metabolite profile of camptothecin and its related alkaloids during seed development, seed

135 Pages·2015·4.74 MB·English

METABOLITE PROFILE OF CAMPTOTHECIN
AND ITS RELATED ALKALOIDS DURING
SEED DEVELOPMENT, SEED GERMINATION
AND ITS ROLE IN SEED DEFENSE

SARIKA GOWDIPERU
PALB 1083

DEPARTMENT OF SEED SCIENCE AND TECHNOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
G.K.V.K CAMPUS, BANGALORE 560 065
2015
METABOLITE PROFILE OF CAMPTOTHECIN
AND ITS RELATED ALKALOIDS DURING
SEED DEVELOPMENT, SEED GERMINATION
AND ITS ROLE IN SEED DEFENSE

SARIKA GOWDIPERU
PALB 1083

Thesis submitted to the
UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE
in partial fulfillment of the requirements
for the award of the Degree of
Doctoral Degree of Science (Agriculture)
in
SEED SCIENCE AND TECHNOLOGY

BANGALORE MAY, 2015
Affectionately
Dedicated to
My Beloved
Parents and My
Husband

ACKNOWLEDGEMENT
First of all I would thank God Almighty for His grace and mercy which made it
possible for me to complete this present venture.
The moment has come to look into the deeper layer of heart, which is filled with
the feelings of togetherness, loveliness, consolation and satisfaction, a sign of relief and
sense of fulfillment. Some are momentary and some are permanent, but both involve a
number of near and dear persons to whom I acknowledge my warm regards and take this
opportunity to express my feelings during the course of my research and it is putting
down in black and white.
At the top of all, I take this opportunity to convey my deep sense of gratitude,
reverence and indebtedness to Dr. G.V. BASAVARAJU, Professor, Department of Seed
Science and Technology and Chairman of my Advisory Committee, for his meticulous
guidance, persistence encouragement, generosity, thoughtfulness and timely advice
extended till the last minute of my research as well as throughout the period of my study
in the department.
I equally avail this opportunity to express my deep sense of gratitude to
Dr. R. UMASHAANKER, Professor, Department of Crop Physiology, University of
Agricultural Sciences, Bangalore for his excellent guidance, constant support, concern
and advice throughout the period of my investigations and sustained interest, help and
encouragement in the preparation of the manuscript. I sincerely and honestly confess that
it has been a rare previlage for me to be one of his students.
I would like to extend my thanks to Dr. B.C. CHANNAKESHAVA, Professor and
Former Head, Seed Science and Technology, University of Agricultural Sciences,
Bangalore, valuable suggestions, affectionate gesture extended support throughout my
venture of this study.
I take this opportunity to It is my pleasure to thank Dr. A.R.V KUMAR,
Professor, Dept. of Agricultural Entomology, for his valuable suggestions and guidance
during my investigation.
I express my heartfelt thanks to Dr. N. NATARAJA KARABA, Professor,
Department of Crop Physiology, University of Agricultural Sciences, Bangalore for his
valuable suggestions and guidance throughout my study period.
I wish to express my heartfelt thanks to Dr. P. BALAKRISHNA, Professor and
Head, Seed Science and Technology, University of Agricultural Sciences, Bangalore of
the department for helping me in conducting research work, their suggestions and
encouragement during my course of study.
I extend my thanks to department teachers Mr. R. PARAMESH, Mr. P.
VENKAPPA, Mr. Venugopal Urs, Department of Seed Science and Technology, for
their generous assistance during my study.
My gratitude also goes to DST for the financial support that I received
throughout my Doctoral Programme.
It seems difficult to use the choicest words to measure the boundless love and
tireless sacrifice of some one. I find no much adequate measures to quantify all that to my
beloved Dad Aseervadam.G, Mom Grace Mary, my Husband Johnson, Brother Nirmal
Vandhan(Late) sister Harika and my mother in law Mrs Pusphavathi who always
encouraged me in all walks of my life.
‘Thanks’ is the worst word in friendship yet, I shall avail this opportunity to
express my sincere gratitude to my senior friends Dr. Ravikanth, Dr. Manjunath, Dr.
Rama, Dr. Vasantha Kumari, Suma, Rajani, Sachita, Manasa, Soujanya, Anu,
Chaitanya and Mohan and to my friends Amrutha. N, Madhuri, T., Sravanthi. K.,
Sravani, Shanti for their care, help, encouragement and the things they do in pleasant
friendly ways to me.
Last but not the least, I wish to express my indebtedness to all those whose names
might have been left over but without whose help my thesis would not have been seen the
light of the day.
Bangalore
May, 2015 (SARIKA GOWDIPERU)
Metabolite profiling of camptothecin and related alkaloids
during seed development, seed germination and seed defense
Sarika Gowdiperu
ABSTRACT
Camptothecin, a potent eukaryotic topoisomerase inhibitor, is an
important anticancer drug. With no synthetic source of this alkaloid and
with an increasing global demand, it is imperative that the demand for
camptothecin is met from a sustainable supply rather than the current
destructive harvesting. Seeds of N. Nimmoniana have been reported to
contain substantial amount of CPT. The present work has been
undertaken to study the accumulation pattern of CPT and its related
alkaloids during seed development, seed hydration, seed germination and
early seedling growth of N.nimmoniana. The highest concentration of CPT
was found in the unripe fruits (stage 3, 0.28 %) compared to either the
young seeds or the ripened seeds. Camptothecin and its derivatives in
the plant tissues were analyzed using high performance liquid
chromatography (HPLC) coupled with LCMS in all parts of the seed, and
it was found out that the maximum accumulation of CPT was seen in the
endosperm (0.458 %). During seed germination, the content of CPT
decreased up to seed hydration (0.12 % to 0.08 %) and thereafter it
increased up to 8-week-old seedling (0.08 % to 0.388 %). CPT was shown
to serve as a defense compound and has the ability to inhibit the fungal
pathogens as well as protecting the seeds from storage pests (100% to
4%).

May, 2015
Department of Seed Science and Technology (G.V. Basavaraju)
UAS, GKVK, Bangalore-65 Major Advisor
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PÀȶ «±Àé«zÁ央AiÀÄ, UÁ.PÀÈ.«.PÉÃ.¨ÉAUÀ¼ÀÆgÀÄ-65 ªÀÄÄRå ¸À®ºÉUÁgÀgÀÄ
CONTENTS
CHAPTER TITLE PAGE No.
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-22
III MATERIAL AND METHODS 23-33
IV EXPERIMENTAL RESULTS 34-73
V DISCUSSION 74-86
VI SUMMARY 87-89
VII REFERENCES 90-115
LIST OF TABLES
Table Page
Title
No. No.
1.1. List of plants producing an anticancer alkaloid CPT 2
2.1. CPT analogues that are in clinical use 11
CPT (% dry weight) content in different plant species and 13,14
2.2.
tissues. & 15
CPT and other related alkaloids during fruit development
4.1. 39
in N. nimmoniana
The presence (+) and absence (-) of CPT and other related
4.2. 42
alkaloids during fruit development in N. nimmoniana
CPT and other related alkaloids in different parts of a
4.3. 45
fruit bunch
CPT and related alkaloids during seed germination of N.
4.4. 50
nimmoniana
The presence (+) and absence (-) of camptothecin and
4.5. other related alkaloids at different time intervals of seed 53
germination of N. nimmoniana
Effect of CPT on growth of endophytic fungi isolates of
4.6. 61
N.nimmoniana
4.7. Effect of CPT on the growth of Aspergillus niger 63
4.8. Effect of CPT on the growth of Pencillium notatum 65
Effect of CPT on the growth of seed borne pathogen
4.9. 67
Rhizoctonia solani
Effect of CPT on the growth of seed borne pathogen
4.10. 69
Colletotichum graminicola
Number of eggs laid by Callasobruchus maculates on
4.11. chick pea seeds treated with different concentrations of 71
CPT
Percent adult emergence from eggs laid by
4.12 Callasobruchus maculates on chick pea seeds treated 72
with CPT