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In vitro culture of Camptotheca acuminata (Decaisne) in Temporary Immersion System (TIS) [Elektronische Ressource] : growth, development and production of secondary metabolites / prepared by Yantree Devi Sankar-Thomas

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Ajouté le : 01 janvier 2009
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In vitro culture of Camptotheca acuminata (Decaisne)
in Temporary Immersion System (TIS): Growth,
development and production of secondary
metabolites




DISSERTATION

A thesis submitted for the degree of Dr. rer.nat. (rerum naturalium)
to the Biology Department,
the Faculty of Mathematics, Informatics and Natural Sciences,
University of Hamburg

prepared by

Yantree Devi Sankar-Thomas
Republic of Guyana

2009





This thesis is dedicated to my
husband and two children
Table of Contents

Page
I. ABSTRACT I
II. Zusammenfassung IV

1. INTRODUCTION 1
1.1. Aims of this Study 3

2. LITERATURE OVERVIEW 5

2.1. Camptotheca acuminata 5
2.1.1. Botany 5
2.1.2. Geographical Distribution 6
2.1.3. Ecology 7

2.2. Camptothecin (CPT) 8
2.2.1. Plant Secondary Metabolites 8
2.2.2. Camptothecin and its Pharmacological Effects 8
2.2.3. Semi-Synthetic Derivatives of Camptothecin 9
2.2.4. Biosynthetic Pathway of Camptothecin 10

2.3. Plant Micropropagation 12
2.3.1. Temporary Immersion System (TIS) 13

173. MATERIAL AND METHODS

3.1. Plant Material 17
3.1.1. Cuttings and Seeds 17

183.2. Plant Culture Media
3.3. Culture Vessels 19
3.3.1. Temporary Immersion System (TIS) Vessels 19
203.4. Sterilisation
3.4.1. Culture Apparatus, Glassware and Equipment 20
3.4.2. Nutrient Media and Substrates 21
3.4.3. Sterilisation of Plant Material 21

213.5. Growth Chambers
3.5.1. Illumination and Temperature 21

3.6. Plant Propagation Methods of C. acuminata 22
3.6.1. Ex Situ Seed Germination 22
3.6.2. Conventional Propagation via Stem Cuttings 23
3.6.3. In vitro Seed Germination 24
3.6.4. Micropropagation via Apical and Axillary Buds 24

3.7. Plant Regeneration via Organogenesis and Embryogenesis 25
3.7.1. Callus Induction in Solid and in Liquid Media 25Table of Contents

3.7.2. Embryogenic Callus Induction in PGR-free Media in TIS 26
3.7.3. Plant Regeneration via Organogenesis in Solid and Liquid Media 27
3.7.4. Plant Regeneration via Somatic Embryo in TIS 28
3.7.5. omabryo on Sterilised Substrates 28

283.8. Shoot Multiplication on Solid Medium and in TIS
3.8.1. Multiple Shoot Induction in Solid Media 28
®3.8.2. le Shn in TIS (DVS and RITA) 29

3.9. Rooting and Acclimatisation 29
3.9.1. In vitro and Ex vitro Rooting 29
3.9.2. Acclimatisation to Greenhouse Conditions 30

3.10. Camptothecin Extraction and Sample Preparation 30
3.10.1. Callus, Greenhouse Plants, Shoots Grown in TIS and Solid Media 30
3.10.2. Cell Suspension Cultures 31
3.10.3. Embryogenic Callus and Different Stages of Somatic Embryos 31
3.10.4. CPT Excretion into Liquid Culture Media 31
3.10.5. CPT Recovery Test 31

313.11. HPLC Analysis of Camptothecin (CPT)
3.12. Plant Selection According to their CPT Content 32
3.13. Fresh and Dry Weight Measurements 32
333.14. Leaf Area
3.15. Chlorophyll Extraction 33
3.16. Determination of Stomata Density 34
3.17. Maintenance of Embryogenic Callus 34
3.17.1. Suspension Cultures and in Solid Media 34
3.17.2. Cell Viability Test with Fluorescein Diacetate (FDA) 34
3.18. Statistical Analysis 35

4. RESULTS 36

4.1. Plant Propagation Methods of C. acuminata 36
4.1.1. Ex situ Seed Germination 36
4.1.2. Conventional Propagation via Stem Cuttings 37
4.1.3. In vitro Seed Germination 38
4.1.4. Micropropagation via Apical and Axillary Buds 40

4.2. Plant Regeneration via Organogenesis and Embryogenesis 42
4.2.1. Embryogenic Callus Induction in Solid Media 42
4.2.2. Plant Regeneration via Organogenesis in Solid Media 43
4.2.3. Embryogenic Callus Induction in Liquid Media 45
4.2.4. Plant Regeneration via Organogenesis in Liquid Media 47
4.2.5. Somatic Embryogenesis Induction in PGR-free Medium in TIS 49
4.2.6. Plant Development via Cotyledonary Embryo in TIS 50Table of Contents

4.2.7. Plant Conversion via Cotyledonary Embryo in Sterilised 52
Substrates

4.3. Shoot Multiplication in Solid Media and in TIS 54
4.3.1. Shoot Multiplication in Solid Media 55
4.3.1.1. Shoot Height in Solid Media 57
4.3.1.2. Shoot Fresh Weight in Solid Media 58
4.3.2. Shoot Multiplication in TIS 60
®4.3.2.1. Multiple Shoot Development in DVS and RITA 62

684.4. Rooting and Acclimatisation
4.4.1. In vitro Rooting in Different Culture Systems 68
4.4.2. Ex vitro Rooting of Microcuttings in Non-sterile Substrates 72
4.4.3. Acclimatisation to Greenhouse Conditions 73

754.5. Maintenance of Embryognenic Calli

4.6. Camptothecin (CPT) distribution in Plant Organs, Tissues and 78
Liquid Media
4.6.1. CPT Comparison between Ex situ and In vitro Seedlings 78
4.6.2. CPT Content in Different Stages of Somatic Embryos 79
4.6.3. CPT Content in Shoots Grown in Solid Media 80
4.6.4. CPT Content in Shoots Grown in TIS Vessels 81
4.6.5. CPT Content in Acclimatised Plantlets 83
®4.6.6. CPT Excretion in Liquid Culture Media in DVS and RITA 84
4.6.7. CPT Content in Callus and Cell Suspension Culture 87

4.7. Plant Selection According to their Camptothecin Content 89

5. Discussion 90
5.1. Plant Propagation Methods of C. acuminata 90
5.2. Plant Regeneration via Organogenesis 93
5.3. Plant Regeneration via Somatic Embryogenesis in TIS in 95
Sterilised Substrates

5.4. Shoot Multiplication on Solid Media and in TIS 97
5.5. Rooting and Acclimatisation in the Greenhouse 98
5.5.1. In vitro Rooting in TIS, in Solid Medium and Sterilised Substrates 98
5.5.2. Ex vitro Rooting of Microcuttings in Non-sterile Substrates 100
5.5.3. Acclimatisation under Greenhouse Conditions 101

5.6. Camptothecin (CPT) distribution in Plant Tissues, Organs and in 102
Liquid Media
5.6.1. Ex situ and In vitro Seedlings 102
5.6.2. Different Stages of Somatic Embryos 104
5.6.3. CPT Content in Shoots Grown in Solid Media, TIS and 104
Acclimatised Plants
5.6.4. CPT Excretion into Liquid Culture Media 106
Table of Contents

1075.7. Plant Selection According to their CPT Content

6. REFERENCES 109

7. APPENDIX 124
7.1. List of Abbreviations 124
7.2. List of Figures 126
7.3. List of Tables 133
1348. LIST OF ORIGINAL PAPERS

ACKNOWLEDGEMENTS 135

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