evaluation of rice transgenic plants expressing regulatory genes for abiotic stress tolerance

89 Pages·2015·5.01 MB·English

EVALUATION OF RICE TRANSGENIC PLANTS
EXPRESSING REGULATORY GENES FOR ABIOTIC
STRESS TOLERANCE

GURUPRASAD, C. S.
PALB 3180

DEPARTMENT OF CROP PHYSIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU-560 065
2015
EVALUATION OF RICE TRANSGENIC PLANTS
EXPRESSING REGULATORY GENES FOR ABIOTIC
STRESS TOLERANCE

GURUPRASAD, C. S.
PALB 3180

Thesis submitted to the
UNIVERSITY OF AGRICULTURAL SCIENCES, BENGALURU
in partial fulfillment of the requirements
for the award of the degree of
MASTER OF SCIENCE (Agriculture)
IN
CROP PHYSIOLOGY

BENGALURU JULY, 2015
Affectionately
Dedicated To My
Beloved Parents

ACKNOWLEDGEMENT
I am extremely happy to take this opportunity to acknowledge my debts of
gratitude to those associated in the preparation of this thesis.
First and foremost, I am happy to portray my deep sense of gratitude and
generous thanks to my chairman Dr. N. Nataraja Karaba, for his adroit guidance,
meticulous care and mellifluous help all along with an ever welcoming heart. His
technical and expert guidance, sustained interest, consistent encouragement, pragmatic
views and constructive ideas made the formidable work to be an easier one.
I feel very proud to bring this research work in a splendid manner with outstanding
efforts taken by the chairman during the course of this research.
I feel immense pleasure in extending my heartfelt gratitude to Dr. R. Uma
Shaanker, Dr. M. S. Sheshshayee and Dr. R. L. Ravi Kumar, for being in my advisory
committee and helping me to shape my research with their valuable suggestions,
scientific counseling, pivotal observation and incessant encouragement in accomplishing
success in this attempt.
I feel extremely happy to thank M. S. Parvathi., for her valuable counseling,
encouragement and constant support rendered when unquestionably were needed.
I wish to place on record my heartfelt thanks to Dr. M. Udaykumar, Dr. T. G. Prasad, Dr. A.
G. Shankar, Dr. B. Mohanraju, Dr. P. C. Reddy, and all the teaching staffs, senior
research fellows, research associates and non-teaching staffs for their support and co-
operation during my learning program.
I have boundless pleasure in placing on record my gratitude and thanks to my
seniors Sajeevan, Mahesh salimath, Dhanya, Sapna, Mahesh Patil, Jalendra, Ashwini,
Madhura, Pruthvi, Amar, Rajashekar Reddy, Shwetha, Nagaveni, Anitha, Pavithra,
Nagarjun, Ugraiah, Pushpa, Basu and all other seniors for their suggestions, timely help
and for a lively and joyful working environment in the lab.
I humbly express my whole hearted thanks to my classmates Pallu, Nagu, Mani,
Shingal, Gowda, Shama, Sahana, Shruthi, Priya and Namratha for their encouragement,
friendliness, help and assured presence in my highs and lows, anytime anywhere.
I felt inadequacy to express my gratitude and indebtness to my beloved parents
Nagalakshmi and Srinivas and my brother Sunil, for their motivation and prayers to keep
me in high sprits to pursue the programme and I dedicate this piece of work to them.
At last, I would like to make prayer of thanks to the Almighty, for being my
guiding light and for showering his blessing on me. I thank him for equipping me with
utmost strength and patience, to combat all the hurdles that came on the way of
completing this task. Above all, zillions of thanks to everyone who directly or indirectly
helped me in successfully completing my course and research

Bengaluru-65 GURUPRASAD, C. S.
July, 2015
EVALUATION OF RICE TRANSGENIC PLANTS EXPRESSING
REGULATORY GENES FOR ABIOTIC STRESS TOLERANCE

GURUPRASAD, C. S.
THESIS ABSTRACT
Plants are sessile organisms which encounter many unfavorable environmental conditions
during their growth and development. Drought is the chief abiotic stress affecting crop
productivity in tropical regions. Plants have developed various strategies to combat the
adverse effect of stresses, among which cellular tolerance is considered to be the major
one. Since cellular tolerance is a multi-component mechanism governed by several genes,
over expression of a candidate transcription factor (TF) to regulate many downstream
genes is an ideal option.
In this study, transgenic rice (genotype AC 39020) plants developed by independently
over expressing TFs CcNF-YB1 and OsSHN1 cloned from hardy species Cenchrus
ciliaris and rice land race Doddiga respectively, were evaluated. The superior lines
identified in T generation were selected and screened in T and T generations under
2 3 4
controlled environmental conditions using antibiotic (hygromycin) and salt (NaCl
250mM) tolerance tests at the seedling level. The select lines exhibited superior
phenotype under salinity and moisture stress conditions both at seedling and whole plant
levels.
The study revealed that the over expression of CcNF-YB1 and OsSHN1 in rice can
improve seedling vigour when compared to wild type, in addition to imparting tolerance
to salinity and drought stresses. However, there is a need to examine the molecular basis
for differences observed between transgenic and wild type plants under stress and non
stress conditions.

July, 2015 (N. Nataraja Karaba)
Department of Crop Physiology Major advisor
UAS, GKVK, Bengaluru
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ಅ$ೈ(cid:19)ಕ ಒತ(ಡಗಳ(cid:31) ಪ(cid:5)ಮುಖ+ಾದ ಶುಷ/ವ0 (ಬರ) ಉಷ3ವಲಯದ (cid:24)ೆ(cid:20)ೆ ಉ5ಾ6ದ"ೆಯ ಕುಂ8ತ9ೆ/
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ಅCವೃEFಪG(cid:9)9ೊಂGದುF, ಅವ0ಗಳ(cid:31) (cid:17)ೕವ9ೋಶಗಳ ಸHಷು35ೆ ಪ(cid:5)ಮುಖ+ಾದು;ಾF(cid:21);ೆ.
ಬಹುAಾಂB(cid:5)ಕ+ಾದ (cid:17)ೕವ9ೋಶಗಳ ಸHಷು35ೆಯನು= ಅ"ೇಕ ವಂಶ+ಾHJಗಳ(cid:15) JಯಂB(cid:5)ಸುತ(+ೆ.
ಆದ(cid:23)Lಂದ ಸೂಕ(+ಾದ JಯಂB(cid:5)ಕ ವಂಶ+ಾHJಗಳನು= ಅCವ(cid:13)ಕ((cid:28)ೊ*(cid:9) ಅ"ೇಕ 9ೆಳವಂಶ+ಾHJಗಳನು=
JಯಂB(cid:5)ಸುವ0ದು ಉತ(ಮ+ೆಂದು Mಾ(cid:19)ಸNಾ(cid:21);ೆ.
ಪ(cid:5)ಸು(ತ ಅಧ(cid:13)ಯನದ(cid:31) ಅBೕಯ+ಾ(cid:21) ಅCವ(cid:13)ಕ((cid:28)ೊ*(cid:9)ದ Jಯಂತ(cid:5)ಕ ವಂಶ+ಾHಗ(cid:20)ಾದ ಹು(cid:31) ನ CcNF-
YB1 Pಾಗೂ ಭತ(ದ (;ೊGR(cid:28)ಾ) OsSHN1 ಗಳನು= ಪ(cid:5)5ೆ(cid:13)ೕಕ+ಾ(cid:21) PೊಂEದ ರೂSಾಂತರ ಭತ(ದ (AC
39020) Tೌಲ(cid:13)Tಾಪನ TಾಡNಾ(cid:21);ೆ. ಎರಡ"ೇ (T2) Vೕ*(cid:28)ೆಯ ಉತ(ಮ (cid:21)ಡಗಳನು= ಆW/TಾG T3
ಮತು ( T4 Vೕ*(cid:28)ೆಯ(cid:31) ಅವ0ಗಳನು= ಹ+ಾTಾನ JಯಂB(cid:5)ತ (cid:9)(cid:16)Bಯ(cid:31) ಪ(cid:5)B(cid:17)ೕ(cid:19)ಕ (Hygromycin)
Pಾಗೂ ಲವಣ ( NaCl 250mM) ಸHಷು35ೆಯ ಪLೕXೆಗಳನು= ಸ(cid:9) ಹಂತದ(cid:31) TಾಡNಾ(cid:21);ೆ. ಆW/
TಾGದ ಸ(cid:9)ಗಳ(cid:15) ಲವಣ Pಾಗೂ JೕLನ ಅMಾವದ ಸHಷು35ೆಯನು= PೊಂEದುF, ಉತ(ಮ YಾLೕLಕ
(cid:24)ೆಳವ(cid:27)(cid:28)ೆಯನು= ಸ(cid:9) Pಾಗೂ (cid:21)ಡದ ಹಂತದ(cid:31) ವ(cid:13)ಕ(ಪG(cid:9)+ೆ.
ಈ ಅಧ(cid:13)ಯನವ0 CcNF-YB1 Pಾಗೂ OsSHN1 JಯಂB(cid:5)ತ ವಂಶ+ಾHಗಳನು= ಅCವ(cid:13)ಕ(ಪGಸುವ0ದLಂದ
ಸ(cid:9)ಗಳ ಲವಣ Pಾಗೂ ಶುಷ/5ೆ ಸHಷು35ೆ ಗುಣವನು= Pೆ[\ಸುವ0ದರ $ೊ5ೆ(cid:28)ೆ ಸ(cid:9) ಹಂತದ ಚುರುಕು
ವೃEFಸುತ(+ೆ ಎಂಬುದನು= (cid:6)ಾ^ೕತುಪG(cid:9);ೆ. ಆದ_ೆ ಒತ(ಡ Pಾಗೂ ಸಹಜ ಸಂದಭaಗಳ(cid:31) ರೂSಾಂತರ
ಮತು (ರೂSಾಂತರವಲ ದ (cid:21)ಡಗಳ ನಡು(cid:19)ನ ವ(cid:13)5ಾ(cid:13)ಸದ ಬ(cid:28)ೆb ಅಣು $ೈ(cid:19)ಕ ಅಧ(cid:13)ಯನದ ಅಗತ(cid:13)(cid:19);ೆ.

ಜುNೈ, ೨೦೧೫
(cid:24)ೆ(cid:20)ೆ ಶLೕರ g(cid:5)AಾYಾಸh (cid:19)Mಾಗ, (ಎj. ನಟ_ಾಜ ಕರಬ)
ಕೃm (cid:19)ಶn(cid:19);ಾ(cid:13)Jಲಯ, (cid:24)ೆಂಗಳoರು- ೫೬೦೦೬೫ ಪ(cid:5)qಾನ ಸಲPೆ(cid:28)ಾರರು

CONTENTS

Sl. No. TITLE PAGE No.
I INTRODUCTION 01-03
II REVIEW OF LITERATURE 04-11
III MATERIAL AND METHODS 12-20
IV RESULTS 21-26
V DISCUSSION 27-30
VI SUMMARY 31-32
VII REFERENCE 32-47
APPENDIX 48-53
LIST OF TABLES

Sl. No. Title Page No.
Different stresses and treatment concentrations used
13
1 to standardize abiotic stress assay to screen transgenic
plants of rice