CHARACTERIZATION OF TRANSGENIC GROUNDNUT (Arachis hypogaea L.)

174 Pages·2017·4.89 MB·English

CHARACTERIZATION OF TRANSGENIC
GROUNDNUT (Arachis hypogaea L.) EXPRESSING
THE ZAT12 TRANSCRIPTION FACTOR GENE
UNDER ABIOTIC STRESSES
By
ABHAY KUMAR
(Registration No-J4-01014-2012)
M. Sc. (Agri. Biotech)
DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH-362 001
FEBRUARY - 2017

CHARACTERIZATION OF TRANSGENIC
GROUNDNUT (Arachis hypogaea L.) EXPRESSING
THE ZAT12 TRANSCRIPTION FACTOR GENE
UNDER ABIOTIC STRESSES

A THESIS SUBMITTED TO
JUNAGADH AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN
PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY

BY
ABHAY KUMAR
(Registration No-J4-01014-2012)
M. Sc. (Agri. Biotech)

DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH-362 001

FEBRUARY - 2017

Abstract

DEPARTMENT OF BIOTECHNOLOGY
Name of the student Major Guide
Abhay Kumar Radhakrishnan T.
CHARACTERIZATION OF TRANSGENIC GROUNDNUT (Arachis
hypogaea L.) EXPRESSING THE ZAT12 TRANSCRIPTION FACTOR
GENE UNDER ABIOTIC STRESSES
ABSTRACT
Key words: transgenic groundnut, ZAT12, abiotic stress, drought, salinity
Groundnut (Arachis hypogaea L.), is an important legume crop and India is
one among the leading producers of groundnut worldwide. The various type of abiotic
stresses ensue major environmental constraints in groundnut production. The present
study was undertaken with the primary objectives to characterize the transgenic
groundnut plants for integration, expression and inheritance of BcZAT12 transcription
factor gene and to evaluate the transgene expression under various abiotic stresses
under in vitro and glasshouse conditions. The experimental transgenic test plants of
Arachis hypogaea, was developed through Agrobacterium-mediated transformation of
groundnut cv. GG20 at Biotechnology Laboratory, ICAR-DGR, Junagadh. PCR
screening of transgenics using BcZAT12 and nptII gene- specific primers gave
expected amplification of approximately 486bp and 750bp, respectively. The presence
of transgene in twelve transgenic events (T3) was confirmed by Dot blot analysis. PCR
analysis of T2 generation plants showed segregation of gene followed Mendelian
pattern of inheritance. RT-PCR results showed that transgenic plants showed induced
expression under soil moisture deficit stress under stress-inducible Lea promoter. The
pattern of gene expression were analyzed at transcript level through real time PCR
(qPCR) showed differential pattern of expression under PEG- induced drought stress,
NaCl- induced salinity stress and progressive soil moisture deficit stress. Under
various abiotic stresses, transgenic events showed delayed and less wilting of leaves,
improved physio-biochemical traits such as accumulation of proline; enhanced
osmotic adjustment; improved water and total chlorophyll retention capacities; less
electrolytic leakage; and speedy recovery. Key biochemical parameters like activity of
antioxidants enzymes, stress metabolites content and growth related parameters were
in significantly better status in transgenic lines. qPCR based differential expression
studies identified the level of induction of downstream target stress-inducible genes.
The better performance of the transgenic lines of BcZAT12 quantified in terms of
physio-biochemical characterization, growth associated parameters and phenotypic
observations under various abiotic stresses gives us the confidence to claim that
BcZAT12 expression improved the tolerance of the transgenic lines compared with the
wild type plants.

ACKNOWLEDGEMENT

I have great privilege to express my deep sense of gratitude to my Major
Advisor, Dr. Radhakrishnan T., Director, ICAR-Directorate of Groundnut Research,
Junagadh for his keen interest, most valuable and inspiring guidance, constructive
criticisms and constant encouragement throughout the course of the investigation. His
special concern for aesthetics and precision are remarkable and giving the student
freedom to satisfy their queries is certainly appreciable.
I would like to express my gratitude to my committee members especially to
Dr. D. N. Vakharia, Professor, Department of Biochemistry for the positive
motivation, encouragement and guidelines provided. I also express gratitude towards
other members Dr. M. K. Mandavia, Professor, Department of Biotechnology,
College of Agriculture, Junagadh Agricultural University, Junagadh and Dr. S. M.
Upadhyay, Professor and Head, Dept. of Agril. Statistics, College of Agriculture,
Junagadh Agriculture University, Junagadh.
I express a deep sense of gratitude to Dr. B. A. Golakiya, Professor and Head,
Department of Biotechnology for his full pledged co-operation, guidance, continued
inspiration and valuable suggestions.
My PhD Thesis would not have seen the right completion and that too in right
time, had it not been the able and untiring support and advices from Dr. G. P. Mishra
and Dr. J. R. Dobaria from whom I learnt a lot. I am indebted to Mr. B. K. Singh who
made the ground work of the present study; he was instrumental in helping me make
the foundation of the study.
It is my great pleasure to extend profuse thanks to the authorities of the
Junagadh Agricultural University, Dr. N. C. Patel, former Vice Chancellor, Dr. A. R.
Pathak, Vice Chancellor, Dr. V. P. Chovatia, Director of Research, Dr. A. Y. Desai,
Former Director of Research, JAU, Junagadh and Dr. A. V. Barad, Principal and
Dean, College of Agriculture, JAU, Junagadh for providing necessary facilities for
conducting the research work.
I want to extend my gratitude to the Library members for their kind and instant
services. I’m also thankful to ‘C’ branch staff specially Gordhanbhai and
Makadiabhai for co-operative and quick processing of academic records.
I am thankful and indebted for the friendly help, guidance and encouragement
rendered to me by my batchmates, Hiren and Abhinandan. My sincere thanks goes to
Tanmoy, Bhagwat, Kiran, Viral, Sahil, Tejas, Suhail, Vivekanand, Binal, Sneha and
Bindu.
The cheerful atmosphere and unconditional support extended by all my lab
staff Mansukhbhai and Shekhbhai helped create a homely environment in the lab.
I am indebted to my parents for their selfless love and faith in me. I would also
like to thank my elder brothers Dr. Ajay Kumar and Dr. Akshay Prakash for
encouraging me at each and every step. I would like to thank Dr. Pratibha Singh for
being my friend, philosopher and guide and for teaching me: patience has its rewards!
Lastly but not the least, I sincerely acknowledge the financial support by
ICAR throughout the period. The other indirect financial helps granted to the
laboratory by DBT and ICAR funded projects also have benefited me and gratefully
acknowledged.
All might have not been mentioned but none is forgotten.

Place: Junagadh
Date: 25/01/2016 (Abhay Kumar)