 2.0 mg/L BAP recorded highest number shoot buds (4.8 in ICPL 87119).

4. The cotyledonary node with cotyledon explants responded to different TDZ

concentration regimes ranged from 0 to 67.0 per cent in case of both test

genotypes. Maximum number of explants responded for shoot bud induction was

at 4.0 mg/L TDZ concentration level. The number of shoot bud induced per

explant ranged from 0 to 41.1 (ICPL 87119) and from 0 to 46.1 (BSMR 736) in

test genotypes.

5. The embryo disc with half cotyledon explants response to increasing TDZ

concentration regimes ranged from 0 to 44.0 per cent in ICPL 87119 and 0 to

50.0 per cent in BSMR 736. The number of shoot bud induced from embryo

discs with half cotyledon explant ranged from 0 to 4.7 (ICPL 87119) and from 0

to 4.5 (BSMR 736). At TDZ concentration regime of 4.0 mg/L, the highest

number shoot buds (4.7) was induced in ICPL 87119.

6. To the effect of zeatin concentration regimes in MS, the cotyledonary node with

cotyledon explants response ranged from 0 to 66.0 per cent in ICPL 87119 and

from 0 to 64.0 per cent in BSMR 736. The maximum number of shoot bud

induced per explant found at 2.0 mg/L zeatin concentration level. The number of

shoot bud induced from embryo discs with half cotyledon explant ranged from 0

to 4.2 in both genotypes.

7. There was an induction of more number of shoot buds in cotyledonary node with

cotyledons explants when compared with that of embryo discs with half

cotyledon explants. The cotyledonary node with cotyledons explants reported

immense potential for multiple shoot bud induction.

8. There was a concomitant decrease in resonance of explants for shoot bud

induction and number of shoot bud induced at higher concentration regimes of

tested cytokinins in growth medium. There was an absence of any artefacts such

as deformed shoots when explants were cultured on MS augmented with

different TDZ concentration regimes.

220

9. Well developed shoots of more than 3 cm long were excised and cultured on MS

medium augmented with increasing IBA concentration regimes. The frequency of

rooting varied with different IBA concentration regimes ranging from 20 to 80% in

both genotypes. The highest root induction was noticed in MS media with 0.5

mg/L IBA.

10. The improvisation of in planta transformation protocol to increased

transformation efficiency was performed using following treatment such as,

Agrobacterium tumefaciens infection alone, A. tumefaciens infection using A.

tumefaciens culture with tobacco extract added in overnight grown culture and A.

tumefaciens infection using air evacuation to increase the proximity between A.

tumefaciens and embryonic axis.

11. There was 80.00 per cent explant response, 53.75 per cent explant survival and

3.0 per cent transformation efficiency recorded in case of A. tumefaciens

infection alone. In case of A. tumefaciens infection using A. tumefaciens culture

along with tobacco extract, recorded 85.00 per cent explant response, 90.00 per

cent explant survival and 6.5 per cent transformation efficiency. The 66.50 per

cent explant response, 90.98 per cent explant survival and 12.0 per cent

increase in transformation efficiency was recorded for the treatment involving air

evacuation to increase the proximity between A. tumefaciens and embryonic

axis.

12. By following improvised in planta transformation protocol, the T1 plant progeny

screening of developed 741 primary transformants identified a set of eighty eight

putative transgenic plants for integration of cry1Ac gene. The in vitro generated

ten transformants (T0 generation) were hardened and grown in transgenic

containment facility.

13. The obtained T2 seeds were sown in plant to row progeny manner to get T2

generation plants. From each parental T1 generation plants up to 40 seeds were

sown and obtained T2 plants were subjected for gene specific PCR assay.

Among developed eighty eight, for forty eight transgenic plant progenies of T1

generations plants recorded chi-square calculated value less than table chi-

square value.

221

14. The larval mortality in case of leaf tissues ranged from 25.0 to 70.0 per cent.

Whereas, it was 22.5 to 52.5 per cent in case of flower tissues and 17.5 to 47.5

per cent in case of pod tissue. The Cry protein level ranged from 0.305 to 0.847

µg/g of fresh leaf tissue, from 0.221 to 0.671 µg/g of fresh flower tissue and

0.250 to 0.685 µg/g of fresh pod tissue.

15. Set of eight transgenic plants viz., Ac16-1, Ac20-2, Ac20-3, Ac29-1, Ac29-3,

Ac31-1, Ac31-2 and Ac50-1 carrying cry1Ac gene were identified and forwarded

to T3 generation. The homozygosity in T2 parental plants viz., 20-2-7, 20-3-2, 29-

1-10, 31-2-9, 31-2-12, 31-1-2 and 31-1-3 belonging to five transgenic lines were

confirmed using cry1Ac gene specific PCR assay.

16. The larval mortality in T3 generation plants ranged from 41.25 to 61.25 per cent

in case of leaf tissue, 32.5 to 47.5 per cent in case of flower tissue and 35.0 to

52.5 per cent in case of pod. In leaf tissues, the Cry1Ac protein level ranged from

0.322 to 0.736 µg/g of fresh tissue weight. Similarly, the Cry protein

accumulation in flower tissues was noticed from 0.254 to 0.646 µg/g and from

0.269 to 0.691 µg/g of fresh pod tissue. The cry1Ac transcript level ranged from

24.6 to 165.1 ng/µl in fresh leaf tissue, from 15.6 to 149.5 ng/µl in flower and

from 18.3 to 152.4 ng/µl in pod tissue.

17. In Sothern blot analysis, an observation of strong signal and the difference in the

hybridization pattern in three selected transgenic plants viz., Ac29-1, Ac20-2 and

Ac20-3, revealed the single copy integration in these plants. Northern blot

analysis indicated hybridization signals in all selected transgenic plant samples

viz., Ac31-2, Ac29-1, Ac31-1, Ac29-3, Ac20-2 and Ac20-3.

18. Juncture region analysis revealed that flanking genomic region recovered from

Ac20-2 and Ac20-3 showed homology with Scaffold130851 of pigeonpea

genome. In case of Ac29-1, the recovered flaking genomic region indicated

homology with Scaffold137204.

19. A set of sixty five putative transformants carrying cry2Aa gene was identified

from 348 primary transformants. The chi-square analysis of observed number of

plants with and without cry2Aa gene indicated that among sixty five transgenic

222

lines, sixteen were following 3:1 (positive: negative) ration for transgene

segregation in T2 generation.

20. The larval mortality in case of leaf tissues ranged from 5.25 to 65.75 per cent,

between 5.25 to 40.5 per cent in case of flower tissue and from 10.25 to 50.25

per cent in case of pod tissue. The transgenic lines, 21A4-21, 21A12-26, 1A2-30,

1A2-42 found consistent in their performance across different tested tissue types.

The Cry2Aa protein level in leaf tissue ranged from 0.013 to 3.231 µg/g of fresh

leaf tissue. Whereas, it was 0.010 to 2.854 µg/g in case of fresh flower tissues

and 0.012 to 2.811 µg/g in case of fresh pod tissue.

21. The transgenic lines viz., 21A2-2, 21A3-4, 21A6-9, 21A5-10, 21A6-12, 21A5-14,

21A5-16, 21A5-19, 21A12-24, 21A12-25, 21A5-59, 21A5-60, 21A4-62, 21A6-68

and 21A4-73 were advanced to T3 generation. Among these fifteen, in four

transgenic lines homozygous nature of cry2Aa locus was detected. The T2

parental plants with homozygous nature were 21A2-2-1, 21A3-4-7, 21A5-16-1

and 21A4-62-6.

22. The bioassay results reported that the larval mortality in case of leaf tissue

ranged from 43.33 to 68.33 per cent, in case of flower tissue from 23.5 to 48.5

per cent and in case of pod from 28.5 to 53.25 per cent. The Cry2Aa protein

levels in leaf tissues ranged from 0.370 to 1.417 µg/g of fresh tissue weight, from

0.312 to 1.021 µg/g in flower tissue and from 0.309 to 0.989 µg/g in in pod.

Further, the cry2Aa transcript level ranged from 48.5 to 134.5 ng/µl in fresh leaf

tissue, from 41.2 to 110.2 ng/µl in flower tissue and from 42.8 to 100.5 ng/µl in

pod.

23. There was a positive correlation between insect morality and Cry protein

accumulation. The frequency distribution analysis of transgenic plants following

3:1 transgene segregation pattern and other than 3:1 segregation pattern

revealed that majority of high Cry protein accumulating transgenic plants were

following 3:1 transgene segregation pattern. The correlation analysis of larval

mortality, Cry protein and cry transcript revealed strong positive in leaf, flower

and pod tissues for these traits of T3 generation transgenic lines.

223

24. The southern bolt analysis revealed a single but weak hybridization signals for

two test samples belonging to 21A2-2 and 21A3-4 transgenic lines with different

prob hybridization pattern. There were strong hybridization signals in other two

other test samples viz., 21A6-9 and 21A6-68 with similar hybridization pattern,

indicating the possibility of same position of T-DNA integration in plant genome.

The northern blot analysis showed hybridization signals in all selected transgenic

plant samples viz., 21A2-2, 21A3-4, 21A6-9, 21A12-24 and 21A6-68.

25. The local blast was set using BioEdit tool for processed contigue sequences and

it was observed that flaking genomic region recovered from 21A2-2 showed

homology with Scaffold137204 of pigeonpea genome. Similarly, the flaking

genomic region recovered from 21A3-4 and 21A12-24 showed homology with

Scaffold134438. In case of 21A6-9 and 21A6-68, the recovered flaking genomic

region indicated with Scaffold127179 of pigeonpea draft genome.

26. The T1 seeds were harvested from well-established 177 primary transformants

and T1 generation was raised. The T1 progeny plant screening using cry1F gene

specific PCR assay identified a set of fourteen putative transgenic plants for

integration of cry1F gene. Among developed fourteen, for seven transgenic

plants the chi-square calculated value was recorded less than table chi-square

value.

27. The larval mortality in case of leaf tissues ranged from 22.5 to 62.5 per cent, it

was 20.0 to 57.5 per cent in case of flower and 12.5 to 47.5 per cent in case of

pod. The Cry protein level ranged from 0.170 to 1.032 µg/g of fresh leaf tissue,

0.113 to 0.870 µg/g of fresh flower tissue and 0.117 to 0.782 µg/g of fresh pod

tissue. The cry1F transcript level ranged from 47.6 to 105.3 ng/µl in fresh leaf

tissue, 45.2 to 101.0 ng/µl in flower tissue and from 48.5 to 103.2 ng/µl in pod

tissue.

28. The correlation analysis of larval mortality, Cry1F protein and cry1F transcript

reported positive in leaf, flower and pod tissues for these traits of T2 generation

transgenic lines.

224

29. An observation of strong Southern hybridization signals and the difference in the

hybridization pattern in the four selected transgenic plants viz., 1F-22, 1F-32, 1F-

38 and 1F-33, revealed the single copy integration in these plants. Whereas, in

two plant samples viz., 1F-25 and 1F-9, two copies of T-DNA integration in plant

genome was noticed. The northern blot analysis indicated hybridization signals in

all selected transgenic plant samples viz., 1F-25, 1F-32, 1F-33, 1F-9, 1F-22 and

1F-38.

30. The T1 progeny plant screening from 120 primary transformants identified a set

of eleven putative transgenic plants carrying cry1Acm gene as revealed by gene

specific PCR assay. The chi-square analysis of observed number of plants with

and without cry1Acm gene indicated that among eleven transgenic lines, seven

were following 3:1 (positive: negative) ration of transgene segregation.

31. The larval mortality in case of leaf tissues ranged from 40.0 to 62.5 per cent, in

case of flower it ranged from 37.5 to 57.5 per cent and in pod from 35.0 to 52.5

per cent. The Cry protein level in leaf tissue ranged from 0.322 to 0.907 µg/g of

fresh leaf tissue; 0.202 to 0.503 µg/g of fresh flower tissue and 0.186 to 0.405

µg/g of fresh pod tissue. The cry1Acm transcript level was ranged from 40.15 to

105.5 ng/µl of fresh leaf tissue; in flower it ranged from 39.80 to 103.0 ng/µl and

in pod from 39.83 to 102.9 ng/µl.

32. A single but weak Southern hybridization signals were noticed for all test

samples M2, M10, M16 and M135 transgenic lines. The Southern hybridization

pattern was different for all samples, indicated different position of T-DNA

insertion in plant genome with single copy integration of transgene. The northern

hybridization signals confirmed production of transgene transcripts in all selected

transgenic plant samples viz., M135, M55, M2, M5 and M7.

33. Among tested plant eight growth parameters, development of non-parental type

phenotypes were noticed for plant height, flower colour and pod colour traits.

34. Based on comparative analysis of different transgenic events expressing cry1Ac,

cry2Aa, cry1F and cry1Acm genes independently, it was indicative that the

225

transgenic events expressing differential levels of cry genes were varying for

their insecticidal activity against H. armigera.

35. Although transgenic lines carrying different cry gene (cry1Ac, cry2Aa, cry1F and

cry1Acm) developed in present study showed insect resistance up to 60.0 to

70.0 per cent, they can be further potentially used for gene pyramiding of

different cry genes for development of broad spectrum and durable resistance.

36. Some lines showed uniform expression (as per bioassay) in different tissue types

which is principally appreciable. Although, the insect mortalities are low in such

lines, this lot will make separate study material for enhancement. The

transformation method followed in present study can be explored to developed

more number of transgenic plants with new cry gene sources that can be

characterised for identification of superior transgenic line with complete

resistance to H. armigera.

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APPENDIX I

a. Yeast Mannitol Agar (YEMA) broth (100 ml)

Mannitol 1.0 g

Orthophosphate (KH2PO4) 20.0 mg

Dipotassium hydrogen phosphate (K2HPO4) 20.0 mg

Yeast extract 100.0 mg

Magnesium sulphate (MgSO4) (1M) 80.0 µl

Calcium chloride (CaCl2) (1M) 40.0 µl

b. Yeast Mannitol Agar (YEMA) (100 ml)

Mannitol 1.0 g

Orthophosphate (KH2PO4) 20.0 mg

Dipotassium hydrogen phosphate (K2HPO4) 20.0 mg

Yeast extract 100.0 mg

Magnesium sulphate (MgSO4) (1M) 80.0 µl

Calcium chloride (CaCl2) (1M) 40.0 µl

Agar agar 1.8 g

259

APPENDIX II

a. Loading dye composition

Loading dye (6X) 0.25% bromophenol blue

40% (w/v) sucrose in water

b. Ethidium bromide

Dissolve 10 mg/ml in distilled water and store at 4 °C in a dark bottle.

c. Recipe for 1% agarose gel (40 ml)

Agarose 400.0 mg

1X Tris-acetate-EDTA (TAE) 40.0 ml

Ethidium bromide (10 mg/ml) 2.0 µl

d. 50X Tris-acetate-EDTA (TAE) composition

Tris base 242.0 g

Glacial acetic acid 57.1 ml

0.5 M Ethylenediamine tetraacetic acid (EDTA) (pH 8.0) 100.0 ml

Make up the volume to 1000 ml with double distilled water

260

APPENDIX III

a. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part A)

Kabuli gram flour 100.0 g

Yeast 30.0 g

Sterile water 400.0 ml

b. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part B)

Agar agar 14.0 g


c. Ingredient for one set of diet to rear Helicoverpa armigera larvae (Part C)

Wesson’s salt 7.0 g

Casein 5.0 g

Ascorbic acid 3.0 g

Multivitamin mix (Polybion SF) 4.0 ml

Sorbic acid 1.0 g

Methyl parahydroxy benzoate (MPHB)* 2.0 g

Streptomycin sulphate 0.25 g

Bavistin 2.0 g

* Dissolve in 5.0 ml of 95.0% Ethanol and added to the diet

261

APPENDIX IV

a. RNA loading dye composition (5X) (10 ml)

Saturated aqueous bromophenol blue solution 16.0 µl

EDTA (0.5M; pH: 8.0) 80.00 µl

Formaldehyde 720.0 µl

Glycerol (100 %) 2000.0 µl

Formamide 3048.0 µl

10X FA gel buffer 4000.0 µl

RNase free water 100.0 µl

b. Ethidium bromide

10 mg/ml in RNase free water. Store at 4°C in a dark bottle.

c. Recipe for 1.2% formaldehyde agarose gel (60 ml)

Agarose 720.0 mg

Formaldehyde 1.0 ml

Ethidium bromide (10 mg/ml) 3.0 µl

1X 3-(N Morpholino) propanesulphonic acid (MOPS) buffer 60.0 ml

d. 10X 3-(N Morpholino) propanesulphonic acid (MOPS) electrophoresis buffer

3-(N Morpholino) propanesulphonic acid (MOPS) 41.8 g

Sodium acetate (DEPC-treated) 20.0 ml

EDTA (DEPC-treated; pH: 8.0) 20.0 ml

Total volume 1000 ml with DEPC treated water

262

APPENDIX V

a. Denaturation Buffer (500 ml)

Sodium hydroxide (0.4M) 8.0 g

Sodium chloride (3M) 87.7g

Sterile water Make up the vol. 500.0 ml

b. Neutralization Buffer (500 ml)

Tris (1M) pH 7.4 78.5 g

Sodium chloride (1.5M) 43.9 g

Sterile water Make up the vol. 500.0 ml

c. Pre-hybridization buffer (100 ml)

20X Saline Sodium Citrate (SSC) 25.0 ml

Formamid 50.0 ml


Casein 0.5 g

SDS 0.1 g

d. Marker preparation (10 µl)

Dig-labelled Marker 3.0 µl

Bromo phenol blue 3.0 µl

Sterile water 4.0 µl

e. Hybridization buffer

Pre-hybridization buffer 10.0 ml

Dig-labelled DNA probe 10.0 µl

263

f. Wash buffer I (200 ml)

20X Saline sodium citrate (SSC) 20.0 ml

10% Sodium dodecyl sulfate (SDS) 2.0 ml


g. Wash buffer II (200 ml)

20X Saline sodium citrate (SSC) 5.0 ml

10% Sodium dodecyl sulfate (SDS) 2.0 ml


h. Wash buffer III (500 ml) pH 7.5

Malic acid (2.0M) 25.0 ml

Sodium chloride (4.0M) 16.15 ml

Twin 20 1.5 ml

Sterile water Vol. made 500 ml

i. Malic acid buffer (500 ml) pH 7.5

Malic acid (2.0M) 25.0 ml

Sodium chloride (4.0M) 16.15 ml


j. Blocking solution (30 ml)

Malic acid buffer pH 7.5 27.0 ml

Blocking reagent 3.0 ml

264

k. Antibody solution (15 ml)

Blocking solution 15.0 ml

Antibody 3.0 µl

l. Detection buffer (100 ml) pH 9.8

Tris-Cl (1M) 10.0 ml

Sodium chloride (4M) 2.5 ml


m. Substrate solution (10 ml)

Detection buffer 10.0 ml

NBT/BCIP 200.0 µl

265

APPENDIX VI

a. Luria agar (LA)

Tryptone 10.0 g/l

Yeast extract 5.0 g/l

Sodium chloride 5.0 g/l

Agar 18.0 g/l

b. Sodium Chloride-Tris-EDTA (STE) buffer

Tris-HCl (pH 8.0) 10 mM

NaCl 0.1 M

EDTA (pH 8.0) 1.0 mM

Autoclaved and stored at 4 °C

c. Alkaline-lysis solution I

Glucose 50 mM

Tris-HCl (pH 8.0) 25 mM

EDTA (pH 8.0) 10 mM


d. Alkaline-lysis solution II

Sodium hydroxide (NaOH) 0.2 N

Sodium dodecyl sulfate (SDS) 1% (w/v)

(Prepared fresh and used at room temperature)

e. Alkaline-lysis solution III

5 M Potassium acetate 60.0 ml

Glacial acetic acid 11.5 ml

Double distilled water 28.5 ml


Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST

Helicoverpa armigera AND MOLECULAR ANALYSES OF SELECTED EVENTS

2014 MAHALE BARKU M. Dr. B. FAKRUDIN

Major Advisor ABSTRACT

In ICPL87119 and BSMR736, MS medium supplemented with 2.0 mg/l BAP,

4.0 mg/l TDZ and 2.0 mg/l zeatin, separately, induced maximum shoot buds, 53.7,

46.1 and 40.9 respectively; any further increase in cytokinins levels resulted in

reduced shoot buds. The MS basal with 0.5 mg/l IBA induced maximum and healthier

roots (4.8±0.7). In planta transformation revealed 80.00, 85.00, 66.50% explant

response, 53.75, 90.00, 90.98% explant survival and 3.0, 6.5, 12.0% transformation

efficiency in Agrobacterium tumefaciens infection alone, A. tumefaciens culture with

tobacco leaf extract and air evacuation, respectively. The 88 putative transformants

carrying cry1Ac were developed, of which 48 showed 3:1 transgene segregation

pattern in T2. Insect mortality ranged from 25.0 to 70.0% whereas, Cry1Ac protein

level from 0.31 to 0.85 µg/g and cry1Ac transcript level from 15.6 to 165.1 ng/µl,

validated through northern blotting in different tissues (leaf, flower and pod). In case

of cry2Aa, 65 transformants developed, of which 16 showed 3:1 transgene

segregation in T2. Insect mortality ranged from 5.25 to 65.75% whereas, Cry2Aa

protein and transcripts ranged from 0.01 to 3.23 µg/g and 41.2 to 134.5 ng/µl,

respectively. Southern and juncture analyses of selected three cry1Ac and five

cry2Aa transformants confirmed T-DNA integration in plant genome. Fourteen

transformants carrying cry1F were developed, of which seven showed 3:1 transgene

segregation pattern in T2, wherein insect mortality ranged from 10.0 to 62.5%, Cry1F

protein level from 0.113 to 1.032 µg/g and transcripts ranged from 45.2 to 105.3

ng/µl. Similarly, eleven cry1Acm transformants were developed, of which seven

showed 3:1 transgene segregation in T2. Insect mortality ranged from 35.0 to 62.5%

whereas, protein level and transcripts ranged from 0.19 to 0.91 µg/g and 41.2 to

134.5 ng/µl respectively, in tested tissues. Pigeonpea transformation procedures and

generated events of present study could be prospected for their further use.

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Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR

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