TRANSFORMATION OF FINGER MILLET

96 Pages·2016·2.76 MB·English

“IN VITRO REGENERATION AND GENETIC
TRANSFORMATION OF FINGER MILLET
(Eleucine coracana L.) GENOTYPE GN-4”

A
THESIS
SUBMITTED TO THE
NAVSARI AGRICULTURAL UNIVERSITY
NAVSARI

IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR
THE AWARD OF THE DEGREE

OF

MASTERS OF SCIENCE
IN
PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY

BY
DABHI KIRTI ARAJANBHAI
B.Sc. (Biotechnology)

DEPARTMENT OF BIOTECHNOLOGY
N.M.COLLEGE OF AGRICULTURE
NAVSARI AGRICULTURAL UNIVERSITY
NAVSARI- 396 450
GUJARAT
November-2015
Registration No. 04-0955-2011
In vitro regeneration and genetic transformation of finger millet
(Eleucine coracana L.) genotype GN-4
Name of Student Major Advisor
Dabhi Kirti A. Dr. Sanjay Jha

ABSTRACT

Finger millet (Eleucine coracana L.) is an important cereal crop
which constitutes about 81% of the minor millets produced in India. An
efficient in vitro plant regeneration and Agrobacterium tumefaciens
mediated genetic transformation protocol has been described for finger
millet (Eleucine coracana L.) using callus as explants. Among different
cytokinins used, kinetin (1.50 mg/l) in combination with BAP (0.50 mg/l)
produced maximum number of shoots (11.00) and shoot length (9.8 cm).
Maximum frequency of rooting and highest number of roots were
produced on half strength MS basal with NAA (1.0 mg/l).
Agrobacterium mediated genetic transformation, of finger millet
genotype GN-4 was developed by using Agrobacterium Rs-AFP2 strain
carrying marker gene hygromycine phosphotransferase II (hpt II) gene
was used. The integration of the gene was confirmed by PCR. When the
effect of the age of the callus and co-cultivation duration were evaluated,
forty five days old co-cultivated callus for five days yielded 1.33%
frequency of transformation. The morphological features of transgenic
plants did not differ from those of non transgenic plants.
Gujarat Agricultural Biotechnology Institute, Surat
Navsari Agricultural University
Navsari - 396 450

Dr. Sanjay Jha Phone no : 7600059128
Associate Professor
DECLARATION

This is to declare that the whole of the research work
reported in the thesis for partial fulfillment of the
requirements for the degree of Master of Science in PLANT
MOLECULAR BIOLOGY AND BIOTECHNOLOGY by the
undersigned is the results of investigation done by her under
direct guidance and supervision of Dr. Sanjay Jha, Associate
Professor, Gujarat Agricultural Biotechnology Institute , Surat,
Navsari Agricultural University, Navsari and that no part of
the work has been submitted for any other so far.

Place : Navsari

Date : (Dabhi K. A.)

Countersigned by

(Sanjay Jha)

Associate Professor
Gujarat Agricultural Biotechnology Institute ,
Surat, Navsari Agricultural University
ACKNOWLEDGEMENT
Writing this thesis has been fascinating and extremely rewarding. I would
like to thank a number of people who have contributed to the final result in many
different ways:
My deepest gratitude goes to Almighty God who has blessed me
abundantly to complete my research work with success. I give him all glory and
honor for all that he had done for me.
I gratefully acknowledge to work under the competent guidance of my
major advisor Dr. Sanjay jha, Associate Professor, G.A.B.I., NAU, Surat for his
advice, supervision, valuable suggestions and crucial contribution, which made
him a backbone of this research and so to this thesis. His involvement with his
originality has triggered and nourished my intellectual maturity that I will
benefit from, for a long time to come.
From the bottom of my heart, with a deep sense of requite, I owe debts of
thanks to members of my advisory committee Dr. Nilima Karmakar, Assistant
Professor, Department of Biochemistry, Dr. R. K. Parikh, Rtd. Professor,
Department of Agril. Statistics and Dr. Taslim Ahmed, Professor and Head,
Department of Plant Molecular Biology and Biotechnology for their guidance and
words of wisdom which guided me to carry out my research in a meaningful
manner.
I deem it honorable pleasure to thank my teachers Dr. R. M. Patel, Mr.
Vipul Parekh, Dr. Diwakar Singh, Dr. Chintan Kapadiya and Mr. Kiran Suthar for
their constant encouragement and for providing the needed facilities to carry out
my research. I take pleasure to acknowledge the sincere help rendered by staffs
of Department of Plant Molecular Biology and Biotechnology, made me realize
that little acts of kindness often add up to noble deeds.
I am also thankful to Director of Research & Dean P. G. Studies Dr. A. N.
Sabalpara, Principal and Dean of N. M. College of Agriculture, Dr. M. K. Arwadiya
and Principal and Dean of ASPEE College of Horticulture and Forestry, Dr. N. L.
Patel for providing me this valuable and golden opportunity to upgrade my
qualifications in the discipline of Plant Molecular Biology and Biotechnology.
I would also like to thank my seniors and juniors for their moral support
during my research work. This journey was not possible without their guidance
and provide friendly environment in lab.
I would like to sincerely thank them not only for their support but also for
welcoming me openheartedly into their communities. My mission would be
incomplete if I fail to acknowledge my beloved friends, Kailu, Chinka, Ami di,
Unnati, Vrunda, Komal, Abhilasha di, Niki di, Manisha, Dipak, suresh, Amit,
Chaitanya , Reena, Pooja, Kajal who helped me in every possible way to make my
efforts worthwhile. I will always cherish your friendship and the times that we
spent together. My special appreciation goes to Sanchit, Bhumi, Rima, Usha and
Samir for their friendship and encouragement.
I would also like to extend huge, warm thanks to my roommates Anku Di
and Pritam Di for their love, care and moral support. I admire your distinguished
helping nature and motivation.
It’s my fortune to gratefully acknowledge the support of some special one.
Words fail me to express my appreciation to my roommate Swati Di for her
support and generous care. She was always beside me during the happy and hard
moments to push me and motivate me. Thank you for your sincere
encouragement and inspiration throughout my research work and lifting me
uphill this phase of life. Thank you doesn’t seem sufficient but it is said with
appreciation and respect to you for your encouragement, understanding and
precious friendship.
Last but not least, This journey would not have been possible without the
huge love, kindness and endless support I enjoyed from my family, my parents
Mr. Arajan Dabhi and Mrs. Varsha Dabhi and my brother Kashyap who are my
pillars of hope and strength. I thank them for bearing with my long absences
away from home. I deeply appreciate their immense input into what I have
become today. Thank you for your encouragement and help when I needed it
most.

Place: Navsari
Date: 06/11/2015 (Dabhi K. A.)
CONTENTS
Page
Sr. No. Chapters Particulars
No.
1. INTRODUCTION 1-4
2. REVIEW OF LITERATURE 5-15
3. MATERIALS AND METHODS 16-37
4. RESULTS AND DISCUSSION 38-60
5. SUMMARY AND CONCLUSION 61-63
6. REFERENCES I-VI
7. APPENDICES I-VI
LIST OF TABLES
Table Title Page
No. No.
4.1 Effect of surface sterilization agent Mercuric Chloride (HgCl2) 40
on contamination and establishment of finger millet seeds
4.2 Effect of days taken for callus formation 40
4.3 Callus formation from seed of finger millet in presence of 41
different growth regulators
4.4 Effect of different concentration of BAP and Kinetin for 44
multiple shoot regeneration through callus
4.5 Effect of different combination of cytokinin and auxin on shoot 45
culture and root cuture through callus
4.6 Effect of serial subculturing on multiple shoot regeneration 47
through callus
4.7 Effect of auxins and strength of media on induction of in vitro 48
rooting in finger millet
4.8 Effect of different potting mixture on survival of in vitro raised 50
plantlets of finger millet
4.9 Effect of different hygromycin concentrations on the callus of 54
control and transformed finger millet plants
4.10 Effect of age of the explants and co cultivation duration with 57
Agrobacterium on survival percentage of the isolated callus
4.11 Percentage generation of PCR positive plants after 60
transformation
LIST OF FIGURES

Fig. Title Page
No. No.
3.1 Schematic procedure for preparation of MS medium 20
3.2 General procedure followed for in vitro regeneration 21
of finger millet
3.3 A schematic diagram depicting the cascade of events 30
leading to Agrobacterium mediated genetic
transformation into plant.
4.1 Comparative real time PCR result expression of gene from 60
finger millet plant after transformation.