ROLE OF PUTRESCINE IN GROWTH, DEVELOPMENT AND ABIOTIC STRESS TOLERANCE OF ...

183 Pages·2016·10.78 MB·English

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ROLE OF PUTRESCINE IN GROWTH, DEVELOPMENT
AND ABIOTIC STRESS TOLERANCE OF RICE
(Oryza sativa L.)

Ms. SINI THOMAS K.

DIVISION OF PLANT PHYSIOLOGY
INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI-110 012

2015
ROLE OF PUTRESCINE IN GROWTH, DEVELOPMENT
AND ABIOTIC STRESS TOLERANCE OF RICE
(Oryza sativa L.)

A Thesis

By

Ms. SINI THOMAS K.

Submitted to the Faculty of Post-Graduate School,
Indian Agricultural Research Institute, New Delhi
In partial fulfillment of requirements for the award of the degree of

DOCTOR OF PHILOSOPHY
IN
PLANT PHYSIOLOGY

NEW DELHI

2015

Approved by:

Chairman : Dr. Madan Pal Singh ________________________________
Co-chairman : Dr. C. Viswanathan ________________________________
Member : Dr. Anjali Anand ________________________________
Member : Dr. A. K. Singh ________________________________
Member : Dr. Jasdeep Padaria ________________________________
Dr. Madan Pal Singh Division of Plant Physiology
Principal Scientist Indian Agricultural Research Institute
New Delhi-110 012, India

CERTIFICATE
This is to certify that the thesis entitled “Role of putrescine in growth,
development and abiotic stress tolerance of rice (Oryza sativa L.)” submitted to
the Faculty of the Post-Graduate School, Indian Agricultural Research Institute,
New Delhi, in partial fulfillment of the requirements for the award of the degree of
DOCTOR OF PHILOSOPHY in PLANT PHYSIOLOGY is a faithful record
of bonafide research work carried out by Ms. Sini Thomas K., Roll No: 10178
under my guidance and supervision. No part of this thesis has been submitted for
any other degree or diploma.
It is further certified that any help or source of information that has been
availed of in this connection has been duly acknowledged by him.

Date: December, 2015 (Madan Pal Singh)
Chairman, Advisory Committee
Place: New Delhi
Dedicated
To my husband
To our son
& to my parents...
ACKNOWLEDGEMENT

It is imperative that I acknowledge all those who enriched and provided a
fertile soil for the relevation of the dissertation. Words are bound and knowledge
is limited to praise ALMIGHTY, the Lord of the world, the Omnipotent, the
Beneficent, Who gave me the requisite potential and diligence for the
successful accomplishment of this task. I could never have accomplished this
without the faith I have in him.
Sincere gratitude to my Chairman, Dr. Madan Pal Singh, Principal
scientist, Division of Plant Physiology, IARI for his sincere guidance, efforts
and involvement, for the successful completion of the proposed research and
preparation of this manuscript. He nurtured my capabilities and gave me ample
freedom to work. His positive attitude, hardworking nature will always
inspire me throughout my life. It is great privilege for me to express my esteem and
profound sense of
gratitude to my Co-chairman Dr. C. Viswanathan, Head, Division of Plant
Physiology, for his constructive and valuable suggestions.
My sincere gratitude towards the members of the advisory committee
comprising of Dr. Anjali Anand, Principal Scientist, Division of Plant
Physiology, Dr. A. K. Singh, Head, Division of Genetics and Dr. Jasdeep
Padaria, Principal Scientist, NRC for Plant Biotechnology, who not only
taught me courses but always helped me in sorting out problems which
occurred during the course of investigation.
I sincerely thankful to Dr. V. P. Singh, Professor, Division of Plant
Physiology for his guidance, help, support and encouragement throughout my
research programme. I sincerely acknowledge and thank all my teachers for
helping me to cope up with my academic stint as a student and for enriching
me with enlightening thoughts and ideas. I would like to give hearfelt thanks
to Dr. Sangeeta Khedarpal for her unstinted support and constant
encouragement.
I sincerely acknowledge and thank for all the co-operation received from
Technical Officers, other staffs members of Division of Plant Physiology Takur Ji,
Banerji sir, Ashok Ji, Naresh Ji, Daya Shankar Ji, Meena Ji, Lakinder ji, Praveen Ji
and the laboratory staff especially Santhosh sir, Pragya, Sadhana mam, Dr.
Divya, Dr. Ashish, Shwetha mam for helping me in successful completion of
research work. I express my deep sense of gratitude for their kind cooperation
during the study.
I am immensely indebted and express my deep sense of gratitude and
heartfelt thanks towards Dr. P. K. Jain, NRCPB; Dr. S. K. Yadav, Division of
SS&T and In charge of National Phytotrone Facility for their support for the
successful completion of my work.
It is beyond my words to express my heartful thanks to Krishna Kumar sir
whose support was beyond words and really inevitable in the completion of the
study. I do take memories to cherish during my Ph. D. Programme with my
classmates Surender, vineeth and shailesh, memorable friends Madhurima,
Kalpana, Vaibhav, Donald, Anshi and Anuja seniors Ravi sir, Thavaprakash
sir, Sivaram sir, Deepika di, Shivani di, Flemine chichi and Krishnapriya di
juniors Gajender, Sukumar, Mukesh, Soumya, Jasna, Renjini, Saju, Manoj for
their moral support and encouragement. Words failed to me to express
appreciation to my dear kerala (PUMA) seniors, juniors and friends for
cheering me up during these three years and giving continuous help and
support. I also extend many thanks to inmates and workers of Varsha hostel
and Saraswati Apartment, for their helps, encouraging words and blessings.
I do express my deep sense of gratitude and indebtedness to my mother, my
father, Aunt, sister and brother, who always picked me up on time & encouraged me
to go on every adventure, especially this one. I could never have done this without
your faith, affection, support, constant encouragement and prays of day and night.
Thank you for teaching me to believe in myself, in God and in my dreams.
At this moment, I can’t forget my beloved husband Robin, sweet son
Cyril, mummy, daddy, Asha chechi and family, Manju chechi and family.. They
were always my source of strength and stood behind me. I also extend my
thanks to Jojo chettan and family for their moral support and encouragement.
My vocabulary utterly fails in expressing my love to my relatives and friends
for their infinite support during all the years of study.
Finally but not the least I would like to remember all those people
whose names were difficult to incorporate but they were directly or indirectly
involved in providing the help and moral support during the entire period of
study.
I am grateful to I.C.A.R. and I.A.R.I. giving me this opportunity for
providing technical support, the financial support and all physical facilities to
undertake this work at this premier institute.

Date: December 2015 (Sini Thomas K.)
Place: New Delhi
CONTENTS

S. No. Chapters Page No.
1 INTRODUCTION 1
2 BACKGROUND 7
3 MATERIALS AND METHODS 25
4 RESULTS 67
5 DISCUSSION 91
6 SUMMARY AND CONCLUSION 99
7 ABSTRACT (ENGLISH) 105
8 ABSTRACT (HINDI) 107
9 BIBLIOGRAPHY i
10 APPENDICES xxiii
LIST OF TABLES

Table Title In/After
No. page
BACKGROUND
2.1 Abiotic stress-induced Put accumulation & biosynthetic enzyme 19
activation in plants
2.2 Enhanced stress tolerance shown by transgenic plants engineered 22
for overproduction of putrescine
MATERIALS AND METHODS
3.1 List of primers used in this study for qPCR analysis 30
3.2 Gene attributes of adc paralogs adc1 and adc2 36
3.3 List of primers used for cloning 37
3.4 Incubation period required for the restriction enzymes used in this 48
study
3.5 List of primers used for colony PCR 50
3.6 Restriction enzyme combination used for restriction digestion 50
analysis
RESULTS
4.1 Representing the features of adc1 and adc2 genes of rice from 72
RGAP
4.2 Primer combinations used for multiplex colony PCR and required 77
product size
4.3 Restriction enzyme combination used for double digestion and 78
the number of recognition sequences present in the binary
construct
4.4 Restriction enzyme combination used for double digestion and 78
theproduct sizes obtained after digestion
4.5 Primer combinations used for plasmid PCR and expected 79
product size
4.6 Restriction enzyme combination used for double digestion and 81
the product sizes obtained after digestion
4.7 Primer combination used for plasmid PCR and expected 81
product size
LIST OF FIGURES

Figure Title In/After
No. page
BACKGROUND
2.1 Schematic representation of polyamine biosynthesis pathway 16
in higher plants
2.2 Polyamines (PAs) mediated abiotic stress tolerance in plants 21
MATERIALS AND METHODS
3.1 Graphical representation of weather data during the rice crop 26
season 2014-15
3.2 Gene structure of adc1 and adc2 36
3.3 Vector map of the TA cloning vector pTZ57R/T. unique 40
restriction sites are indicated
3.4 DNA sequence of MCS region of the TA cloning vector 40
pTZ57R/T
3.5 pCAMBIA1300 vector with drought inducible AtRD29A 48
promoter
3.6 Procedure for RNAi vector construction 62
3.7 Vector map of pENTR/D-TOPO entry vector 66
3.8 Vector map of pANDA vector 66
RESULTS
4.1 Effect of different abiotic stresses on relative water content 68
(RWC; % ) in contrasting rice cultivars at seedling stage
4.2 Effect of different abiotic stresses on leaf membrane stability 68
index (MSI; %) in contrasting rice cultivars at seedling stage
4.3 Effect of different abiotic stresses on a) net CO assimilation 68
2
rate; b) stomatal conductance and c) transpiration rate in
contrasting rice cultivars at seedling stage
4.4 Effect of different abiotic stresses on a) chlorophyll a; b) 70
chlorophyll b and c) total chlorophyll content in contrasting
rice cultivars at seedling stage
4.5 Effect of different abiotic stresses on carotenoids (car) content 70
in contrasting rice cultivars at seedling stage
4.6 Effect of different abiotic stresses on leaf lipid peroxidation/ 70
TBARS content in contrasting rice cultivars at seedling stage
4.7 Effect of different abiotic stresses on leaf hydrogen peroxide 70
(H O ) content in contrasting rice cultivars at seedling stage
2 2
4.8 Expression of Arginine Decarboxylase 1(Osadc1) gene in root, 72
stem and leaves of contrasting rice cultivars under different
abiotic stresses at panicle initiation stage
4.9 Expression of Arginine Decarboxylase 2 (Osadc2) gene in 72
root, stem and leaves of contrasting rice cultivars under
different abiotic stresses at panicle initiation stage
4.10 Expression of gene Arginine Decarboxylase 1(Osadc1) in root, 72
stem leaves and panicle of contrasting rice cultivars under
different abiotic stresses at anthesis stage
4.11 Expression of Arginine Decarboxylase 2 (Osadc2) gene in 72
root, stem leaves and panicle of contrasting rice cultivars
under different abiotic stresses at anthesis stage
4.12 Sequence of events followed during cloning: a) Amplification 74
with proofreading enzyme; b) confirmation of isolated gene
insert; c) ligation of insert with cloning vector pTZ57R/T
4.13 Blue-white screening of transformed E. coli strain XL-1 Blue 74
a) OSADC1 b) OSADC1 replica plating
c) OSADC2 d) OSADC2 replica plating
4.14 Confirmation of transformed colonies by a) colony PCR of 74
OSADC1; b) colony PCR of OSADC2; c) restriction digestion
with EcoRI and BamHI; d) plasmid PCR
4.15 Alignment of OsADC1 and OsADC2 with other homologous 76
ADC proteins using ClustalW2 and represented by BoxShade
4.16 Multiple sequence alignment of OsADC1 between the cloned 76
gene from NL-44 and reference sequence from database by
clustal omega
4.17 Multiple sequence alignment of OsADC2 between the cloned 76
gene from NL-44 and reference sequence from database by
clustal omega
4.18 Phylogenetic tree constructed by the neighbor-joining method 76
of MEGA 6.06 software with 500 bootstrap replicates of the
ClustalW2 alignment
4.19 Predicted 3D structure of OsADC1 and OsADC2 proteins by 76
Phyre2 server
4.20 Mobilization of gene insert into binary vector 78