Physiological and Genetic Diversity Studies on Regeneration of Santalum Album L.

174 Pages·2011·3.64 MB·English

PHYSIOLOGICAL AND GENETIC DIVERSITY 
STUDIES ON REGENERATION OF 
SANTALUM ALBUM L. 
THESIS 
SUBMITTED TO 
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY 
IN PARTIAL FULFILMENT OF THE DEGREE OF 
DOCTOR OF PHILOSOPHY 
IN ENVIRONMENTAL BIOTECHNOLOGY 
UNDER THE FACULTY OF 
ENVIRONMENTAL STUDIES 
RAMYA. R 
KERALA FOREST RESEARCH INSTITUTE 
PEECHI, THRISSUR ­ 680653, KERALA, INDIA 
JULY 2010
CERTIFICATE 
This  is  to  certify  that  the  thesis  entitled,  “PHYSIOLOGICAL  AND  GENETIC 
DIVERSITY  STUDIES  ON  REGENERATION  OF  SANTALUM  ALBUM  L.” 
embodies the result of original work carried out by Ms. Ramya. R (Reg. No. 2701), 
under our guidance and supervision. No part of this thesis has previously formed the 
basis for the award of any degree, diploma, associateship or other similar titles of this or 
other institutes or any University or Society. 
Supervising Guide  Co­supervising Guide 
Dr. M. Balasundaran  Dr. Jose Kallarackal 
Former Scientist F & Programme Coordinator  Emeritus Scientist, CSIR 
Forest Genetics and Biotechnology Division  Kerala Forest Research Institute 
Kerala Forest Research Institute  Peechi­680653, Thrissur, Kerala 
Peechi­680653, Thrissur, Kerala
DECLARATION 
I  hereby  declare  that  the  thesis  entitled  “PHYSIOLOGICAL  AND  GENETIC 
DIVERSITY  STUDIES  ON  REGENERATION  OF  SANTALUM  ALBUM  L.”, 
submitted for the degree of Doctor of Philosophy in Environmental Biotechnology 
under the  Faculty  of  Environmental  Studies  of  Cochin  University  of  Science  and 
Technology is my original work and has not previously formed the basis for the award 
of any degree, diploma, associateship, fellowship or other similar titles or recognition. 
Peechi 
28­07­2010  Ramya. R
ACKNOWLEDGEMENTS 
I express my profound sense of gratitude to my Guide and teacher Dr. M. Balasundaran, 
Former Scientist F and Programme Co­ordinator, FG & B Division, KFRI, for his efficient 
guidance, constant encouragement, and support during the entire period of work at KFRI and 
in the preparation of the thesis. I thank my Co­Guide Dr. Jose Kallarackal, Emeritus Scientist, 
CSIR, KFRI for his efficient guidance, valuable advice and support. 
I thank Dr. R. V. Varma, former Chairman, Ph.D Programme Advisory Committee, 
Dr. E. A. Jayson, present Chairman, Ph.D Programme Advisory Committee and  Dr. K. 
Swarupanandan, Research Co­ordinator for their support. I thank Dr. J. K. Sharma, former 
Director and Dr. K. V. Sankaran, present Director KFRI for their support and for permitting me 
to use the laboratory facilities of this Institute. My sincere thanks to Kerala State Council for 
Science,  Technology  and  Environment  (KSCSTE)  for  the  award  of  KSCSTE  Research 
Fellowship in Botany. 
I thank Dr. T. B. Suma, Scientist, SCMS, Cochin for her valuable advice and support 
during my work. I thank my colleagues Dr. Sreekanth. P. M, Dr. Mrs. Haseena. A, Mrs. 
Anupama. C, Mr. Nishad, V. M, Mr. Arun Sebastian, Ms. Divya Varghese Pattath, Mrs. Arathy. 
K, Mrs. Brinda, C. M, Mrs. Shilpa Mathew and Ms. Sheena. K. for their valuable help and 
support. I thank all fellow co­workers at KFRI who have rendered their assistance and timely 
help during the period of my work. I thank Mr. M. M. Roy for the help rendered to computerize 
my thesis. I thank Mr. K. Mohandas, and Ms. T. G. Chandrika for their help and assistance 
during the period of nursery studies at KFRI nursery. I especially thank Rev. Sr. Mary Ann, SIC, 
Sisters  and hostel mates at  Bethany Convent,  Vilanganoor,  Peechi  for  their support and 
prayers. 
I indeed owe a lot to my husband and both of our families for their moral support, constant 
encouragement and prayers. I bow before God, without his grace nothing would have been 
possible. 
Ramya. R
CONTENTS
LIST OF TABLES v
LIST OF FIGURES vii
ABBREVIATIONS x
1. INTRODUCTION 1
1.1. Sandal and its importance 1
1.2. Distribution of sandal 3
1.3. Studies conducted on sandal 4
1.4. Problems facing sandal growth 4
1.4.1. Lack of regeneration 4
1.4.2. Low seed setting in seed stands 5
1.4.3. Low seed germination and seedling mortality 6
1.4.4. Shade requirement 7
1.4.5. Host plant requirement 7
1.5. Objectives of the present study 8
2. REVIEW OF LITERATURE 9
2.1. Sandal and its importance 9
2.2. Major problems facing sandal 9
2.2.1. Lack of sandal regeneration 12
2.2.2. Loss of genetic diversity 12
2.2.3. Light/shade requirement at early seedling stage 13
2.2.4. Absence of appropriate host 14
2.2.5. Fungal disease and seedling mortality 14
2.2.6. Clonality and inbreeding in seed stand 14
2.3. Ecophysiology of host-parasite relationship 15
2.3.1. Parasitism 16
2.3.2. Host preference of parasites 16
2.3.3. Host influence on parasites 17
2.3.4. Santalum spp. and their host plants 18
i
2.4. Shade requirement of seedlings 19
2.4.1. Seed reserves and seedling development 19
2.4.2. Chlorophyll fluorescence 21
2.4.3. F /F 22
v m
2.4.4. Photoinhibition 24
2.5. Genetic diversity of trees 25
2.5.1. Methods of genetic diversity study 27
2.5.2. Randomly Amplified Polymorphic DNA 27
2.5.3. Inter Simple Sequence Repeats 28
2.5.4. Genetic diversity of sandal 30
2.6. Sandal seedling mortality and low germination percentage: genetic
diversity of Fusarium 31
2.6.1. The genus Fusarium 31
2.6.2. Diversity of fungal isolates and adaptability 33
2.6.3. Method of study of Fusarium diversity 33
3. AUTOTROPHIC AND PARASITIC PHASE OF SANDAL
SEEDLING GROWTH 36
3.1. Introduction 36
3.1.1. Host dependency of sandal plants 37
3.2. Materials and methods 38
3.2.1. Autotrophic phase of sandal seedlings 38
3.2.2. Host dependency of sandal plants 39
3.2.3. Sturdiness Quotient 40
3.2.4. Dickson Quality Index 40
3.2.5. Physiological measurements 41
3.2.6. Chlorophyll content estimation 42
3.3. Results 43
3.3.1. Autotrophic phase of sandal seedlings 43
3.3.2. Host dependency of sandal plants 43
3.3.3. Chlorophyll content estimation 52
3.4. Discussion 59
3.5. Conclusion 62
ii
4. EFFECT OF SHADE ON MORPHOLOGY, CHLOROPHYLL
CONCENTRATION AND CHLOROPHYLL FLUORESCENCE OF
SANDAL SEEDLINGS 63
4.1. Introduction 63
4.2. Materials and methods 64
4.2.1. Plant material 64
4.2.2. Shade treatments 65
4.2.3. Morphological measurements 66
4.2.4. Experimental design and statistical analysis 67
4.3. Results 67
4.4. Discussion 77
4.5. Conclusion 79
5. GENETIC DIVERSITY OF FUSARIUM OXYSPORUM Schlechtend
Fr ISOLATES CAUSING SANDAL SEEDLING WILT 80
5.1. Introduction 80
5.1.1. Fusarium wilt of sandal 80
5.1.2. Variability and spore formation in Fusarium 80
5.1.3. Genetic variability study of pathogenic fungi 81
5.1.4. Genetic variability of F. oxysporum 81
5.1.5. Objective of the present study 82
5.2. Materials and methods 82
5.2.1. Fungal isolates 82
5.2.2. Total genomic DNA extraction and purification 84
5.2.3. RAPD primers and PCR amplification 85
5.2.4. Data analysis 86
5.3. Results 87
5.4. Discussion 91
5.5. Conclusion 92
6. STUDIES ON GENETIC DIVERSITY OF SANDAL IN SEED STAND 93
6.1. Introduction 93
6.1.1. Establishment of seed stands 93
6.1.2. Poor seed setting in seed stand 94
iii
6.1.3. Importance of genetic diversity in seed stand 95
6.1.4. Clonality in sandal forests and its effect on seed production 95
6.1.5. Tools to study genetic diversity in natural forest and seed stands 95
6.1.6. ISSR marker as a tool to study genetic diversity 96
6.1.7. Objective of the present study 96
6.2. Materials and methods 96
6.2.1. Plant material 96
6.2.2. Genomic DNA extraction 99
6.2.3. ISSR primers and PCR amplification 100
6.2.4. Data analysis 101
6.3. Results 101
6.4. Discussion 108
6.5. Conclusion 109
7. SUMMARY 111
7.1. Autotrophic and parasitic phase of sandal seedling growth 112
7.2. Effects of shade on morphology, chlorophyll concentration and
chlorophyll fluorescence of sandal seedlings 113
7.3. Genetic diversity of Fusarium oxysporum Schlechtend Fr isolates
causing sandal seedling wilt 114
7.4. Studies on genetic diversity of sandal in seed stand 115
7.5. Conclusions 116
REFERENCES 118

iv
LIST OF TABLES
Page
Table No. Title
No.
Autotrophic phase of nutrition during developmental stages of
Table 3.1. 43
sandal seedling after germination
Final growth parameters of sandal plants with annual hosts
Table 3.2. 45
(mean ± SE)
Final growth parameters of sandal plants with perennial hosts
Table 3.3 45
(mean ± SE)
ANOVA-Final growth parameters of sandal plants with annual
Table 3.4. 46
hosts
Evaluation of annual host species’ growth based on root:shoot
Table 3.5. 47
ratio (mean ± SE)
Evaluation of perennial host species’ growth based on
Table 3.6. 47
root:shoot ratio (mean ± SE)
ANOVA - Evaluation of annual host species based on root
Table 3.7. 48
fresh weight, shoot fresh weight and root:shoot ratio
Fv/Fm of sandal grown along with best annual hosts (mean ±
Table 3.8. 49
SE)
Fv/Fm of sandal grown along with best perennial hosts (mean
Table 3.9. 51
± SE)
Performance Index of sandal with and without annual host
Table 3.10. 51
plants (mean ± SE)
Performance Index of sandal with perennial host plants (mean
Table 3.11. 52
± SE)
Chlorophyll content of sandal leaves grown with two annual
Table 3.12. 52
host plants (mean ± SE)
Chlorophyll content of sandal leaves grown with two perennial
Table 3.13. 52
host plants (mean ± SE)
Height (cm) of sandal seedlings under four shade treatments
Table 4.1. monitored for six months from two months after germination 68
(mean ± SE)
v
ANOVA-Height of sandal seedlings under four shade
Table 4.2. treatments monitored for six months from two months after 69
germination
Final morphological characteristics of sandal seedlings under
Table 4.3. 69
three shade treatments (mean ± SE)
ANOVA-Final morphological characteristics of sandal
Table 4.4. 70
seedlings under three shade treatments
Mortality percentage of sandal seedlings under four shade
Table 4.5. treatments monitored for six months from two months after 71
germination
Effect of shade on chlorophyll fluorescence (F /F ) of four
v m
Table 4.6. shade treatments monitored for five months from three months 72
after germination
ANOVA-Variation in F /F under four shade treatments
v m
Table 4.7. monitored for five months from three months after 73
germination
Performance Index (PI) of sandal seedlings under four shade
Table 4.8. treatments monitored for five months from three months after 75
germination
ANOVA-Variation in PI under four shade treatments
Table 4.9. monitored for five months from three months after 75
germination
Effect of shade on total chlorophyll content of sandal
Table 4.10. 76
seedlings under four shade treatments
Table 5.1. Primers used for RAPD analysis and their sequences 86
Table 5.2. Number of RAPD products from six isolates of F. oxysporum 90
Nei’s (1978) genetic distance coefficients from six different
Table 5.3. 90
F. oxysporum isolates
Table 6.1. Girth of sandal trees growing in the experimental plots 99
Primers used for the study and size range of amplicons
Table 6.2. 100
generated from S. album DNA
Table 6.3. Dice genetic similarity coefficients for 17 sandal trees in plot I 106
Table 6.4. Dice genetic similarity coefficients for 17 sandal trees in plot II 106

vi