IN VITRO STUDIES ON STEVIA REBAUDIANA FOR STEVIOL GLYCOSIDE PRODUCTION
PRATIBHA GUPTA
CENTRE FOR RURAL DEVELOPMENT AND TECHNOLOGY INDIAN INSTITUTE OF TECHNOLOGY DELHI
HAUZ KHAS, NEW DELHI-110016 (INDIA)
APRIL 2012
©Indian Institute of Technology Delhi (IITD), New Delhi, 2012
IN VITRO STUDIES ON STEVIA REBAUDIANA FOR STEVIOL GLYCOSIDE PRODUCTION
PRATIBHA GUPTA
CENTRE FOR RURAL DEVELOPMENT AND TECHNOLOGY
Submitted
In fulfillment of the requirements of the degree of
DOCTOR OF PHILOSOPHY
to the
INDIAN INSTITUTE OF TECHNOLOGY DELHI HAUZ KHAS, NEW DELHI-110016 (INDIA)
APRIL 2012
Dedicated to
My family
CERTIFICATE
This is to certify that the thesis entitled "In vitro studies on Stevia rebaudiana for Steviol glycoside Production" submitted by Pratibha Gupta has been prepared under our guidance in accordance to the rules and regulation of Indian Institute of Technology Delhi, India. The research report and results presented in thesis have not been submitted for any degree or diploma in any other institute or university.
(Prof. Satyawati Sharma)
Centre for Rural Development and Technology Indian Institute of Technology Delhi
Hauz Khas, New Delhi INDIA
(Dr. Sanjay Saxena)
Micropropagation Technology Park The Energy and Resources Institute (TERI)
TERI Gram, Gual Pahari Gurgaon, Haryana
INDIA
ACKNOWLEDGEMENTS
It gives me great pleasure and moment of immense satisfaction and opportunity for me to express my sense of deep gratitude towards my supervisor Prof. Satyawati Sharma, who has given me wonderful opportunity to do research work under her guidance. I am deeply indebted to her for expert supervision, precious suggestions, constant motivation and invaluable help throughout the course of present work and successful completion of this thesis.
I take this esteemed opportunity in expressing my profound regards to my co- supervisor Dr. Sanjay Saxena (Micropropagation Technology Park, The Energy and Resources Institute (TERI), Gual Pahari, Haryana) for providing me green house facility. I am indebted for his valuable guidance and untiring attention which he bestowed on me right from the inception to the successful completion of this endeavor.
It gives me great pleasure to express my sincerest gratitude to Prof. S. N. Naik (Head), Centre for Rural Development and Technology, IIT Delhi for providing me required facilities and encouragement throughout the completion of research work. I am very much grateful to faculty members, Prof. P. L. Dhar, Prof. Rajendra Prasad, Prof. Santosh Satya, Prof. V. K. Vijay, Dr. V. M.
Chariyar and other faculty members of the centre for their constant encouragement and moral support. I would like to extend my gratitude to the committee members Prof. A. K. Srivastava (Chairperson), Dr. Anushree Malik (Internal expert) and Prof. S. K. Khare (External expert) for their valuable time, comments and expert suggestions during discussions to improve my thesis.
I sincerely thanks to Arbro Pharmaceutical Ltd. Kirti Nagar, Delhi (India) for providing me their lab facilities for HPLC analysis. Without their support I would not have been able to complete my thesis.
I wish to express my cordial thanks to staff members of CRDT, IIT Delhi for their assistance and help related to equipment, information, services and resources.
I owe special thanks to Dr. Kirti Srivastava, Dr. Manoj Goyal, Dr. Seema
Mishra and Dr. Ashutosh Murkute for their valuable suggestions, assistance
and moral support at various stages of my research experiments.
I am indeed very grateful to my seniors and colleagues Dr. Prashant Rout, Dr.
Parvaiz Ahmad, Dr. Kaviraj Pradhan, Dr. Ashwani Kumar, Dr. Ram Chandra Pradhan, Ms. Monica Verma, Ms. Kalpana Arora, Ms. Aditi Gupta, Mr.
Abhishek Sharma, Ms. Nishu Khurana, Ms. Lalita Sharma, Mr. Lalit Mohan Bal, Mr. Narendra Sahoo, Ms. Surbhi Goyal, Ram Kumar and Mewa Lal for their suggestions and help during my experiments.
I am extremely grateful to my friend Mr. Navin Kumar, whose unconditional support, valuable suggestions and encouragement enabled me not only to complete my work in time but has also given a wide horizon and vision for
future.
I am very much grateful to my friends Ms. Priya Sonkar, Dr. Leela Kaur, Mr.
Vivek, Mr. Madhav, Dr. Sukhdeep Kaur, Ms. Neelam Rani, Ms. Mayuri Chabukdhara, Ms. Shruti Trivedi, Ms. Hashmi Fatima, Ms. Anamica Tripathi, and Ms. Shashikala Chauhan and all my friends who always made me feel happy when everything went wrong. Their support and love has major contribution in wading through all the difficult circumstances of my work.
I am grateful to the Department of Science and Technology (DST) for providing me financial help in the form of Young Scientist and Ministry of Human Resource and Development (MHRD) to provide me institute fellowship
from IIT Delhi.
I am forever indebted to my entire family for their encouragement and support. I am very much thankful to my respected parents (Smt. Sursati Devi and Dr. Harish Chandra Gupta) for their endless love and belief in me. The love showered by my brothers (Mr. Kedar Nath Gupta, Mr. Gyan Prakash Gupta and Mr. Ved Prakash Gupta) and Bhabhi's (Ms. Deepmala Sahu and Ms.
Rohini Gupta) always encouraged me to overcome the hardship in my life. It's their love, affection and blessed care that has helped me to move ahead in difficult time and complete my work successfully.
Finally, I thank the almighty God for his blessings which made this thesis possible.
(Pratibha Gupta)
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ABSTRACT
Stevia rebaudiana (Bertoni) has attracted economic and scientific interests due to its sweetness and therapeutic properties present in leaves. The sweetening compounds found mainly in the leaves are Steviol glycosides (SGs), with Stevioside being the most abundant, followed by Rebaudioside A. These SGs are non-fermentative, low-calorific, non-toxic and flavor enhancing sweeteners. This shows the advantages of Stevia over other artificial sweeteners as an ingredient for the food industry, thereby making Stevia a more suitable substitute for saccharine in different drinks, beverages and bakery products. Stevia also offers therapeutic benefits having anti-hyperglycemic, anti-hypertensive, and immuno-modulatory effects. These beneficial effects focused the importance of Stevia, however, plants raised through seeds varied widely due to its heterozygous nature, therefore, affecting the overall Stevioside content.
To avoid such problems and to improve the yield of Stevioside, it is necessary to propagate a genetically homogeneous population from a selected elite plant of desirable characters. For this, plant tissue culture (PTC) is found to have potential alternative approach for the production of advantageous medicinal compounds from plants and cell culture.
Keeping all these facts in consideration, the present study was conducted on "In vitro studies on Stevia rebaudiana for SGs production". The work was focused on; (1) to develop an efficient protocol for micropropagation using axillary branching (2) the development and optimization of culture conditions for callus and suspension culture (3) the effect of elicitors (NaCl, Na2CO3, Proline and Polyethylene glycol (PEG)) on in vitro cultured shoots, callus and suspension culture for their growth (biomass yield i.e., fresh weight and dry weight) and on SGs production (4) evaluation of Stevioside and Rebaudioside A in in vitro cultured shoots, callus and suspension culture using HPLC technique.
For micropropagation studies, the application of growth regulators (cytokinin alone and in combination with auxin) showed a promoting effect on shoot multiplication and rooting. It was noticed that cytokinins especially kinetin (Kn) produced best results. Hence, a protocol was standardized taking different concentrations of Kn (1-10 mgl-1) and it was observed that nodal explants cultured on Murashige and Skoog (MS) medium + 4.0 mgl-1 Kn was the best medium for shoot multiplication. Also, for rooting V2 strength MS medium + 0.05 mgl-1 IBA + 0.05% activated charcoal produced best results. For hardening, mixture of soil and agropeat (3:1) was found to be best. Among different explants (leaves, nodes and in vitro raised roots) tested for callus induction, leaves explant showed better response.
However, combination of auxins (0.75 mgl-1 NAA + 1.0 mgl-1 2,4-D) with MS media were found to be suitable for callus induction while for development and multiplication, MS medium with 2.0 mgl-1 NAA (i.e. maintenance media) showed superior results. Further, suspension culture developed through calli obtained from maintenance media demonstrated good growth followed by S-shaped growth curve.
The effect of different elicitors (NaCl, Na2CO3, Proline and PEG) on in vitro cultured shoots, callus and suspension culture were studied. Stevia shoots showed maximum stress with PEG followed by Na2CO3. However, lower concentrations of NaCl and Proline could be tolerated by explants with shoot growth. A significant reduction in shoot growth (i.e., shoot length, number of shoots/node and multiplication fold) was noticed with increased concentrations of elicitors tested. Similarly, a significant reduction in callus biomass (i.e., fresh weight and dry weight) and growth index (GI) was observed with different concentrations of elicitors tested. However, in case of salts (NaCl, Na2CO3), continuous decline in biomass and GI was observed with their increased concentrations. In contrast, with optimum concentrations of Proline (5 mM) both biomass and GI were increased while at further higher concentrations (?7.5 mM) negative effect was seen. However, in case of PEG, only fresh weight content increased upto 5% PEG, whereas dry weight and GI reduced
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significantly at all concentrations of PEG. In case of suspension culture, all parameters studied (i.e., fresh weight, dry weight and moisture content) were affected with increased concentrations of salts (NaCI and Na2CO3). Proline upto 5 mM concentration increased the fresh weight, dry weight and moisture content and after that negatively affected the results.
Similarly, fresh weight increased upto 5% PEG while at further higher concentrations significant reduction was observed. Also, with all concentrations of PEG moisture content increased while dry weight decreased.
SGs analysis of in vivo and in vitro grown plants, callus and suspension culture (with and without elicitors) was done. In case of field grown plants, maximum production of total SGs (Stevioside and Rebaudioside A) were found in the cultivar procured from Maharashtra (9.26%) followed by Haryana A (8.74%), Haryana B (7.82%) and Uttarakhand (6.33%) plants. Leaves obtained from in vitro shoots showed maximum amount of SGs (2.60%) with 0.025% Na2CO3 followed by 5 mM Proline (1.65%), 0.10% NaCl (1.25%) and 5% PEG (1.15%), which were 3.3, 2, 1.6 and 1.5 times higher than control (0.79%), respectively.
Further, in case of callus, optimum concentrations of each salt i.e., 0.10% NaCl and 0.025%
Na2CO3 produced 5.3 and 5.6 times higher SGs (1.43% and 1.51%) while 2.5 mM Proline and 5% PEG produced 4.0 and 7.0 times higher SGs (1.09% and 1.83%) than control (0.27%), respectively. Similarly, in case of suspension culture, with optimum concentrations of each, 0.10% NaCl and 0.025% Na2CO3, 5 mM Proline and 5% PEG produced 2.61%, 5.14% 5.03% and 6.38% SGs respectively, which were 1.9, 3.8, 3.7 and 4.7 times higher than control, respectively.
Hence, it can be concluded that protocol developed for mass propagation and callusing will be helpful for high frequency regeneration, somaclonal variation and genetic engineering experiments. Also, it was found that the application of abiotic elicitors and appropriate culture conditions can enhance the production of secondary metabolites in in vitro raised Stevia shoots, callus and suspension culture.
V
CONTENTS
ACKNOWLEDGEMENTS i-ii
ABSTRACT iii-v
CONTENTS vi-x
LIST OF FIGURES xi-xiii
LIST OF TABLES xiv-xvi
LIST OF PLATES xvii-xix
ABBREVIATIONS xx
CHAPTER 1: INTRODUCTION 1-11
1.1. Scope of work 8
1.2. Objectives 11
CHAPTER 2: REVIEW OF LITERATURE 12-56
2.1. Stevia plant description and related aspects 12
2.1.1. Botanical distribution and Taxonomy 12
2.1.2. Chemical constituents 14
2.1.3. Physical factors affecting Stevia cultivation 16
2.1.4. Applications of Stevia 18
2.1.5. Biosynthesis of Steviol glycosides 19
2.1.6. Transportation and storage of Steviol glycosides 23
2.1.7. Safety of Stevia sweeteners 24
2.2. In vitro studies of Stevia 25
2.2.1. Micropropagation 25
2.2.2. Callus and suspension culture 31
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2.3. In vitro secondary metabolite production in Stevia and other medicinal plants 34
2.4. Effect of stress on plants, callus and suspension culture on growth
and secondary metabolite production 36
2.4.1. Effect of salts 42
2.4.2. Effect of Proline and Polyethylene glycol 44 2.5. Biochemical analysis for SGs production in Stevia shoots, callus and suspension
culture 51
2.5.1. Extraction of SGs 51
2.5.2. Techniques used for SGs analysis 53
CHAPTER 3: MATERIALS AND METHODS 57-73
3.1. In vitro studies 57
3.1.1. Explants/ Study material 57
3.1.2. Micropropagation studies 57
3.1.3. Callus culture studies 63
3.1.3.1. Callus induction 64
3.1.3.1.1. Callus induction from leaves 64
3.1.3.1.2. Callus induction from nodal explants 64 3.1.3.1.3. Callus induction from root explants 65
3.1.3.2. Callus development! multiplication 65
3.1.4. Suspension culture studies 66
3.1.5. Effect of elicitors (NaCl, Na2CO3, Proline and PEG) on Stevia shoots,
callus and suspension culture 66
3.1.5.1. Micropropagation 66
3.1.5.2. Callus and suspension culture 67
VII
3.1.6. Data analysis 67 3.2. Biochemical analysis for Steviol glycosides (SGs) 70
3.2.1. Extraction procedure 70
3.2.1.1. Extraction of Stevia leaves grown in field for SGs analysis 70 3.2.1.2. Extraction from in vitro leaves, callus and suspension culture 71
3.2.2. Preparation of standard 72
3.2.3. Preparation of mobile phase 72
3.2.4. HPLC analysis 73
3.2.4.1. Chromatographic system 73
3.2.4.2. Analytical condition 73
3.2.5. Quantitative estimation 73
CHAPTER 4: RESULTS AND DISCUSSIONS 74-173
4.1. In vitro studies 74
4.1.1 Development of Micropropagation protocol 74 4.1.1.1. Surface sterilization and culture initiation 74
4.1.1.2. Shoot multiplication 78
4.1.1.2.1. Effect of Cytokinins 78
4.1.1.2.2. Effect of cytokinin and auxin in combination 83
4.1.1.3. Rooting 85
4.1.1.3.1. Effect of IBA on rooting 86
4.1.1.3.2. Effect ofActivated charcoal (AC) on rooting 88
4.1.1.4. Hardening and transplantation 90
4.1.2. Callus and Suspension culture 92
4.1.2.1. Callus induction 93
VIII
4.1.2.1.1. From Stevia leaves 94
4.1.2.1.1.1. Effect ofAuxins 94
4.1.2.1.1.2. Effect of Cytokinin 96
4.1.2.1.1.3. Effect of combination of cytokinins and auxin 97 4.1.2.1.1.4. Effect of combination of auxins 99 4.1.2.1.1.5. Standardization of 2, 4-D and NAA 100
4.1.2.1.2. From nodal explants 101
4.1.2.1.2.1. Effect ofAuxins 101
4.1.2.1.2.2. Effect of cytokinins 103
4.1.2.1.2.3. Effect of cytokinins and auxin combination 104
4.1.2.1.3. From root explants 105
4.1.2.2. Callus development and multiplication 107
4.1.2.2.1. Effect of auxins 107
4.1.2.2. 1.1. Auxins on leaf induced callus 107
4.1.2.2.1.2. Auxins on node induced callus 108
4.1.2.2.1.3. Auxins on root induced callus 109
4.1.2.2.2. Effect of different strength of MS medium 110
4.1.2.3. Suspension culture development 111
4.1.3. Effect of elicitors (NaCl, Na2CO3, Proline and PEG) on Stevia shoots,
callus and suspension culture 113
4.1.3.1. Effect of elicitors on in vitro raised Stevia shoots 114
4.1.3.1.1. Effect of Salts (NaCl and Na2CO3) 114
4.1.3.1.2. Effect of Proline and Polyethylene glycol 120
4.1.3.2. Effect of elicitors on callus culture 125
4.1.3.2.1. Effect of salts (NaCl and Na2CO3) 126
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4.1.3.2.2. Effect of Proline and Polyethylene glycol 130 4.1.3.3. Effect of salts and elicitors on suspension culture 134
4.1.3.3.1. Effect of Salts (NaCl and Na2CO3) 134
4.1.3.3.2. Effect ofProline and PEG 139
4.2. Biochemical analysis for Steviol glycosides (SGs) 144 4.2.1. Steviol glycosides (SGs) in Stevia plants grown in vivo 146 4.2.2. SGs in in vitro raised leaves treated with and without elicitors (NaCl,
Na2CO3, Proline and Polyethylene glycol) 148
4.2.2.1. Effect of Salts (NaCl and Na2CO3) 148
4.2.2.2. Effect ofProline and PEG 151
4.2.3. Production of SGs in callus and suspension culture treated with and
without elicitors (NaCl, Na2CO3, Proline and Polyethylene glycol) 154 4.2.3.1. SGs in callus culture treated with and without elicitors 155 4.2.3. 1.1. Effect of salts (NaCl and Na2CO3) 155
4.2.3.1.2. Effect ofProline and PEG 158
4.2.3.2. SGs in suspension culture treated with and without elicitors 161 4.2.3.2.1. Effect of salts (NaCl and Na2CO3) 164
4.2.3.2.2. Effect of Proline and PEG 166
4.2.4. SGs content in in vivo and in vitro cultured Stevia leaves, callus
and suspension culture 170
CHAPTER 5: SUMMARY, CONCLUSIONS AND FUTURE SCOPE 174-183
REFERENCES 184-229
APPENDICES 232-252
CURRICULUM VITAE
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