In vitro studies on morphogenesis and conservation of some threatened medicinal plants
THESIS
SUBMITTED FOR THE AWARD OF THE DEGREE OF
DOCTOR of Philosophy IN BOTANY
BY
SHIWALI SHARMA
Department of Botany Aligarh Muslim University Aligarh (INDIA) 2011
Plant Biotechnology Lab., Dr. Anwar Shahzad Ph.D. (AMU) Department of Botany,
Assistant Professor Aligarh Muslim University DST Young Scientist Aligarh-202002, UP, India Member Academic Council, [email protected] AMU [email protected]
Dated: ……………….
Certificate
This is to certify that the thesis entitled “In vitro studies on morphogenesis and conservation of some threatened medicinal plants” submitted for the award of the degree of Doctor of Philosophy, embodies the original research work carried out in the Plant
Biotechnology Laboratory at the Department of Botany, Aligarh Muslim University, Aligarh by Ms. Shiwali Sharma under my guidance and supervision and has not been submitted in part or full for the award of any other degree or diploma of this or any other university.
(DR. ANWAR SHAHZAD) (Research Supervisor)
Contact no. +91- 9837061683 Acknowledgement
Begins with the name of GOD who is our creator and sustainer my all praise goes to almighty GOD, the most beneficent and merciful for bestowing and blessing me during the completion of this work. I consider it my right to communicate profound and overwhelmed instinct of gratitude and indebtedness to my esteemed Supervisor Dr. Anwar Shahzad, Assistant Professor, Plant Biotechnology Laboratory, Department of Botany for his insight sagacity, insinuation of belief, compassion, commiseration, critical suggestions, persuasion and continued interest during the present work. Despite his busy schedule, he gave me liberty to encroach his valuable time. It is immense pleasure for me to express my thanks to Prof. M. Anis, the Chairman and Head of Plant Biotechnology Laboratory, Department of Botany for his indispensible guidance and providing all necessary and possible facilities required to conduct the experiments. He has been a constant source of strength and inspiration to me throughout the execution of this study. My special “Vote of Thanks” goes to Prof. Sayeed A. Siddiqui, founder of Plant Tissue Culture & Experimental Embryology Laboratory in our Department for his best wishes from beginning to the completion of this thesis. I wholeheartedly acknowledge my seniors, Dr. Naseem Ahmad (Young Scientist, DST- Fast-Track), Dr. Iram Siddique (RA-CSIR), Ankita Varshney and Anushi A. Jahan for their help and support during the crucial stages of my research work. Continuous encouragement and valuable suggestion I received from my friends, Aastha Sahai (SRF-CSIR), Shahina Parveen (SRF-MANF UGC), Neelu Singh, Kavita Parihar, Nigar Fatima, Ruphi Naaz and Shahnaz Parveen. They always stimulated me to work hard. My colleagues, Taiba Saeed (RA-UPCST) and Arjumend Shaheen (PF-UGC) have striven hard to make the thesis error-free as possible. I am most thankful to both of them for their untiring help and support. In true sense, it will be unjustified if I would not acknowledge my friend, Pratush Raj Singh (B. Tech. & M.Tech., IIT-Delhi) for boosting my moral, strength and being a source of
i never ending inspiration. Without his valuable cooperation at each stride during writing, this task could not have been possible. My sincere and heartiest thanks go to my Parents, brothers, Sachin & Arjun and nephew, Abhishek whose moral support, inspiration and affection sustained me during this work. I sincerely acknowledge various authors and publishers both inside and outside the country, to whom I have referred to in the text. Thus, my sincere gratitude goes to many personalities who inspired me to shape this thesis. Finally, financial support in the form of Research Fellowship in Science under the scheme of UGC, New Delhi for Meritorious student is greatly acknowledged.
Dated: …….. (Shiwali Sharma)
ii
ABBREVIATIONS
ADS : Adenine sulphate ABA : Abscissic acid + NH4 : Ammonium ion BA : 6-Benzyladenine ++ : Ca2 Calcium ion CA : Chlorengenic acid
CaCl2·2H2O : Calcium chloride (dihydrated) CAT : Catalase CH : Casein hydrolysate Chl : Chlorophyll CCC : Chlorocholine chloride
CKOx : Cytokinin oxidase CPPU : 1-(2-chloro-4-pyridyl)-3-phenylurea cm : Centimeter °C : Degree centigrade 2, 4-D : 2, 4-Dichlorophenoxyacetic acid DDW : Double distilled water DPU : Diphenylurea EDTA : Ethylene diamine tetra acetic acid FAA : Formalin acetic acid
B5 : Gamborg’s medium (1968) GA : Gibberellic acid g : Gram Glu : Glutamine g l-1 : Gram per litre h : Hour Ha : Hectare H+ : Hydrogen ion HCl : Hydrochloric acid OH- : Hydroxyl ion IAA : Indole-3-acetic acid IBA : Indole-3-butyric acid
iii
IRGA : Infra Red Gas Analyzer 2-iP : (∆2-Isopentenyl) adenine Km : Kilometer Kn : Kinetin LS : Linsmaier and Skoog’s medium (1965)
HgCl2 : Mercuric chloride µg : Microgram µl : Microlitre µm : Micrometer µM : Micromolar mM : Millimolar mg : Milligram ml : Millilitre mm : Millimeter mg dm-3 : Milligram per decimeter cube mg g-1 : Milligram per gram mg l-1 : Milligram per litre min : Minute M : Molarity MS : Murashige and Skoog’s medium (1962) NAA : α-Naphthalene acetic acid
PN : Net photosynthetic rate NN : Nitsch and Nitsch medium (1969) - NO3 : Nitrate ion N : Normality % : Percentage PAA : Phenyl acetic acid PG : Phloroglucinol PGR : Plant growth regulator PPM : Plant preservative medium Polyox : Polyoxyethyelene glycol PLBs : Protocorm-like bodies RH : Relative humidity SA : Salicyclic acid iv
SH : Schenk and Hilderbrandt’s medium (1972) NaCl : Sodium chloride
AgNO3 : Silver nitrate 2, 4, 5-T : 2, 4, 5-Trichlorophenoxyacetic acid TDZ : Thidiazuron [N-phenyl-(1, 2, 3- thidiazol)-5-ylurea] TRIA : Triacontanol UV : Ultraviolet V : Volt v/v : Volume by volume W : Watt w/v : Weight by volume WPM : Woody plant medium (Lloyd and McCown 1980) YE : Yeast extract
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CONTENT
Page
ACKNOWLEDGEMENT i-ii
ABBREVIATIONS iii-v
CHAPTER 1 INTRODUCTION 1-20
1.1 Biodiversity loss: crisis facing medicinal plants 1.2 Conservation strategies 1.3 Plant tissue culture 1.4 Synseed technology
1.5 Spilanthes acmella (L.) Murr. 1.5.1 Habit 1.5.2 Habitat 1.5.3 Botanical description 1.5.4 Chemical constituents 1.5.5 Medicinal importance 1.5.5.1 Traditional uses 1.5.5.2 Larvicidal and insecticidal activities 1.5.5.3 Antiobesity activity 1.5.5.4 Antifungal activity 1.5.5.5 Transmucosal behavior 1.5.5.6 Antiaging activity 1.5.5.7 Antiinflammatory and analgesic activities 1.5.6 Causes of its extinction and need of micropropagation 1.5.7 Work conducted and lacuna for further research
1.6 Spilanthes mauritiana DC. 1.6.1 Habit 1.6.2 Habitat 1.6.3 Botanical description 1.6.4 Chemical constituents 1.6.5 Medicinal importance 1.6.5.1 Traditional uses 1.6.5.2 Larvicidal and insecticidal activities 1.6.5.3 Antifungal activity 1.6.6 Causes of its extinction and need of micropropagation 1.6.7 Work conducted and lacuna for further research
1.7 Decalepis hamiltonii Wight and Arn. 1.7.1 Habit 1.7.2 Habitat 1.7.3 Botanical description 1.7.4 Chemical constituents 1.7.5 Medicinal importance 1.7.5.1 Traditional uses 1.7.5.2 Antimicrobial activity 1.7.5.3 Insecticidal activity 1.7.5.4 Other properties 1.7.6 Causes of its extinction and need of micropropagation 1.7.7 Work conducted and lacuna for further research
1.8 Objectives
CHAPTER 2 REVIEW OF LITERATURE 21-51
2.1 Organogenesis
2.1.1 Meristem, shoot tip and nodal segment culture 2.1.1.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.1.2 Effect of urea-based cytokinins on shoot regeneration
2.1.2 Leaf culture 2.1.2.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.2.2 Effect of urea-based cytokinins on shoot regeneration
2.1.3 Cotyledon culture 2.1.3.1 Effect of adenine-based cytokinins on shoot regeneration 2.1.3.2 Effect of urea-based cytokinins on shoot regeneration
2.2 Indirect organogenesis
2.3 Other factors influencing regeneration 2.3.1 Effect of different culture media on shoot regeneration 2.3.2 Effect of different carbon sources on shoot regeneration 2.3.3 Effect of different pH on shoot regeneration
2.4 In vitro rooting of microshoots 2.5 Synseed production 2.6 Acclimatization of plantlets
CHAPTER 3 MATERIALS AND METHODS 52-62
3.1 Plant material and explant source 3.2 Culture media
3.3 Preparation of culture medium 3.3.1 Preparation of stock solutions 3.3.2 Plant growth regulators (PGRs) 3.3.3 Carbon and energy sources 3.3.4 pH adjustment and gelling of the medium 3.3.5 Medium filling
3.4 Sterilization 3.4.1 Sterilization of the medium 3.4.2 Sterilization of glass-wares, DDW and instruments 3.4.3 Sterilization of laminar airflow hood 3.4.4 Sterilization of seeds
3.5 Inoculation of sterilized seeds and germination 3.6 Culture establishment and shoot regeneration 3.7 Sub-culturing and shoot proliferation 3.8 In vitro rooting 3.9 Culture room conditions
3.10 Synseed production 3.10.1 Plant material 3.10.2 Encapsulation matrix 3.10.3 Encapsulation and in vitro germination 3.10.4 Low temperature storage 3.10.5 Direct or ex vitro sowing
3.11 Acclimatization of plantlets
3.12 Physiological study 3.12.1 Chlorophyll (a, b and total) and carotenoids content estimation 3.12.1.1 Procedure 3.12.1.2 Estimation
3.12.2 Net photosynthetic rate (PN) estimation
3.13 Histological study 3.13.1 Fixation and storage 3.13.2 Embedding and sectioning 3.13.3 Staining
3.14 Chemicals and glass-wares used 3.15 Statistical analysis
CHAPTER 4 OBSERVATIONS AND RESULTS 63-115
4.1 Spilanthes acmella 4.1.1 Seed germination and collection of explants
4.1.2 Nodal segment culture 4.1.2.1 Effect of adenine-based cytokinins on shoot regeneration 4.1.2.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.1.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.1.3 Shoot tip culture 4.1.3.1 Effect of adenine-based cytokinins on shoot regeneration 4.1.3.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.1.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration 4.1.3.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue 4.1.3.3.2 Effect of different nutrient strengths on shoot proliferation from stock derived culture 4.1.3.4 Effect of different pH on shoot regeneration 4.1.3.5 Effect of different carbon sources on shoot regeneration 4.1.3.6 Effect of different culture media on shoot regeneration
4.1.4 Leaf culture 4.1.4.1 Effect of adenine-based cytokinins on shoot regeneration 4.1.4.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.1.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration 4.1.4.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue 4.1.4.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture 4.1.4.4 Effect of auxin (2,4-D) on callus induction
4.1.5 Cotyledon culture 4.1.5.1 Effect of adenine-based cytokinins on shoot regeneration 4.1.5.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.1.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.1.6 Effect of sub-culturing on shoot proliferation 4.1.7 In vitro rooting of microshoots 4.1.8 Acclimatization of plantlets
4.1.9 Synseed production 4.1.9.1 Effect of Na2-alginate concentration on synseed formation 4.1.9.2 Effect of CaCl2·2H2O concentration on synseed formation 4.1.9.3 In vitro plantlet regeneration from synseeds on culture medium 4.1.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage 4.1.9.5 Acclimatization of plantlets 4.1.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
4.1.10 Physiological study 4.1.10.1 Cholrophyll a, b and total chlorophyll content during acclimatization 4.1.10.2 Carotenoids content during acclimatization
4.1.10.3 Net photosynthetic rate (PN) during acclimatization
4.1.11 Histological study
4.2 Spilanthes mauritiana 4.2.1 Seed germination and collection of explants
4.2.2 Nodal segment culture 4.2.2.1 Effect of adenine-based cytokinins on shoot regeneration 4.2.2.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.2.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.2.3 Shoot tip culture 4.2.3.1 Effect of adenine-based cytokinins on shoot regeneration 4.2.3.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.2.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration 4.2.3.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue 4.2.3.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture 4.2.3.4 Effect of different pH on shoot regeneration 4.2.3.5 Effect of different carbon sources on shoot regeneration 4.2.3.6 Effect of different culture media on shoot regeneration
4.2.4 Leaf culture 4.2.4.1 Effect of adenine-based cytokinins on shoot regeneration 4.2.4.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.2.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration 4.2.4.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue 4.2.4.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture 4.2.4.4 Effect of auxin (2,4-D) on callus induction
4.2.5 Cotyledon culture 4.2.5.1 Effect of adenine-based cytokinins on shoot regeneration 4.2.5.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.2.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.2.6 Effect of sub-culturing on shoot proliferation 4.2.7 In vitro rooting of microshoots 4.2.8 Acclimatization of plantlets
4.2.9 Synseed production 4.2.9.1 Effect of Na2-alginate concentration on synseed formation 4.2.9.2 Effect of CaCl2·2H2O concentration on synseed formation 4.2.9.3 In vitro plantlet regeneration from synseeds on culture medium 4.2.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage 4.2.9.5 Acclimatization of plantlets 4.2.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
4.2.10 Physiological study 4.2.10.1 Chlorophyll a, b and total chlorophyll content during acclimatization 4.2.10.2 Carotenoids content during acclimatization
4.2.10.3 Net photosynthetic rate (PN) during acclimatization
4.2.11 Histological study
4.3 Decalepis hamiltonii 4.3.1 Seed germination and collection of explant
4.3.2 Nodal segment culture 4.3.2.1 Effect of adenine-based cytokinins on shoot regeneration 4.3.2.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.3.2.2.1 Effect of growth additives on shoot proliferation and growth 4.3.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration 4.3.2.4 Effect of different pH on shoot regeneration 4.3.2.5 Effect of different carbon sources on shoot regeneration 4.3.2.6 Effect of different culture media on shoot regeneration
4.3.3 Shoot tip culture 4.3.3.1 Effect of adenine-based cytokinins on shoot regeneration 4.3.3.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.3.3.2.1 Effect of growth additives on shoot proliferation and growth 4.3.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.3.4 Leaf culture 4.3.4.1 Effect of adenine-based cytokinins on shoot regeneration 4.3.4.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.3.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.3.5 Cotyledon culture 4.3.5.1 Effect of adenine-based cytokinins on shoot regeneration 4.3.5.2 Effect of cytokinin-auxin combinations on shoot regeneration 4.3.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
4.3.6 Effect of sub-culturing on shoot proliferation 4.3.7 In vitro rooting of microshoots 4.3.8 Acclimatization of plantlets
4.3.9 Synseed production 4.3.9.1 Effect of Na2-alginate concentration on synseed formation 4.3.9.2 Effect of CaCl2·2H2O concentration on synseed formation 4.3.9.3 In vitro plantlet regeneration from synseeds on culture medium 4.3.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage 4.3.9.5 Acclimatization of plantlets 4.3.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
4.3.10 Physiological study 4.3.10.1 Chlorophyll a, b and total chlorophyll content during acclimatization 4.3.10.2 Carotenoids content during acclimatization
4.3.10.3 Net photosynthetic rate (PN) during acclimatization
4.3.11 Histological study
CHAPTER 5 DISCUSSION 116-139
5.1 Seedling establishment 5.2 Direct organogenesis through shoot tip explants and nodal segments 5.3 Direct organogenesis through leaves and cotyledons 5.4 Indirect organogenesis 5.5 In vitro rooting of microshoots 5.6 Acclimatization of plantlets 5.7 Synseeds development and their germination under in vitro and ex vitro conditions 5.8 Physiological study of acclimatized plantlets 5.9 Histological study of morphogenic tissues
CHAPTER 6 SUMMARY AND CONCLUSIONS 140-150
6.1 Spilanthes acmella 6.2 Spilanthes mauritiana 6.3 Decalepis hamiltonii
REFERENCES 151-202
LIST OF TABLES
Table 1 Distribution of medicinal plants
Table 2 Nutritional composition of different stock solutions for MS medium in mg l-1
Table 3 Nutritional composition of different stock solutions for MS medium
Table 4 Effect of pre-soaking of seeds in GA on in vivo seed germination in S. acmella after 3 weeks of sowing
Table 5 Effect of different GA concentrations on in vitro seed germination in S. acmella after 3 weeks of culture
Table 6 Effect of different cytokinins on shoot multiplication through nodal segments of S. acmella after 4 weeks of culture
Table 7 Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through nodal segments of S. acmella after 4 weeks of culture
Table 8 Effect of different cytokinins on shoot multiplication through shoot tip explants of S. acmella after 4 weeks of culture
Table 9 Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through shoot tip explants of S. acmella after 4 weeks of culture
Table 10 Effect of different concentrations of TDZ on multiple shoot formation through shoot tip explants of S. acmella after 4 weeks of culture
Table 11 Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated shoot tip explants of S. acmella after 4 weeks of transfer
Table 12 Effect of PGRs and PGR-free nutrient media on growth and hyperhydricity of TDZ regenerated shoots through shoot tip explants of S. acmella after 4 weeks of transfer
Table 13 Efficacy of cytokinins on direct shoot organogenesis through leaf explants of S. acmella after 4 weeks of culture
Table 14 Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on shoot multiplication through leaf explants of S. acmella after 4 weeks of culture
Table 15 Effect of different concentrations of TDZ on callus induction through leaf explants of S. acmella after 4 weeks of culture
Table 16 Effect of different combinations of TDZ with BA on shoot induction through TDZ derived leaf calli of S. acmella after 4 weeks of culture
Table 17 Shoot proliferation through TDZ derived leaf calli of S. acmella after 4 weeks of transfer
Table 18 Effect of different concentrations of 2,4-D and BA on callus induction through leaf explants of S. acmella after 4 weeks of incubation
Table 19 Efficacy of cytokinins on direct shoot organogenesis through cotyledons of S. acmella after 4 weeks of culture
Table 20 Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on shoot multiplication through cotyledons of S. acmella after 4 weeks of culture
Table 21 Effect of different concentrations of TDZ on callus formation through cotyledons of S. acmella after 4 weeks of culture
Table 22 Effect of different auxins augmented to half-strength basal MS media on in vitro rooting in S. acmella after 4 weeks of transfer
Table 23 Effect of sodium alginate concentration on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium
Table 24 Effect of calcium chloride concentration on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium
Table 25 Effect of different treatments on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium
Table 26 Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of S. acmella into plantlets
Table 27 Effect of different planting substrates on ex vitro conversion of encapsulated nodal segments of S. acmella into plantlet after 6 weeks of sowing
Table 28 Effect of pre-soaking of seeds in GA on in vivo seed germination in S. mauritiana after 3 weeks of sowing
Table 29 Effect of different GA concentrations on in vitro seed germination of S. mauritiana after 3 weeks of culture
Table 30 Effect of different cytokinins on shoot multiplication through nodal segments of S. mauritiana after 4 weeks of culture
Table 31 Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through nodal segments of S. mauritiana after 4 weeks of culture Table 32 Effect of different cytokinins on shoot multiplication through shoot tip explants of S. mauritiana after 4 weeks of culture
Table 33 Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through shoot tip explants of S. mauritiana after 4 weeks of culture
Table 34 Effect of different concentrations of TDZ on multiple shoot formation through shoot tip explants of S. mauritiana after 4 weeks of culture
Table 35 Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated shoot tip explants of S. mauritiana after 4 weeks of transfer
Table 36 Effect of PGR combination and PGR free nutrient media on growth and hyperhydricity of TDZ treated shoot tip explants of S. mauritiana after 4 weeks of transfer
Table 37 Effect of different cytokinins on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture
Table 38 Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture
Table 39 Effect of different concentrations of TDZ on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture
Table 40 Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated leaf explants of S. mauritiana after 4 weeks of transfer
Table 41 Effect of PGR combination and PGR-free nutrient medium on growth and hyperhydricity of TDZ derived tissue through leaf explants of S. mauritiana after 4 weeks of transfer
Table 42 Effect of different concentrations of 2,4-D and BA on callus induction through leaf explants of S. mauritiana after 4 weeks of incubation
Table 43 Effect of different cytokinins on direct shoot organogenesis through cotyledons of S. mauritiana after 4 weeks of culture
Table 44 Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on direct shoot organogenesis through cotyledons of S. mauritiana after 4 weeks of culture
Table 45 Effect of different concentrations of TDZ on callus formation through cotyledons of S. mauritiana after 4 weeks of culture Table 46 Effect of different auxins augmented to half-strength basal MS medium on in vitro rooting in S. mauritiana after 4 weeks of transfer
Table 47 Effect of sodium alginate concentration on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium
Table 48 Effect of calcium chloride concentration on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium
Table 49 Effect of different treatments on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium
Table 50 Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of S. mauritiana into plantlets
Table 51 Effect of different planting substrates on ex vitro germination of encapsulated nodal segments of S. mauritiana after 6 weeks of sowing
Table 52 Effect of pre-soaking of seeds in GA on in vivo seed germination in D. hamiltonii after 3 weeks of sowing
Table 53 Effect of different GA concentrations on in vitro seed germination of D. hamiltonii after 3 weeks of culture
Table 54 Effect of different cytokinins on shoot induction through nodal segments of D. hamiltonii after 4 weeks of culture
Table 55 Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through nodal segments of D. hamiltonii after 4 weeks of culture
Table 56 Effect of glutamine (Glu), adenine sulphate (ADS) and phlouroglucinol (PG) supplemented with 5.0 µM BA and 0.5 µM IAA on shoot multiplication through nodal segments of D. hamiltonii on MS medium after 4 weeks of transfer
Table 57 Effect of different cytokinins on shoot induction through shoot tip explants of D. hamiltonii after 4 weeks of culture
Table 58 Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through shoot tip explants of D. hamiltonii after 4 weeks of culture
Table 59 Effect of glutamine (Glu), adenine sulphate (ADS) and phlouroglucinol (PG) supplemented with 5.0 µM BA and 0.5 µM IAA on shoot multiplication through shoot tip explants of D. hamiltonii on MS medium after 4 weeks of transfer
Table 60 Effect of PGR, GA and ADS on shoot regeneration through shoot tips derived nodular tissues of D. hamiltonii after 4 weeks of transfer
Table 61 Effect of different cytokinin concentrations on shoot induction through leaf explants of D. hamiltonii after 4 weeks of culture
Table 62 Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through leaf explants of D. hamiltonii after 4 weeks of culture
Table 63 Effect of different auxins concentrations on in vitro root induction in D. hamiltonii after 4 weeks of transfer
Table 64 Effect of sodium alginate concentration on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture on MS medium
Table 65 Effect of calcium chloride concentration on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture on MS medium
Table 66 Effect of different treatments on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture
Table 67 Effect of different NAA concentrations supplemented to half-strength MS basal medium on in vitro root induction in microshoots recovered through synseeds of D. hamiltonii after 4 weeks of incubation
Table 68 Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of D. hamiltonii into plantlets
LIST OF FIGURES
Figure 1 Bio-geographical distribution of medicinal plants
Figure 2 Strategies for biodiversity conservation
Figure 3 Scope of synseeds
Figure 4 A twig of field grown plant of S. acmella showing flower heads
Figure 5 A twig of field grown plant of S. acmella showing infested leaves
Figure 6 A twig of field grown plant of S. mauritiana showing flower heads
Figure 7 Structural modifications in some adenine and urea-based cytokinins
Figure 8 Seed germination in S. acmella
Figure 9-12 Shoot regeneration through shoot tip explants
Figure 13 Effect of different pH of the culture medium on shoot proliferation efficiency of shoot tip explants of S. acmella after 4 weeks of culture
Figure 14 Effect of different carbon sources on shoot proliferation efficiency of shoot tip explants of S. acmella after 4 weeks of culture
Figure 15 Effect of different culture media on shoot proliferation efficiency of shoot tip explants of S. acmella after 4 weeks of culture
Figure 16-18 Shoot regeneration through leaf explants
Figure 19 Callus induction through leaf explants
Figure 20-21 Shoot regeneration through cotyledons
Figure 22 Effect of sub-culture passage on shoot proliferation efficiency of shoot tip explants
Figure 23 Sub-culturing of shoot tip derived tissues
Figure 24 Effect of sub-culture passage on shoot proliferation efficiency of leaf explants
Figure 25 Sub-culturing of leaf derived tissues
Figure 26 Effect of sub-culture passage on shoot proliferation efficiency of cotyledons
Figure 27 Sub-culturing of cotyledon derived tissues
Figure 28 In vitro root induction of microshoots Figure 29 Effect of different planting substrates on survival percentage of regenerated plantlets after 2 months of field transfer
Figure 30 Successfully hardened micropropagted plantlets
Figure 31-32 Synseeds production and germination
Figure 33 Successfully hardened plantlets obtained from synseeds
Figure 34 Change in chlorophyll content (a, b & Total) (mg g-1) during acclimatization
Figure 35 Change in carotenoids content (mg g-1) during acclimatization
-2 -1 Figure 36 Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization
Figure 37-38 Histological study of regenerating tissues
Figure 39 Seed germination in S. mauritiana
Figure 40 Shoot regeneration through nodal segments
Figure 41-44 Shoot regeneration through shoot tip explants
Figure 45 Effect of different pH of the culture medium on shoot proliferation efficiency of shoot tip explants of S. mauritiana after 4 weeks of culture
Figure 46 Effect of different carbon sources on shoot proliferation efficiency of shoot tip explants of S. mauritiana after 4 weeks of culture
Figure 47 Effect of different culture media on shoot proliferation efficiency of shoot tip explants of S. mauritiana after 4 weeks of culture
Figure 48-51 Shoot regeneration through leaf explants
Figure 52 Callus induction through leaf explants
Figure 53-54 Shoot regeneration through cotyledons
Figure 55 Effect of sub-culture passage on shoot proliferation efficiency of shoot tip explants of S. mauritiana
Figure 56 Effect of sub-culture passage on shoot proliferation efficiency of leaf of S. mauritiana
Figure 57 Effect of sub-culture passage on shoot proliferation efficiency form cotyledons of S. mauritiana
Figure 58 Sub-culturing of shoot tip, leaf and cotyledon derived tissues Figure 59 In vitro root induction of microshoots
Figure 60 Effect of different planting substrates on survival percentage of regenerated plantlets of S. mauritiana after 2 months of field transfer
Figure 61 Plantlets transferred in field soil mixed with farmyard showing flowering
Figure 62-63 Synseed production and germination
Figure 64 Successfully hardened plantlets obtained from synseeds
Figure 65 Change in chlorophyll content (a, b & Total) (mg g-1) during acclimatization.
Figure 66 Change in carotenoids content (mg g-1) during acclimatization
-2 -1 Figure 67 Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization.
Figure 68 Histological study of regenerating tissues
Figure 69 Seed germination in D. hamiltonii
Figure 70-72 Shoot regeneration through nodal segments
Figure 73 Effect of different pH of the culture medium on shoot regeneration through nodal segments of D. hamiltonii supplemented with BA (5.0 µM) + IAA (0.5 µM) + ADS (30 µM) after 4 weeks of culture.
Figure 74 Effect of different carbon sources on shoot regeneration through nodal segments of D. hamiltonii supplemented with BA (5.0 µM) + IAA (0.5 µM) + ADS (30 µM) after 4 weeks of culture.
Figure 75 Effect of different culture media on shoot regeneration through nodal segments of D. hamiltonii supplemented with BA (5.0 µM) + IAA (0.5 µM) + ADS (30 µM) after 4 weeks of culture.
Figure 76 Shoot regeneration through shoot tip explants
Figure 77 Regeneration of basal nodular tissue through shoot tip explants
Figure 78 Shoot regeneration through nodular tissue
Figure 79 Leaf culture
Figure 80 Effect of sub-culturing on shoot proliferation through nodal and shoot tip explants of D. hamiltonii.
Figure 81 Effect of sub-culturing on shoot proliferation through shoot tip derived N1 and N2 tissues of D. hamiltonii. Figure 82 In vitro root induction
Figure 83 Acclimatization of micropropagated plantlet
Figure 84 Effect of different planting substrates on survival percentage of regenerated plantlets after 2 months of field transfer
Figure 85 Synseed production and their germination
Figure 86 Change in chlorophyll content (a, b & Total) (mg g-1) during acclimatization
Figure 87 Change in carotenoids content (mg g-1) during acclimatization
-2 -1 Figure 88 Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization
Figure 89 Histological study of regenerating tissues
Figure 90 Flow chart showing overall design of conducted experiments in the present study
Chapter 1 Introduction
Chapter 1
INTRODUCTION
India has a characteristic geographic location at the junction of the three major bio- geographic realms, namely, the Indo-Malayan, the Eurasian and the Afro-tropical (6°45' to 37°6' N latitude and 68°7' to 97°25' E longitude) with a land frontier of about 15,200 km and a coastline of 7,516 km. Its geographical area is 3287.263 sq. km (3,287 million Ha). It is the seventh largest country in the world and second largest in Asia. It is one of the 17 megadiversity countries and has all the 13 biomes found in the world, with two major hotspots out of a total of 34 (Bapat et al. 2008). It has been reputed as the treasure house of a wide range of valuable medicinal and aromatic plants on account of vast diversity in climatic condition (Fig. 1). Most of them used in Ayurvedic, Unani, Siddha, Homeopathic, Allopathic and other alternate medicinal practices such as folk medicine, household remedies, naturopathy, Amchi and Tribal medicines. It is estimated that more than 7500 higher plant species forming about 44% of the higher plant diversity of the country are used in its codified and folk healthcare traditions. This proportion of medicinal plants is the highest proportion of plants known for their medicinal purposes in any country of the world for the existing flora of that respective country (Shiva 1996, Samant et al. 1998, Schippmann et al. 2002) (Table 1).
Table 1. Distribution of medicinal plants
Country or Total number of Number of % of medicinal Source region native medicinal plant plants species in flora species reported World 29,7000 52,885 10 Schippmann et al. (2002) India 17,000 7,500 44 Shiva (1996) Indian 8,000 1,748 22 Samant et al. Himalayas (1998)
Herbs are staging a comeback and herbal renaissance is happening all over the globe. The blind dependence on synthetics is now over and people are returning to the naturals with hope of safety and security. The World Health Organization (WHO) has estimated that at least 80% of the world population relies on traditional systems of
1
Western & N. Western Trans Himalayas Himalayas (1700 sp.) Central & Eastern (700 sp.) Himalayas (1200 sp.)
Coastal (500 sp.)
Semi Arid zone Desert zone (1000 sp.) (500 sp.) MEDICINAL PLANTS
Western Ghat Malabar Bay Islands Coast (2000 sp.) (1000 sp.) Deccan Peninsula (3000 sp.)
North East India Gangetic Plain (2000 sp.) (1000 sp.)
Figure 1. Bio-geographical distribution of medicinal plants
2 medicine for their primary health needs despite the introduction of antibiotics since 1940’s. Moreover, the introduction of a single new synthetic drug into the market would take about 10 to 15 years of time and expenditure in the scale of about US $ 100 to 300 million (Abelson 1990). Plant based drug would take a comparatively much lesser time and expense than synthetic drugs. Hence, plant based medicine would be far inexpensive, unless the market price are inflated by other considerations. Medicinal plants occupy an important place in India not only in the medicinal arena but also in socio-cultural and spiritual areas. The use of traditional medicine and medicinal plants in most developing countries, as a normative basis for the maintenance of good health, has been widely observed (UNESCO 1996). Furthermore, an increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs and chemotherapeutics from these plants as well as from traditionally used rural herbal remedies (UNESCO 1998). The WHO has estimated the present demand for medicinal plants is approximately US $14 billion per year (Sharma 2004). The demand for medicinal plant-based raw materials is growing at the rate of 15 to 25% annually and according to an estimate of WHO, the demand for medicinal plants is likely to increase more than US $5 trillion in 2050. In India, the medicinal plant related trade is estimated to be approximately US $1 billion per year (Joshi et al. 2004). According to an estimate, the quantity of export of Ayurvedic products produced in India has tripled between last two financial years (2001-2002 and 2002-2003). Population rise, inadequate supply of drugs, prohibitive cost of treatments, side effects of several allopathic drugs and development of resistance to currently used drugs for infectious disease have led to increased emphasis on the use of herbal products. Additionally, the indiscriminate use of chemical pesticides has given rise to serious environmental pollution, genetic resistance of pests, toxic residues in stored products and hazards from handling etc. warranted to develop eco-friendly herbal pesticides which would effective even at low concentrations, biodegradable and having broad spectrum activity (Rani and Murty 2006).
1.1 Biodiversity loss: crisis facing medicinal plants
In the light of undeniable importance of herbal products over synthetics, growing demand for herbal product has led to a quantum jump in volume of plant materials traded within and across the countries results in a concomitant increase in the demand for raw
3 material. More than 95% of the 400 plant species used in preparing medicine by various industries are harvested from wild populations in India (Uniyal et al. 2000). Harvesting medicinal plants for commercial use, coupled with the destructive harvest of underground parts of slow reproducing, slow growing and habitat-specific species, are the crucial factors in meeting the goal of sustainability (Kala 2005, Ghimire 2005). Harvesting shoots and leaves of medicinal plants may decline their photosynthetic capacity and as well as the potential for survival and effective propagation. Medicinal plants tolerance to harvest varies with climatic conditions as the temperate herbs become highly vulnerable to harvest of individuals (Ticktin 2004). Furthermore, rising demand with shrinking habitats may lead to the local extinction of many medicinal plant species. India harbors about 47,000 species of plants of which 17,000 are angiosperms (Bapat et al. 2008). A total of 560 plant species of India have been included in the International Union for Conservation of Nature and Natural Resources (IUCN) Red List of Threatened species, out of which 247 species are in the Threatened category. On a global basis, the IUCN has estimated that about 12.5% of the world’s vascular plants, totaling about 34,000 species are under varying degree of threat (Phartyal et al. 2002). However, according to the report of Hamilton (2004) an estimated 4,000 to 10,000 species of medicinal plants face potential local, national, regional or global extinction, with subsequent serious consequences for livelihoods, economies and health care systems.
1.2 Conservation strategies
Conservation of biodiversity is a matter of utmost concern worldwide. As a result efforts are dedicated domestically as well as internationally to protect and conserve existing spectrum of plant species, threatened as well as endangered plants. In India, there are several organizations which are engaged in the protection and preservation of such plants. Some of these organizations are Indian Council of Agricultural Research, New Delhi; National Bureau of Plant Genetic Resources, Pusa Campus New Delhi and Council of Scientific and Industrial Research, New Delhi (Batra 2001). The laudable attempts made (Singh et al. 2006) for biodiversity conservation are as follows (Fig. 2); • In situ strategy • Ex situ strategy • Reduction of anthropogenic pressures • Rehabilitation of endangered species.
4
Single use wild land Strict Nature Reserves. management Wildlife Sanctuaries.
National Parks. In Situ Biosphere Reserves. Multiple uses of wild Grand Forest Parks. Nature Recreation Parks.
Area other than wild Consorted land specially managed for genetic diversity
Reduction in anthropogenic pressures
Conservation
Rehabilitation of endangered species
Botanic Gardens Arboreta Whole organism Botanic Gardens Arboreta
Ex Situ Organ Banks. Seed and Pollen Banks. Gene Banks, Tissue OrganismOrganism partsparts Culture.
Figure 2. Strategies for biodiversity conservation
5 In situ strategy involves the preservation of germplasm in their natural environment by establishing biosphere reserves, national parks, gene sanctuaries etc. In situ preservation facilitates evolution and ecological balance, but it prone to natural calamities and requires a large space. While, ex situ conservation provides a better degree of protection to germplasm compared to in situ conservation. However, both ex situ and in situ conservation are complementary but should not be viewed as alternatives (Wang et al. 1993). Ex situ conservation includes germplasm banks, common garden archives, seed banks, DNA banks and techniques involve tissue culture, cryopreservation, incorporation of diseases, pest and stress tolerance traits through genetic transformation and ecological restoration of rare plant species and their population. In vitro propagation of rare and threatened plants is generally undertaken to enhance the biomass and conserve the germplasm especially when population numbers are low in wild, difficult to regenerate by conventional methods and where population has decreased due to over exploitation by destructive harvesting. The modern technology of micropropagation provides numerous advantages over conventional propagation methods like mass production of true-to-type and disease free plants of elite species in a highly speedy manner irrespective of the season within smaller space and tissue source. Additionally, when species have been over collected by herbalist for medicine, food or fragrance, in vitro propagation can provide an alternative source of plants and alleviate pressures on wild populations. Tissue culture raised plantlets can also be used as a source of seed for long-term storage and if seed is not produced, the tissue culture line themselves can be cryopreserved (Tripathi et al. 2007).
1.3 Plant tissue culture
With the advances in fundamental and applied science, human thinking became more lucid and directed towards conservation of germplasm as well as production of novel eco- friendly plants. Consequently the birth of ‘BIOTECHNOLOGY’ took place – a link between the biological sciences, physical sciences and technological science. Biotechnology finds its major application in the field of agriculture. Improving and increasing global yield of good grains as well as commercially important crops emerged as a major task to be accomplished. In recent years, it has become increasingly important to achieve a target in a relatively short period of time without impairing the quality.
6 To achieve these objectives, tissue culture technology has brought a revolutionary breakthrough and is perhaps the most sought after because of its long term possibilities in rapid production of useful plants and their products throughout the year. Plant tissue culture, an off-shoot of human curiosity has presently become an essential component of plant biotechnology. Through the efforts of pioneers in France, USA, U.K. and Germany, plant tissue culture spread to various parts of the world. It was Maheshwari who started the first tissue culture laboratory in India at Delhi University in the 1950s as foresaw the value of this technique in experimental embryology (Batra 2001). The term “tissue culture” is an umbrella term which covers a spectrum of techniques involving in vitro culture of plants, seeds, plant parts (tissues, organs, embryos, single cells, protoplasts etc.) on nutrient media under aseptic conditions. Its versatile advantages like morphological, physiological and biochemical investigation of cells, investigation of phytopathological problems, raising virus-free individuals, plant breeding, micropropagation, somatic embryogenesis, production of synseeds, somaclonal variation, protoplast isolation, production of secondary metabolites, cryopreservation, genetic engineering etc. has made it a paragon for today’s world (Normah and Makeen 2008, Pandey et al. 2010). Micropropagation is one of the most useful aspects where plant tissue culture technique has found its widest practical application. It refers as the rapid production of large number of identical clones within a short duration in available small space. Bhatt (1997) has also described micropropagation as a rapid and successful technique for asexual propagation of plants. This is generally obtained by in vitro methods involving culturing of meristem, shoot tip culture, stimulation of axillary or lateral meristems or through culture of non- meristematic explants (leaf, petiole, root etc.). The process of micropropagation involves the following four distinct stages; • Stage 0: Culture initiation • Stage I: Shoot multiplication • Stage II: Rooting • Stage III: Acclimatization and transfer of plantlets to field environment The first stage; culture initiation depends on explants type or the donor plant at the time of excision. Explants from actively growing shoots are generally used for mass scale multiplication. The second stage; shoot multiplication is crucial and achieved by using plant growth regulators (PGRs) generally auxin and cytokinin. In the third stage, elongated shoots
7 are subsequently rooted either ex vitro or in vitro. The fourth stage; acclimatization of in vitro grown plants is an important step in micropropagation. Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, due to their sessile nature and long life span, have developed a greater ability to endure extreme conditions and predation than have animals. This plasticity allows plants to alter their metabolisms, growth and development to best suit their environment. Particularly important aspects of these adaptations, as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organ or undergo different developmental pathways in response to particular stimuli. This inherent regenerative capacity of the somatic cells was termed as totipotency by Steward (1968). Tissue culture is a very influential component of plant biotechnology, which has opened an existing frontier in the field of agriculture and offers opportunity for the increase in productivity, profitability, stability, and sustainability (Batra 2001). It has advanced the knowledge of fundamental botany especially in the field of agriculture, horticulture, plant breeding, forestry, somatic cell hybridization, phytopathology and industrial production of plant metabolites (Normah et al. 1997, Batra 2001, Misra and Chakrabarty 2009, Chatterjee et al. 2010). The modern technology of micropropagation provides numerous advantages over conventional propagation methods like mass production of true-to-type and disease free plants of elite species in highly speedy manner irrespective of the season requiring smaller space and tissue source. Thus, it provides a reliable technique for in vitro conservation of various rare, endangered threatened germplasm (Sharma et al. 2010, Krishnan et al. 2011). Plant tissue culture offers tremendous advantages for the conservation of vegetatively propagated plant species with recalcitrant seeds or having with long vegetative period prior to seed set and sterile individuals possessing useful traits. Furthermore, development in genetic engineering with new tools for transferring foreign genes into plants, combined with progress in the identification and isolation of genes have allowed specific alternatives of single traits in already successful variety. Therefore, there is a pre-requisite of the establishment of in vitro micropropagation system to facilitate successful genetic engineering experiment for the development of newer economic variants.
1.4 Synseed technology
8 In order to achieve germplasm conservation through plant tissue culture, it is necessary to reduce the frequency of sub-cultures so as to reduce the chances of contamination. This could be achieved by cryopreservation. The technique of cryopreservation has found its wide applicability for preserving biological materials and has good potential for long-term storage of germplasm and referred as “in vitro based gene bank” (Cho et al. 2002, Tripathi et al. 2007, Normah and Makeen 2008). The long-term storage of plants in liquid nitrogen, without regular sub-cultures allows one to rationalize the production of nuclear stocks and maintains gene collections as storage tissue (Withers and Engelmann 1999) but it cannot be applied to all genotypes. In this regard, tissue culture technique namely, ‘artificial seed’ technology has been first publicly addressed by Murashige in 1977 at a conference, since then different approaches have been actively pursued to make it a viable technology for practical use. Now, this technology has been extended by several research groups throughout the world for a variety of plant species including cereals, fruits, vegetables, medicinal plants, forest trees, orchids and other ornamentals (Mandal et al. 2000, Chand and Singh 2004, Manjkhola et al. 2005, Rai et al. 2008, Roy and Mandal 2008, Utomo et al. 2008, Ahmad and Anis 2010, Ozudogru et al. 2011). The term “artificial seed” which was first coined by Murashige, now is also known by other names like manufactured seed, synthetic seed or synseed. The original definition of an artificial seed as given by Murashige (1978) was “an encapsulated single somatic embryo”. Gray and Purohit (1991) also defined synseed as “a somatic embryo that is engineered for the practical use in commercial plant production”. However, Bapat et al. (1987) proposed the making of synseeds from in vitro derived propagules other than somatic embryos. Later, Pond and Cameron (2003) suggested that the term “artificial seed” can also be referred to un- encapsulated (naked) somatic embryos (either hydrated or desiccated). Encapsulation technology is an exciting and rapidly growing area of plant biotechnology research. It has considerable impact on conservation and delivery of tissue cultured plants in a more economical and convenient way (Rao et al. 1998). The scope of synseeds is presented in Fig. 3. The synseed technology is highly promising for conservation and propagation of valuable rare hybrids, elite genotypes, sterile unstable genotypes and genetically engineered plants for which the seeds are either not available or require mycorrhizal-fungal association for their germination (as in case of orchids).
9 Synseeds
Propagation Conservation Transport Analytical tool
• Cost effective approach for ex • Large scale mono culture • Comparative study aid for • Easy, cost effective disease situ germplasm conservation of rare, endangered, zygotic embryogeny free germplasm genetically engineered • Ensure economy of space, • Determining the role of transportation elite genotype medium, storage period endosperm in embryo • Direct greenhouse and • Protection from environmental • Mixed genotype development and field delivery plantation disaster germination • Germplasm exchange • Extra protection to the explants • Uniform genetic • Seed coat formation between the countries constitution of plantlets against pest and disease due to studies without obligations form presence of antibiotics to the gel • Study of somaclonal quarantine department ti
Short to medium- Long-term storage term storage
Slow-growth Cryopreservation conservation
• Maintenance under reduced • Two-step freezing temperature and/or reduced light • Simple desiccation intensity Encapsulation-dehydration • Use of growth retardants such as • Vitrification ABA • Encapsulation-vitrification • Use of minimal growth medium • Use of osmoticum • Reduction in oxygen concentration • Combination of one than one 10 treatment Figure 3. Scope of synseeds
As the production cost for hybrids or other conventional propagation methods are very high, synseeds offer low cost alternatives. Some other potential advantages of encapsulation technology include ease in handling (due to small size of capsule), genetic uniformity of plants and their direct delivery to field or green house (Maruyama et al. 1997). Synseeds can be made available throughout the year whereas most of tree plants produce natural seeds only in certain months of the year (Bapat and Mhatre 2005). The direct delivery of encapsulated material will save many subcultures to obtain plantlets. Conservation is an important aspect of encapsulation technology. In vitro conservation involves the maintenance of explants in a pathogen-free environment for short to medium or long-term (Engleman et al. 2003). Additionally, many commercially important plants particularly cereals, fruits, medicinally important plants are studied worldwide for breeding, genetic engineering, propagation and pharmaceutical purposes. In this context, the most important application of synseeds for these plants could be in exchange of elite and axenic plant material between countries (Hasan and Takagi 1995, Danso and Ford-Lloyd 2003, Naik and Chand 2006, Rai et al. 2008). As in vitro plant regeneration is often the most important step for successful implementation of various biotechnological tools used for plant improvement programs, in the present study three important threatened or endangered medicinal plant species viz., Spilanthes acmella, Spilanthes mauritiana and Decalepis hamiltonii have been selected for their in vitro regeneration, multiplication and re-vegetation in natural environment. The relevant description of all the selected species is as follows;
11
1.5 Spilanthes acmella (L.) Murr.
Synonymous: Acmella oleracea (L.) R. K. Jansen Common name: Akarkara English name: Toothache plant, Pelliatry, Eyeball Plant, Spot Plant, Para Cress Family: Asteraceae (Compositae) Native: Tropics of Brazil Plant part used: Whole plant Threat status: Threatened (Rao and Reddy 1983) Propagation: Through stem cuttings and seeds
1.5.1 Habit
An annual herb.
1.5.2 Habitat
In India, it is reported from South India, Chhattisgarh and Jharkhand. It is also reported from Jhalarapatan in Jhalawar district in Rajasthan. S. acmella prefers moist or very damp soil. It must be keep watered and should be planted in rich soil (Sharma et al. 2010).
1.5.3 Botanical description
It is up to 90 cm tall, stem usually decumbent to ascending, green to red, glabrous. Petioles 2-6.5 cm long, narrowly winged, glabrous or very sparsely pilose; leaf blades broadly ovate to deltate, base truncate to short attenuate, glabrous on both sides. Peduncles 3.5-12.5 cm long, sparsely to very sparsely pilose. Capitula discoid, solitary, terminal, cylindrical, 10.5-23.5 mm high, 11-17 mm in diameter; involucral bracts 15-18, triseriate, margins entire to sinuate; flower heads yellow in color with rust-red centre, ray florets absent, disc florets yellow (Fig. 4), flowers are non-fragrant mostly bisexual; Achenes black, 2- 2.5×0.9-1.1 mm, ciliated at the edge (Chung et al. 2008).
1.5.4 Chemical constituents
12 S. acmella is reported to contain alkaloids, carbohydrates, pungent amides, tannins, steroids, carotenoids, essential oils, sesquiterpens, amino acids etc. (Tiwari and Kakkar 1990, Lemos et al. 1991, Nagashima 1991, Nagashima and Nakatani 1992, Amal and Sudhenden 1998). The flower heads and roots of the plant have been known to be especially rich in the active principle contents which contribute acrid flavor to them (Nayak 2002). The plant species is attributed with immense medicinal properties because of the presence of spilanthol (88.8%) which is found as the main lipidic component (Gerber 1903). Eight N-isobutyl amide, two 2-methylbutyl amides and one 2-phenylethyl amide were also detected with spilanthol [(2E, 6Z, 8E)-deca-2, 6, 8-trienoic acid]. Till now, two of these N-isobutyl amides were not yet described in Spilanthes extract (Boonen et al. 2010). More recent work has found acetylenic-alkyl amide such as undeca-2E-en-10-diynoic acid isobutyl amide (UDA) in lower quantities (Bae et al. 2010). Lemos et al. (1992) reported β-caryophyllene (30.2 %), γ- cadinese (30.3 %) and thymol (18.3 %) as major constituents of S. acmella.
13 Figure 4
Explanation of Figure 4
A twig of field grown plant of S. acmella showing flower heads
14 1.5.5 Medicinal importance
1.5.5.1 Traditional uses
S. acmella is largely grown for its ornamental and medicinal purpose (Sharma et al. 2010). Since generation, it is used as folk medicine. Leaves and flower heads are chewed to relieve toothache and affections of throat and gums, flue, cough and tuberculosis (Haw and Keng 2003, Sharma et al. 2010). They are also said to be a popular remedy for stammering in children in Western India (Sharma et al. 2010). A tincture made from the flower heads is used as a substitute for the tincture of pyrethrum to treat the inflammation of jaw-bones and caries. Leaves are regarded as powerful stimulant, sialogogue and local anesthetic; the seeds also are chewed for this purpose (Anonymous 2003b, Sharma et al. 2010). The leaves may be used topically to treat bacterial and fungal skin disease such as ringworm scan. Internally the decoction of leaves and roots is given as diuretic and lithotriptic. The herb exhibits general immnunomodulator properties when used internally, boosting the production of leukocytes and antiviral interferon, as well as promoting phagocytosis (Kirtikar and Basu 1980, Yoganarasimhan and Chelladuri 2000, Anonymous 2003b). Besides, the leaves of S. acmella have also been used as salad ingredient (Sharma et al. 2010). Sri Lankan traditional physicians especially in the Uva province claimed that the cold infusion of the flowers of Spilanthes acmella has potent diuretic activity and the ability to dissolve urinary calculi (Jayaweera 1981).
1.5.5.2 Larvicidal and insecticidal activities
Fragmentary studies have been carried out on its larvicidal and insecticidal activities using the flower head extract against Anopheles stephensi, A. culicifacies, Aedes aegypti and Culex quinquefasciatus (Pendse et al. 1946, Ramsewak et al. 1999, Saraf and Dixit 2002, Pandey et al. 2007). Pendse et. al. (1946) found the ethanolic extract of S. acmella one tenth active as compared to DDT against Anopheles larva. Pandey et al. (2007) achieved hundred percent mortalities against the late third/early fourth instar larvae of A. stephensi, A. culicifacies and C. quinquefasciatus using crude hexane extract obtained from flower heads. Spilanthes is effective at extremely low concentrations against blood parasites and is a poison to most invertebrates, while remaining harmless to warm blooded creatures. Thus, Spilanthes is effective against blood parasites, specifically malarial spirochetes, either as a prophylactic or as a treatment for malarial paroxysms (Richard 1996). While, Prasad and Seenayya (2000) reported that S. acmella possessed an excellent anti-microbial activity against red halophillic 15 cocci from salt cured fish. Additionally, there are reports that S. acmella contains alkaloids which act as insecticide (Krishnaswamy et al. 1975, Borges-Del-Castillo et al. 1984).
1.5.5.3 Antiobesity activity
Ethanolic extract of seeds of S. acmella produce pancreatic inhibitory activity by pancreatic lipase inhibition (Ekanem et al. 2007).
1.5.5.4 Antifungal activity
Different concentrations of S. acmella flower head extract were evaluated for antifungal activity. The diameter of inhibition zones ranged from 0.1 to 2.3 cm with the increase in concentration of the tested solution. Among different fungal species highest inhibition zone was observed against Fusarium oxysporium (2.3 cm) and F. moniliformis (2.1 cm) followed by Aspergillus niger (2.0 cm) and A. paraciticus (Rani and Murty 2006).
1.5.5.5 Transmucosal behavior
Besides acting as a flavoring agent and its topical use against fungal infections, Spilanthes extracts are formulated in local buccal mucosa preparations indicated for painful mouth issues and minor ulcers. In addition to local effects, the buccal mucosa is a good alternative way to reach effective systemic concentrations, thus bypassing different disadvantages of oral administration. The rich microcirculation with direct drainage of blood into the internal jugular vein permits systemic effects of permeated molecules (Boonen et al. 2010).
1.5.5.6 Antiaging activity
Spilanthol inhibits contractions in subcutaneous muscles, notably those of the face and can be used as an anti-wrinkle product. A cosmetic treatment procedure for wrinkles consists of locally or subcutaneously applying an effective quantity of a composition based on spilanthol pure or in the form of a crude extract of S. acmella extracts (Anonymous 2003b).
1.5.5.7 Antiinflammatory and analgesic activities
16 Anti-inflammatory and analgesic activities of the aqueous extract of S. acmella in experimental animal models have also been evaluated (Chakraborty et al. 2004, Wu et al. 2008). Carrageen induced rat paw edema model, Granuloma pouch method and Adjuvent arthritis method were used for acute, sub-acute and chronic inflammation respectively (Braman et al. 2009).
1.5.6 Causes of its extinction and need of micropropagation
Due to immense medicinal importance and pesticidal activity, S. acmella has increased its demand. It is conventionally propagated through seeds which lose their viability within a short period of time (Singh and Chaturvedi 2010). Moreover, germination of S. acmella seeds under natural conditions is very poor (Pandey and Agrawal 2009). Moreover, due to its preference to grow near moist and damp places it is vulnerable to pest and diseases (Fig. 5). Damp and cool conditions are held responsible for rotting of seeds (Chandra et al. 2007). Therefore, in order to stop using contaminated pharmaceutical raw materials and fulfill the demands of pharmaceutical industries, it is necessary to develop an efficient method of propagation of S. acmella.
1.5.7 Work conducted and lacuna for further research
Fragmentary reports are available on direct organogenesis of S. acmella using different propagaules such as hypocotyls (Saritha et al. 2002), axillary buds (Haw and Keng 2003, Deka and Kalita 2005) and leaf explants (Saritha and Naidu 2008) while Pandey and Agrawal (2009) reported indirect organogenesis from leaf derived calli. All these workers used a variety of PGRs including adenine-based cytokinins and auxins; still no report is available on the effect of TDZ on regeneration potential of the explants. Besides, there is no report related to the effect of variable pH, carbon sources and culture media composition on in vitro explants’ growth. Moreover, previous results are not much satisfactory as multiplication rate is comparatively less as compared to the pharmaceutical demands. Besides, a high shoot regeneration potential with consistency for several sub-culture passages leading to the production of a large number of propagules, in addition describing a way for in vitro conservation, but previous workers did not performed any experiment for sub-culturing in order to enhance the multiplication rate.
17
Figure 5
Explanation of Figure 5
A twig of field grown plant of S. acmella showing infested leaves
18 On the other hand, Singh et al. (2009b) used shoot tip explants for the synseed production in this medicinal herb and achieved complete plantlet recovery from synseeds. But ex vitro sowing is still require as direct sowing of synseeds in the soil or other type of substrates helps in escaping acclimatization. Histological evidences are also lacking to explain organogenesis from different plant tissues; single report is available in this regard which provide the basis for organogenesis through leaf explant only (Saritha and Naidu 2008).
1.6 Spilanthes mauritiana DC.
Common name: Akarkara English name: Toothache plant Family: Asteraceae (Compositae) Native: African and the South American tropics Plant part used: Whole plant Threat status: Endangered (Anonymous 2003b) Propagation: Through stem cuttings and seeds
1.6.1 Habit
A perennial herb.
1.6.2 Habitat
It occurs in high rainfall areas and elsewhere in wet habitats such as nearby streams and rivers up to an altitude range of 600-2000 m.
1.6.3 Botanical description
Taxonomically it is 20-50 cm tall creeping or trailing herb; leaves opposite to lanceolate, dentate or almost entire; flower head axillary or in terminal panicles, involucral bracts, florets yellow, both disc and ray florets are presents in flower heads, ray florets few, disc florets campanulate (Fig. 6, Chung et al. 2008).
19
Figure 6
Explanation of Figure 6
A twig of field grown plant of S. mauritiana showing flower heads
20 1.6.4 Chemical constituents
Spilanthol, the only value added secondary metabolite alkaloid characterized from S. mauritiana is present at a concentration of as much as 1.25% in the flowers (Watt and Brayer-Brandwijk 1962).
1.6.5 Medicinal importance
1.6.5.1 Traditional uses
The plant has been reported to have many medicinal properties (Fabry et al. 1996 & 1998). It is used in the local pharmacopeia to cure infections of throat and mouth (Watt and Brayer-Brandwijk 1962) and as a remedy for stomachache and diarrhea (Kokwaro 1976). Kamba tribes in Kenya chew the flower of S. mauritiana for the relief of toothache and the treatment of pyorrhea (Watt and Brayer-Brandwijk 1962) and an infusion of the herb is used as a febrifuge (Dalziel 1937). In the Cameroons plant is used as a snake-bite remedy and in the treatment of articular rheumatism (Dalziel 1937). It has also been used against malaria, pneumonia and tonsillitis (Baerts and Lehmann 1989, Rwangabo 1993). In India the plant has been used a remedy for kidney stones and bladder infections. In addition, the flowering head is reported to produce stupefaction of fish and to be used as a fish poison (Drangendorff 1898).
1.6.5.2 Larvicidal and insecticidal activities
S. mauritiana extracts, as potential insecticides, have not been extensively studied. Jondiko (1989) and Hassanali and Lwande (1989) reported its larvicidal and pesticidal properties respectively under laboratory conditions. The hexane extracts have been shown to be active against Aedes aegypti larvae and Helicoverpa zea neonates (Ramsewak et al. 1999). Recently, larvicidal activity against Anopheles gambiae and Culex quinquefasciatus was reported by Ohaga et al. (2007). Hassanali and Lwande (1989) reported its anti-pest activity.
1.6.5.3 Antifungal activity
Extract from both roots and flower heads exhibited antifungal activities against Aspergillus spp. but no activity against Candida spp. (Fabry et al. 1996).
1.6.6 Causes of its extinction and need of micropropagation
21 Plant is under severe pressure and is constantly shrinking due to over exploitation and habitat loss. Thus, there is an urgent need of its conservation.
1.6.7 Work conducted and lacuna for further research
To date, single report is available on in vitro micropropagation in S. mauritiana through axillary buds (Bais et al. 2002). They reported few shoots, insufficient to adopt the protocol for a large-scale plantation programme. Further, they mentioned very slow rooting during experimentation. Thus, an efficient, rapid and practical protocol for mass propagation of this endangered herb is still lacking. No report is available on the application of synseed technology for this plant. Therefore, there is indeed an urgent need for the establishment of an efficient micropropagation with histological proof in S. mauritiana.
1.7 Decalepis hamiltonii Wight and Arn.
Common name: Maredugeddalu, Nannari and Sariba English name: Shallow root Family: Asclepiadaceae Native: Native of the Deccan peninsula and forest areas of Western Ghats of India Plant part used: Root Threat status: Endangered (Anonymous 2003a) Propagation: Through seeds, stem cutting and root suckers
1.7.1 Habit
A monogeneric woody climbing shrub.
1.7.2 Habitat
Prefer to grow along open rocky slopes and rocky cervices of dry, moist deciduous forest at an altitude from 300-1200 meters (Anonymous 2003a).
1.7.3 Botanical description
It is a large hairless extensively climbing shrub, latex sticky and milky. The stem and its branches are articulated, slightly angled with swollen nodes. The roots are tuberous, fleshy, strongly fragrant and pale brown in color and grow up to 160 cm in length and 2.5 cm
22 width and characterized by a sarsaparilla (Hemidesmus indicus) like taste accompanied by a tingling sensation on the tongue. Leaves opposite, oval to round shaped, about 7×5 cm, base gradually tapering to transcate, apex sub-acute to rounded, margin entire to wavy, petiole about 1 cm long. Flowers are yellow and small. Seeds are many and egg-shaped about 6×4 mm, with long white shiny hairs. Root is fleshy and cylindrical 1-6 cm diameter (Anonymous 2003a).
1.7.4 Chemical constituents
It contains quercetin, kaempferol, coumarin and rutin, lupeol, β-amyrin, ferulic and 2- hydroxy-4-methoxy benzaldehyde (2H4MB) (Bias et al. 2000). Hydro-distillation of D. hamiltonii roots yielded an essential oil (0.33% v/w) that contained 2-hydoxy-4- methoxybenzaldehyde (37.45%), 2-hydoxybenzaldehyde (31.01%), 4-O- methylresorcylaldehyde (9.12%), benzyl alcohol (3.16%) and R-atlantone (2.06%) as major constituents, with aromatic aldehydes constituting the main fraction of its roots’ essential oil (George et al. 1998, Thangadurai et al. 2002).
1.7.5 Medicinal importance
1.7.5.1 Traditional uses
The roots of D. hamiltonii are also used in folk medicine and in Ayurvedic preparations (Nayar et al. 1978). Dried samples are available in the country drug stores and local tribal markets. Ancient tribes in the Western Ghats of India use its roots particularly for inflammation (Ashalatha et al. 2010). Tuberous roots are used as a flavoring principle (Murti and Seshadri 1941) and a blood purifier (Jacob 1937). Roots are used to cure indigestion, dysentery, cough, bronchitis, leucorrhoea, uterine hemorrhage, skin disease, fever, vomiting, chronic rheumatism, anemia and blood diseases. Roots are also consumed as pickles and as a popular cool drink in the forest areas of the Eastern and Western Ghats, known as Nannari which has a cooling effect without any toxic effects in human beings (Vijaykumar and Pullaiah 1998, Harish et al. 2005). It finds its use as culinary spice because of its high priced aromatic roots (Ahmedulla and Nayar 1986). Recently, Naveen and Khanum (2010) suggested that root extract could be used not only as food preservative (to replace for the toxic butylated hydoxy anisole and butylated hydoxy toluene currently under use) but also can be used in the preparation of nutraceuticals and pharmaceutical products.
23 1.7.5.2 Antimicrobial activity
Roots show antimicrobial activity against food borne pathogens responsible for food spoilage and human pathologies. The results suggest that methanolic and petroleum ether extracts can be use against noble antimicrobials which prevent food spoilage. Root extract exhibited strong antimicrobial activity against Bacillus cereus, B. megaterium, Candida albicans, Escherichia coli, Micrococcus luteus, Micrococcus roseus and Staphylococcus aureus (George et al. 1999, Thangaduari et al. 2002, Thangaduari et al. 2004). Recently, Thangavel et al. (2011) investigated the antibacterial potential of D. hamiltonii callus extract against Salmonella tyhphi, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and E. coli.
1.7.5.3 Insecticidal activity
Many researchers investigated its roots for insectidial activity against three coleopteran stored product pests, viz., rice weevil, the lesser grain borer and red-rust flour beetle (George et al. 1998 & 1999). Further, the residual deposits of 2H4MB has been assayed for contact toxicity on rice weevil (Sitophilus oryzae), Rhyzopartha dominica and Tribolium castaneum. Thus, the aromatic chemicals of D. hamiltonii have been established with insecticidal and pesticidal properties.
1.7.5.4 Other properties
Recently, antidiabetic, hepatoprotective and antiatherosclerotic properties of root extract of D. hamiltonii have been evaluated in rats and reported that the tuber extract could be able to protect the rats from oxidative stress and also inhibits the activity of antioxidant enzymes causing liver damage (Naveen and Khanum 2010, Harish and Shivanandappa 2010). The roots possess potent antioxidant properties, which could be responsible for their health benefits (Murthy et al. 2006, Srivastava et al. 2007).
1.7.6 Causes of its extinction and need of micropropagation
In D. hamiltonii, extended flowering pattern, self-incompatibility, pollinator limitation, absence of seed dormancy, abortion of a considerable percentage of seedlings prior to establishment are contributing factors for the regulation of its population size (Raju 2010). The highly aromatic roots have been subjected to overexploitation by destructive
24 harvesting that affect the survival of this plant in its wild habitat. Moreover, the absence of any organized cultivation of this plant (Reddy et al. 2002) calls for immediate conservation measures. Therefore, the development of an efficient in vitro regeneration system is of great importance for rehabilitation of this valuable endangered woody climber.
1.7.7 Work conducted and lacuna for further research
There are limited efforts on micropropagation which support the cultivation of this endangered plant species. Bais et al. (2000), Anita and Pullaiah (2002) used nodal segments for direct multiplication whereas Giridhar et al. (2003 & 2005a) exploited shoot tip explants for this purpose. Indirect organogenesis through leaf derived calli has been documented by George et al. (2000). Later, they induced somatic embryogenesis from leaf cultures in 2004. For successful in vitro rooting, a protocol has been reported by Reddy et al. (2001). There are so many lacuna exist in the above said studies out of which low regeneration potential (particularly for indirect organogenesis as only 3 shoots per culture was reported by George et al. in 2000) and presence of intervening callus during direct caulogenesis are the main constraints which require further refinement in the existing protocols. As far as the literature is concerned, no report is available on the synseed production. Besides, histological study is still lacking to reveal the morphogentic pathways in D. hamiltonii.
1.8 Objectives
Considering the medicinal importance of the above mentioned plants, the present study has been carried out to improve the regeneration potential with the following objectives;
• To raise the seedlings under in vivo and in vitro regimes. • To establish aseptic cultures from different somatic tissues (meristematic and non- meristematic). • To see the effect of various cytokinins on shoot induction and multiplication. • To evaluate the synergistic effect of cytokinin-auxin combinations on shoot regeneration and multiplication. • To observe the effect of sub-culture passages on shoot proliferation efficiency. • To optimized the auxin concentration for best in vitro rooting.
25 • Hardening, acclimatization and field transfer of the complete plantlets. • To optimized the conditions for an ideal synseed production. • To evaluate best suited conditions for synseed germination under in vitro and ex vitro. • To carry out the histological examinations of regenerating tissues. • To study physiological parameters during successive ex vitro establishment of in vitro raised plantlets.
26
Chapter 2 Review Of Literature
Chapter 2 REVIEW OF LITERATURE
The science of plant tissue culture takes its roots from path breaking research in botany like discovery of cell followed by propounding of cell theory by Schleiden (1838) and Schwann (1839). They proposed that a cell is the basic unit of an organism and capable to regenerate into whole plant if an appropriate environment is given. But Schleiden and Schwann had no experimental evidence to prove it. The Cell Theory received much impetus from the famous aphorism of Virchow (1858), ‘‘Omnis cellula e cellula’’ (All new cells arise from pre-existing cells) and by the very prescient observation of Vötching (1878) that the whole plant body can be built up from ever so small fragments of plant organs. An important approach of tissue culture was discovered by Rechinger (1893) who tried to determine experimentally the limit of plant divisibility permitting tissue proliferation. He used isolated buds, slices of roots, stems and other materials. The explants were placed on sand moistened with tap water. But he did not use nutrients or aseptic conditions; his culture could scarcely be called tissue culture. However, Rechinger’s experiments were suggested by a concept related to the tissue culture principles; thus he was recognized as a true pioneer in this field (Gautheret 1983). In 1902 Haberlandt, a German physiologist was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf mesophyll cells of Lamium purpureum, glandular hairs of Pulmonaria and cells from petioles of Eicchornia crassipes on diluted Knop’s (1865) salt solution enriched with glucose. Unfortunately, he failed largely because of the poor choice of experimental materials, inadequate nutrients and infection (Vasil and Vasil 1972). But, he boldly predicted that it should be possible to generate artificial embryos (somatic embryos) from vegetative cells which encouraged subsequent attempts to regenerate whole plants from cultured cells. This potential of a cell is known as ‘totipotency’, a term coined by Steward in 1968. Despite lack of success, Haberlandt made several predictions about the nutrients’ requirement in experimental conditions which could possibly induce cell division, proliferation and embryo induction. Haberlandt is thus regarded as ‘father of tissue culture’.
21
Initial progress in plant tissue culture came from the work of Molliard (1921) in France, Kotte (1922) in Germany, Robbins (1922) in the United States, who successfully cultured the fragments of embryos and roots. Unfortunately, the growth of those cultured tissues could not be sustained for long even if they were transferred to fresh medium. Innovative plant tissue culture techniques progressed rapidly during
the 1930s due to the discovery of natural auxin and vitamin B5 which were necessary for the growth of isolated tissues containing meristem. In 1926, Went discovered the first plant growth regulator, indole-3-acteic acid (IAA) which is a naturally occurring member of a class of plant growth regulator (PGR) termed as ‘auxin’. The breakthrough progress came from White (1934) who was the first to demonstrate continuous culture of excised tomato root-tips on a medium containing inorganic salts, sucrose and yeast extract (YE). Later, he (1937) replaced YE by vitamin B namely pyridoxine, thiamine and proved their growth promoting effect. One of the main thrust in the history of tissue culture is the induction of callus. Gautheret (1934) is credited with the first successful attempt of callus induction from cambial cells of some tree species. This was followed by the formation of continuous callus cultures in carrot and tobacco independently endorsed by Gautheret, White and Nobécourt in 1939. Adding to the ongoing improvements in the culture media, van Overbeek (1941) used coconut milk besides usual salts, vitamins and other nutrients for embryo culture. After 1950, there was an immense advancement in the area of PGR. Skoog and Tsui (1951) demonstrated continued induction of cell division and bud formation in tobacco by adenine and high levels of phosphate. This led to further investigations by Miller et al. (1955) who isolated ‘kinetin’ (Kn), a derivative of adenine (6-furyl amino purine). They worked further and proposed the concept of hormonal control for organ formation in 1957. Their experiment on tobacco pith culture showed that the high concentration of auxin promoted rooting; whereas high kinetin induced bud formation. Later studies led to the isolation of other naturally occurring as well as synthetic cytokinins, elucidation of their role in cell division and bud development and their extensive use in the micropropagation industry related to their suppression of apical dominance resulting in the development of many axillary shoots. In early 1960s, the most significant breakthrough in the field of plant tissue culture was the development of a defined culture medium by Murashige and Skoog (1962), prepared by increasing the concentration of salts twenty-five times higher than 22
Knop’s solution. Today MS medium has been proved as the most effective and widely used culture medium for various plant species. The role of tissue culture in plant genetic engineering was first exemplified by Kanta et al. (1962). They developed a technique of test tube fertilization which involved growing of excised ovules and pollen grains in the medium thus overcoming the incompatibility barriers at sexual level. In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos which developed into haploid plants initiating androgenesis. Recently, tissue culture technology gained unbeatable recognition in plant science for successful micropropagation and improvement of plant species, leading to its commercial application. A number of plant species have been micropropagted around the globe, out of which the review of some important medicinal plants has been categorized below;
2.1 Organogenesis
Organogenesis is a complex phenomenon involving the de novo formation of organs (shoots or roots). Shoots can be derived either through pre-existing meristematic tissues known as ‘axillary shoot formation’ or through differentiation of non-meristematic tissues known as ‘adventitious shoot formation’. Both these approaches require synergistic interaction of physical and chemical factors. A successful plant regeneration protocol requires appropriate choice of explant, age of the explant, definite media formulation, specific growth regulators, genotypes, energy source, gelling agent and other physical factors including light regime, temperature and humidity (Bhojwani and Razdan 1983).
2.1.1 Meristem, shoot tip and nodal segment culture
Meristem culture is based on suppressing the shoot apical dominance by addition of cytokinins to the growth medium followed by axillary bud sprouting in multiple shoots. As the cells of apical and axillary bud are uniformly diploid and least susceptible to genotypic changes, they produce a large number of genetically stable plants in a short span of time, thus it is regarded as the most common technique for mass production of useful plant species. The history of meristem culture began with the first successful shoot tip culture of Nasturtium (Tropaeolium majus) by Ball in 1946. Since then meristem
23
culture has attracted much attention of the plant scientists. However, demonstration of practical utilisation of this important technique must be credited to Morel and Martin (1952) who for the first time produced virus-free Dahlia plants from infected individual by excising and culturing their shoot tips in vitro. Later, Morel extended this approach for the production of virus free plants of orchids (Morel 1960). That was the beginning of tissue culture. Thereafter, in the 1970s developed countries began commercial exploitation of this technology. The meristem tip must be small enough to eradicate viruses and other pathogens, yet large enough to develop into a shoot. In case of meristematic propagation, elimination of virus particles in explant cells is reached within a short time. In many cases meristematic cells do not contain virus particles due to absence of vascular connection with other plant parts. Now, meristem culture is considered as a unique technique to produce pathogen-free (bacteria, fungi, viruses, viroides and mycoplasma) plants of many species (Morel and Martin 1955, Walkey 1978, Bhojwani and Razdan 1983, Biswas et al. 2007). Recently, Banerjee et al. (2010) used apical meristem for reducing phytoplasma infection in Artemisia roxburghiana. Plant tissue culture entered in the developing world during the 1980s. It was earlier used to develop ornamental plants for export. With tree species, the technique of tissue culture remained confined for many years to laboratory stage and had generally invited only academic interest. But in most developing countries, the shortage of biomass and the ever-increasing energy requirements created the need to explore possibilities of mass propagation of trees by tissue culture. Using this approach of micropropagation, significant achievements in in vitro cloning has been made for various herbaceous and woody plants of medicinal, horticultural and ornamental values (Sharma et al. 2002, Tripathi and Tripathi 2003, da Silva 2003, Zhou and Wu 2006, Rout et al. 2006, Chaturvedi et al. 2007).
2.1.1.1 Effect of adenine-based cytokinins on shoot regeneration
Cytokinins are plant hormones promoting cell division and differentiation. Since the discovery of first cytokinin i.e., Kn, a number of chemicals suited to the definition of cytokinin has grown to include a large array of natural and synthetic compounds, adenine and phenylurea derivatives. The natural cytokinins are adenine derivatives and can be classified by their configuration of N6-side chain as isoprenoid
24
or aromatic cytokinins (Fig. 7). Cytokinins with an unsaturated isoprenoid side chain are the most prevalent, in particular those with a trans-hydroxylated N6-side chain i.e., trans-zeatin. However, cis-zeatin and N6-(∆2-isopentenyl) adenine (2-iP) are generally minor components although exceptions exist (Durand et al. 1994, Emery et al. 1998). Kn and N6-benzyladenine (BA) are the best known cytokinins with ring substitutions at N6-position. In the early years of cytokinin research, only cytokinins with an isoprenoid side chain were thought to be endogenous compounds; however in the mid 1970s BA derivatives were also identified as natural cytokinins (Horgan et al. 1973 & 1975). Three adenine-based cytokinins viz., BA, Kn and 2-iP have been commonly used for different approaches of micropropagation; however, zeatin rarely screened for shoot multiplication. It is now well established that cytokinins are effective only at optimum concentrations. Higher concentrations are not recommended for shoot proliferation as callus production interferes with subsequent rooting and acclimatization. Among all the adenine-based cytokinins tested, BA has been found the most effective for axillary shoot proliferation, but it is recommended that a range of concentrations should be tested to optimize shoot production. Ault (2002) reported that in Hymenoxys acaulis var. glabra, BA at 20 µM induced significantly more axillary shoots (10.3 shoots per explant) than did other cytokinins. In comparison, 23.3 shoots per node at 5.0 µM BA in Mucuna pruriens (Faisal et al. 2006d), 6.3 shoots per node at 8.87 µM BA in Tinospora cordifolia (Raghu et al. 2006), 12.9 shoots per node at 5.0 µM BA in Ocimum basilicum (Siddique and Anis 2008), 8.6 shoots per shoot tip in Spilanthes mauritiana (Sharma et al. 2009b), 20.7 shoots per node in Veronica anagallis-aquatica (Shahzad et al. 2011), 14.37 shoots per node at 2.22 µM BA in Ceropegia spiralis (Murthy et al. 2010) have been reported. The important role of BA in shoot proliferation has been reported for various taxa of Asteraceae family, such as Wedelia calendulacea (Emmanuel et al. 2000), Echinacea purpurea (Koroch et al. 2002), Eclipta alba (Dhaka and Kothari 2005, Husain and Anis 2006), Stevia rebaundiana (Debnath 2008, Sharma and Shahzad 2011), Centaurea ultreiae (Mallóon et al. 2011) etc. Similarly, the superiority of BA over other cytokinins has also been reported for various Asclepiads like Gymnema sylvestre (Komavalli and Rao 2000), Hemidesmus indicus (Sreekumar et al. 2000), Decalepis hamiltonii (Anitha and Pullaiah 2002), Holostemma ada-kodien (Martin
25
Figure 7. Structural modifications in some adenine and urea-based cytokinins
26
2002), Ceropegia spp. (Beena et al. 2003, Nikam et al. 2008, Murthy et al. 2010, Chavan et al. 2011), Marsdenia brunoniana (Ugraiah et al. 2010). Borthakur et al. (2000) reported Kn as the best cytokinin for shoot proliferation in Eclipta alba and Eupatorium adenophorum. Similarly, Özel et al. (2006) found Kn as more effective cytokinin for regeneration in Centaurea tchihatcheffii on optimal concentration of 4.5 mg l-1. While, on 2.5 mg l-1 Kn growth in regenerants was very slow which improved considerably when the regenerants were transferred to BA (1.0 mg l-1), NAA (2.0 mg l-1) and glutamic acid (50 mg l-1) combination. While, the promotive role of 2-iP on shoot regeneration has been reported by Cellarova and Hocariv (2004) in Digitalis purpurea and Sujatha and Ranjitha Kumari (2007 & 2008) in Artemisia vulgaris. In several studies, combination of two cytokinins proved to be advantageous over single cytokinins treatment for apical and axillary bud sprouting. High frequency shoot multiplication of Eclipta alba was obtained with MS medium containing BA combined with Kn or 2-iP. The shoots regenerated on a combination of BA (4.4 µM) and 2-iP (14.7 µM) grew faster than those initiated in BA and Kn combination (Baskaran and Jayabalan 2007). In Decalepis hamiltonii, the MS medium composed of 9.1 µM zeatin, 4.7 µM Kn and 0.6 µM IAA proved to optimal for maximum shoot regeneration and multiplication (5.4 shoots per shoot tip). Further multiplication and elongation was achieved on medium containing 2.5 µM 2-iP and 0.3 µM GA (Giridhar et al. 2005). Whereas, Rani and Raja (2010) reported callus-free multiple shoot formation in Tylophora indica as a function of BA activity alone, but internode elongation was dependent on the synergistic effect of GA. Similarly, the shoot buds of Eclipta alba were multiplied and maintained on BA and GA containing MS medium (Dhaka and Kothari 2005). Generally, inclusion of low concentration of auxin to the cytokinin containing medium results in high frequency shoot production through axillary or apical buds and indicates the synergistic effect of a cytokinin and an auxin. The presence of NAA and BA has increased shoot multiplication in Gymnema sylvestre (Reddy et al. 1998), Mentha arvensis (Shahzad et al. 1999), Spilanthes mauritiana (Bais et al. 2002), Santolina canescens (Casado et al. 2002), Clitoria ternatea (Shahzad et al. 2007), Tylophora indica (Faisal et al. 2007), Gynura procumbens (Keng et al. 2009), Carlina acaulis (Trejgell et al. 2009) etc. Corral et al. (2011) also investigated that the addition of NAA with BA significantly improved the shoot proliferation efficiency in 27
Crepis novena as compared to the combination of NAA and Kn. They reported that an average of 49.77 shoots per axillary bud were produced in 100% of cultures on MS medium comprised of 0.54 µM NAA and 4.44 µM BA. In contrast, Banerjee et al. (2010) reported maximum shoot regeneration (38.0 shoots per explant) on the combination of 13.95 µM Kn and 0.27 µM NAA in Artemisia roxburghiana. They have advocated that the combination of 8.88 µM BA and 0.27 µM NAA was found to be more stimulative for further multiplication and elongation during sub-culturing. For Gymnema sylvestre, Komalavalli and Rao (2000) reported a maximum number of shoots (57.2) induced from axillary node explants on MS medium containing BA (1 mg l-1), Kn (0.5 mg l-1), NAA (0.1 mg l-1), malt extract (100 mg l-1) and citric acid (100 mg l-1). Maximum shoot proliferation on BA and IAA combination has been reported by Sivaram and Mukundan (2003), Debnath (2008) and Sharma et al. (2009b). The combination of BA and IAA in addition to additives like adenine sulphate (ADS), arginine, citric acid and ascorbic acid used to establish the aseptic cultures of Leptadenia reticulata (Arya et al. 2003). Whereas, Devi and Srinivasan (2008) found optimal response for micropropagation of Gymnema sylvestre on MS medium containing 1 mg l-1 BA, 0.5 mg l-1 IAA, 100 mg l-1 vitamins -1 B2 and 100 mg l citric acid using nodal explants. In contrast, Beena et al. (2003) established a protocol for in vitro propagation of Ceropegia candelabrum through axillary bud multiplication using 8.87 µM BA in combination with 2.46 µM IBA. Gantait et al. (2010) reported an elite protocol for accelerated quality-cloning in Gerbera jamesonii using shoot tips in which MS medium supplemented with 0.5 mg l-1 NAA and 1.5 mg l-1 BA promoted earliest axillary bud initiation within 5 day in 91.6% of the inoculants. They achieved very high rate of shoot multiplication (14 shoots per explant) when MS medium was fortified with a relatively higher level of BA (2 mg l-1) and 60 mg l-1 ADS within 27 day of incubation. According to Gantait and Mandal (2010), ADS acts as an elicitor or enhancer of growth in synergism with endogenous and exogenously supplemented PGRs in Anthurium anderanum. Many researchers studied the comparative efficiency of different explants for maximum shoot production. Sivanesan and Jeong (2007) achieved more number of shoots from nodal segments as compared to shoot tips in Pentanema indicum. They also revealed the additive effect of ADS when added to the BA and IAA containing MS medium. Trejgell et al. (2009 & 2010) reported maximum shoot regeneration form the seedling derived shoot tips in comparison to the hypocotyl, cotyledon and 28
root explants of Carlina acaulis and Senecio macrophyllus. Similarly, in Stevia rebaudiana shoot tips were proved to be the most effective explants for shoot regeneration over nodal segments and leaf explants when cultured on MS medium supplemented with 1.0 mg l-1 BA and 0.5 mg l-1 IAA (Anbazhagan et al. 2010). However, Gonçlaves et al. (2010) reported maximum shoot proliferation from nodal segments than apical shoot tips in Tuberaria major. The proliferation frequency was not differed by cytokinin type when nodal segments were used. More than 6 shoots were obtained on BA and zeatin supplemented MS media. However, a differential proliferation was noticed for shoot tips depending upon the cytokinins. The difference in shoot multiplication among different explants in response to exogenous PGR could be a reflection of probable variation of endogenous PGR level (Yucesan et al. 2007) or different tissue sensitivity to PGR (Lisowska and Wysokinska 2000).
2.1.1.2 Effect of urea-based cytokinins on shoot regeneration
Diphenylurea (DPU) was the first cytokinin-active phenyl urea identified (Shantz and Steward 1955). Although, this discovery was linked to the detection of a compound in liquid coconut endosperm, it was later found to be a contaminant from prior chemical analysis of DPU. This fortuitous discovery however led to the synthesis of a number of potent analogues such as forochlorfenuron [1-(2-chloro-4- pyridyl)-3-phenylurea, CPPU] and thidiazuron [N-phenyl-(1, 2, 3- thidiazol)-5-ylurea TDZ] (Fig. 7) with cytokinin activity exceeding that of natural cytokinins (Takahashi et al. 1978, Mok et al. 1982). Synthetic phenylureas are less susceptible to the plants’ degrading enzymes than endogenous cytokinins and can persist in the plant tissues for long periods of time (Mok and Mok 1985, Mok et al. 1987). Besides, there is no evidence that any phenylurea cytokinin occurs naturally in plant tissues. TDZ and CPPU have proven advantageous for micropropagation of a wide range of recalcitrant plant species that do not respond well to amino puirne because of their tremendous ability to stimulate shoot proliferation (Huetteman and Preece 1993). The mode of action of these urea derivatives is still unclear even though it appears quite sure that they inhibit cytokinin oxidase (CKOx) activity (Hare and van Staden 1994) and thus induce cytokinins accumulation within the cells (Victor et al. 1999). ++ TDZ mediated response has been reported to be influenced by Ca2 (Yip and Yang 1986, Hosseini and Rashid 2000). Mundhara and Rashid (2006) and Sharma et al.
29
(2011) reported an enhancement in number of responding explants when transient ++ Ca2 was provided. Application of TDZ induces a diverse array of culture response in plant tissues. These range from induction of callus to the formation of somatic embryos. The activity of TDZ varies widely depending on its concentration, exposure time, cultured explant and species (Murthy et al. 1998). The concentration at which TDZ is most effective is 10-1000 times lower than the other PGRs (Huetteman and Preece 1993). Therefore, direct comparison between TDZ and purine-based cytokinins at equimolar concentrations or at similar durations of the treatment is complicated. Short duration exposure to TDZ has been proved very effective for morphogenesis (Tulać et al. 2002). Higher levels, on the other hand, promote callus and somatic embryo fromation (Huetteaman and Preece 1993, Rida et al. 2001, Fengyen and Han 2002). In most of the studies, continuous or more than critical exposure with TDZ resulted in stunted or abnormal shoot development. Its deleterious effect has also been well documented in several plant species (Huetteman and Preece 1993, Faisal et al. 2005, Khurana et al. 2005, Ahmad and Anis 2007). Primarily TDZ was used as a cotton defoliant (Arndt et al. 1976) and later found to mimic cytokinin like activity that was 20 times more effective in dormancy breaking (Wang et al. 1986). However, extended research showed that TDZ, unlike traditional cytokinins is capable of fulfilling both the cytokinin and auxin requirement of various regenerative responses (Mok et al. 1982, Murthy et al. 1998). The list of plant species exhibiting morphogenesis in the presence of individual TDZ has continued to increase over the years, facilitating the improvement of tissue culture technology (Murthy et al. 1998). For some species, the combination of TDZ and purine-based cytokinins (usually BA) has been found more effective to induce morphogenetic response than either TDZ or BA alone as reported by Mohamed- Yasseen (2002) in Hylocereus undatus. The exploitation of TDZ for regeneration has been reported vastly superior over adenine-based cytokinin for a number of woody plant species such as Hydrangea quercifolia (Ledbetter and Preece 2004), Cassia angustifolia (Siddique and Anis 2007), Pterocarpus marsupium (Husain et al. 2007a) and Vitex negundo (Ahmad and Anis 2007). Apart from woody plant species, TDZ has also shown a promise role for regeneration in many other plants (including herbs and shrubs) belonging to diverse
30
groups of families such as Hypericum perforatum (Murch et al. 2006), Bacopa monniera (Tiwari et al. 2001), Artemisia judaica (Liu et al. 2003), Hordeum vulgare (Ganeshan et al. 2003), Cineraria maritime (Banerjee et al. 2004), Oryza sativa (Gairi and Rashid 2004), Hyoscyamus niger (Uranbey 2005), Psoralea corylifolia (Faisal and Anis 2006).
2.1.2 Leaf culture
Direct regeneration from leaf is another alternative step for clonal propagation and germplasm conservation. Direct de novo or adventitious shoot regeneration is most preferred if Agrobacterium-mediated gene transfer is to be achieved and leaf explants are the best suited for both adventitious shoot formation and Agrobacterium- mediated gene transfer experiments. Hildebrandt et al. (1946) and Hildebrandt and Ricker (1947) were the first who cultured the excised leaf tissues of tobacco and sunflower under aseptic conditions. In a large number of studies, leaf has been proved as one of the most potent explant for a number of plant species (Misra and Datta 2001, Beegum et al. 2007, Saritha and Naidu 2008, Zheng et al. 2009, Sahai and Shazad 2010). Leaf explants have been found to be the most regenerative at their proximal or petiolar end as compared to leaf margin and mid rib portion. In view of this, high frequency shoot induction at proximal region may be due to higher accumulation of PGRs (Rajasekharan et al. 1987). There is a physiological gradient in the leaf explant from proximal to distal end for de novo regeneration of shoot buds. In some reports, excised petioles have been found to be more effective than leaf segments to exert shoot organogenesis. The leaf vein is an extension of xylem and phloem of the stem through the petiole which is surrounded by one or more layers of parenchymatous cells, but not well specialized for a particular function yet having the capacity for cell division. These parenchymatous cells are very sensitive to different growth stimuli, such as PGRs and environmental conditions; therefore they are easier to be differentiated to new organs than other cells in tissue culture (Gahan and George 2008). Multiple shoot regeneration from proximal part of the leaves than distal ends has been reported in a number of plant species including Tagetes erecta (Misra and Datta 2001), Anthurium andraeanum (Martin et al. 2003), Euphorbia nivulia (Martin 31 et al. 2005), Spilanthes mauritiana (Sharma et al. 2009b), Lysimachia species (Zheng et al. 2009). However, Sreedhar et al. (2008) reported direct regeneration of shoot buds on both side of midrib and rarely from regions of smaller veins, but never from lamina indicating the presence of vascular cells appear to be crucial for de novo organogenesis from immature leaf explants of Stevia rebaudiana.
2.1.2.1 Effect of adenine-based cytokinins on shoot regeneration
Among the adenine-based cytokinins, BA was found to be the most effective to induce direct shoot bud regeneration from the leaf explants in a variety of plant species. BA alone at 8.87 µM induced 38.0 shoot per leaf explant in Ophiorrhiza prostrata (Beegum et al. 2007). For Chicorium intybus, BA was found nearly twice more successful than Kn (Yucesan et al. 2007). Sreedhar et al. (2008) reported that a combination of BA and Kn was found to be an ideal combination for de novo shoot regeneration from leaf explants of Stevia rebaudiana. The MS medium containing 8.88 µM BA and 4.65 µM Kn resulted in the formation of highest number of shoots per explant at the end of 7 week of incubation. Hedayat et al. (2009) evaluated direct organogenesis from leaf and petiole segments of Tanacetum cinerariifolium and reported that the leaf segments were highly responsive than petiole cuttings and produced a maximum shoot regeneration (70%) on MS medium supplemented with 4.0 mg l-1 BA and 0.2 mg l-1 2, 4-D. The highest proliferation rate was observed on MS medium supplemented with 1.5 mg l-1 BA and 2.0 mg l-1 NAA. For Coleus forskohlii, Sahai and Shahzad (2010) evaluated leaf size, position, orientation and season of collection to select the most regenerative explant condition. Enhanced shoot production and proliferation has been achieved on medium containing 2.0 μM BA and 0.1 μM NAA wherein, a highest number of 35.0 shoots per explant were produced. In the same year, Krishna et al. (2010) provided a rapid in vitro regeneration protocol using leaf explant of C. forskohlii. They used three different segments of leaf i.e. proximal, middle and distal and cultured on MS basal medium supplemented with different cytokinins. Comparison of shoot regeneration response from different leaf segments at 5.0 mg l-1 BAP showed that the distal end was comparatively most regenerative as induced 45.0 shoots followed by 23.0 and 21.0 shoots from middle and proximal ends. Further elongation was achieved on MS medium augmented with 0.1 mg l-1 BA and 0.1 mg l-1 IAA combination. For Populus
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tremula, Huang and Dai (2011) reported a high frequency of shoot regeneration on 10-20 µM zeatin as compared to other cytokinins. A maximum of 92.6 shoots were induced from each petiole explant when culture on 20 µM zeatin added to MS medium, while an average of 60.9 shoots were induced from leaf explants on similar concentration of zeatin. The existence of synergistic and additive interaction of auxin and cytokinins combination involves a complex web of signal interactions such as increased sensitization, receptivity, feedback inhibition and modulation of gene expression resulting in variable translation of mRNA population (Cline 1991, Eklof et al. 1997, Schmulling et al. 1997, Armstrong et al. 2004). In most of the studies, combination of BA and NAA was found to be most effective for regeneration through leaf explants such as in Chaememelum species (Echeverrigaray et al. 2000), Phellodendron species (Azad et al. 2005), Saussurea species (Dhar and Joshi 2005). Through leaf disc cultures of Sansevieria cylindrica, highest shoot regeneration frequency (80%) and mean number of shoots per explant (13.5) were obtained in MS medium supplemented with 10.0 µM BA and 0.1 µM NAA (Anis and Shahzad 2005). Mohapatra et al. (2008) reported a maximum shoot (8.3 shoots per leaf) and shoot length (2.1 cm) in 81.6% of cultures of Centella asiatica on MS medium supplemented with 3.0 mg dm-3 BA and 0.05 mg dm-3 NAA. In Lysimachia nummularia a maximum of 12.73 shoots per leaf explant were induced in 100% of cultures on MS medium supplemented with 1.0 mg l-1 BA and 0.1 mg l-1 NAA (Zheng et al. 2009). Similarly, Corral et al. (2011) reported mean number of 2.48 shoots per explant on 2.22 µM BA and 2.69 µM NAA combination from leaf explant of Crepis novoana. Superiority of NAA with BA over other auxins might be due to the fact that NAA has more affinity for easy penetration through plasma membrane even without active uptake as suggested by Nordstrom et al. (2004). In contrast to BA and NAA combination, in Tagetes erecta leaf culture highest shoot regeneration established on BA (13.3 μM) and IAA (17.1 μM) combination (Vanegas et al. 2002). While in Chicorium intybus, 0.5 mg l-1 Kn combined with 0.3 mg l-1 IAA gave optimum response with a mean of 19.7 shoots per lamina explant (Yucesan et al. 2007) and in Ophiorrhiza prostrata combination of 8.87 µM BA and 2.46 µM IBA yielded maximum number of shoots per leaf explant (76.0) (Beegum et al. 2007). Whereas, Saritha and Naidu (2008) reported direct organogenesis from juvenile leaf explants of Spilanthes acmella on the medium augmented with BA and 33
IAA. Similarly, the combination of BA (2.0 mg l-1) and IAA (0.5 mg l-1) produced maximum number of shoots (32.8) from leaf explants of field grown plants of Solanum nigrum, whereas from in vitro derived leaf explants maximum number of shoots (38.0) were obtained on BA (3.0 mg l-1) and IAA (0.5 mg l-1) combination (Sridhar and Naidu 2011). As an additive, GA also plays a very significant role for the induction of shoot buds from leaf explants. In this context, Sekioka and Tanaka (1981) were of the opinion that GA can act as a replacement for auxin in shoot induction and thus a ratio of cytokinins-GA may be decisive for differentiation in certain plant tissues. GA has also found conducive for promotion of biomass production and enhanced xylem fibre length in transgenic aspen (Eriksson et al. 2000). A combination of 14.43 µM GA and 4.44 µM BA in the absence of any auxin induced multiple shoot bud differentiation from the leaf segments of Tagetes erecta (Misra and Datta 2001). Pre-treatment with low temperature improved the regeneration potential of plant tissues. The enhancement of plant regeneration by low temperature treatment was related to the alternation of endogenous auxin-cytokinin balance and redox-state which played a key role in the plant growth and development (Hou et al. 1997, Merce et al. 2003, Andersone and Levinah 2005). Guo et al. (2007) reported an efficient micropropagation system for Saussurea involucrata, an endangered Chinese medicinal plant through leaf explants. A maximum of 66.0% of shoot regeneration frequency and 5.2 shoots per explant were achieved when explants cultured on a medium containing 10.0 µM BA and 2.5 µM NAA. Shoot organogenesis was improved further when the leaf explants were pre-incubated at low temperature and 80.6% of shoot regeneration frequency was recorded with 9.3 shoots per leaf explant at 4 °C by 5-day pre-treatment period.
2.1.2.2 Effect of urea-based cytokinins on shoot regeneration
Similar to the meristem culture, TDZ has been successfully exploited for direct regeneration of shoot buds from leaf explant in a number of plant species (Feyissa et al. 2005). Like adenine-based cytokinins, TDZ in combination with a suitable auxin also favoured high frequency of shoot regeneration from leaf explants (Orlikowska and Dyer 1993). Radhika et al. (2006) reported high frequency of shoot regeneration and high number of shoots per regenerating leaf explant on a wide range
34
of TDZ and NAA combinations in Carthamus tinctorius. Later, Sujatha and Dinesh Kumar (2007) compared the efficacy of TDZ plus NAA and BA plus NAA combination for the leaf explants of eleven Carthamus species. They observed highly prolific adventitious shoot regeneration on MS medium supplemented with 0.2 mg dm-3 TDZ and 0.2 mg dm-3 NAA in C. tinctorius whereas 0.2 mg dm-3 TDZ plus 1.0 mg dm-3 NAA was found effective for shoot regeneration in C. arborescens. Zeng et al. (2008) reported an efficient micropropagation system using leaves as explants for Tigridiopalma magnifica. Up to 7.6 adventitious buds formed per leaf explant after a 40-day culture on MS medium containing 2.0 mg l-1 BA and 0.1 mg l-1 TDZ. During 30-day subculture, the proliferation rate of adventitious bud in cluster was 5.7 on MS medium supplemented with 2.0 mg l-1 BA and 0.5 mg l-1 NAA. Ma et al. (2011) established an efficient propagation and regeneration system via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia. Among various PGRs tested, 2.5 µM TDZ was found to be the most effective to induce a maximum of 36.7 shoots per leaf explant. Shoot regeneration capacity was further enhanced when auxin was added to TDZ. Among a wide range of cytokinin (Kn, BA and TDZ) and auxin combinations, 5.0 µM TDZ along with 0.5 µM NAA induced a maximum of 79.1 adventitious shoots from each leaf explant.
2.1.3 Cotyledon culture
Cotyledons are a potential source of regeneration because of their year-round availability, ease of culture initiation and applicability to a number of genotypes (Burger and Hackett 1982, Baker et al. 1999) and represent a good source not only for micropropagation studies but also serve as a target tissue for transformation studies (Franklin et al. 2004). Organogenesis from cotyledons was successfully obtained in Citrullus lanatus (Chaturvedi and Bhatnagar 2001), Dalbergia sissoo (Singh et al. 2002a), Glycine max (Sairam 2003), Capsicum annuum (Joshi and Kothari 2007), Pongamia pinnata (Sujatha et al. 2008). As far as the literature is concerned, single cotyledon explant can produce multiple shoot buds from the proximal cut ends due to the presence of highly meristematic cells (Guerra and Handro 1988). Similar results were also reported by Hisajima (1982) who found that up to 10 million shoots of almond species could be obtained from a single seed explant within a year after several subcultures. This type
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of response has been initiated from the seeds of many species, particularly legumes (Vasanth et al. 2004, Maina et al. 2010).
2.1.3.1 Effect of adenine-based cytokinins on shoot regeneration
Webb et al. (1984) showed that cotyledon age can influence the regeneration response; with older cotyledons has less ability for direct shoot regeneration than younger ones. According to Hunter and Burritt (2002), cotyledon age influences the shoot-forming ability of cotyledon explants. Zhang and Cui (2001) studied the stimulatory effect of different cytokinins on direct plant regeneration from 5-day old cotyledon explants in Cucumis sativus. 1.0 mg l-1 zeatin had a highest efficiency (85%) over BA, Kn, TDZ. Singh et al. (2002a) compared the regeneration potential of semi-mature and mature cotyledons lacking embryonic axes in Dalbergia sissoo. Shoot buds were induced from the proximal region of semi-mature cotyledons on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA. Adventitious shoot bud formation was also noticed from the mature cotyledons. However, unlike the semi- mature explants, the mature cotyledons exhibited shoot bud differentiation on MS medium containing 22.20 μM BA without NAA. Pre-culture of mature cotyledons in liquid MS medium containing 8.88 μM BA for 48 h improved shoot bud regeneration up to six-fold. Vega et al. (2006) examined regeneration efficacy from three different regions (proximal, middle and distal) of cotyledon explants in six sun flower inbred lines. A decreasing regeneration was observed from proximal to distal sections for all inbred lines. Shoot differentiation depends upon the presence of proximal region of explant regardless of the genotype. Maximum regeneration frequency (87.1%) was noticed for
N 834 genotype. This was in accordance with other studies in which regenerated plants were obtained from cotyledons at high level of cytokinins (Joshi and Kothari 2007). Rashid et al. (2010) assessed the in vitro response of two genotypes of tomato (Lycopersicon esculentum) viz., Punjab Upma and IPA-3 for direct regeneration from cotyledon explants. They noticed that direct regeneration was significantly influenced by the genotype. The MS medium supplemented with Kn (0.5 mg l-1) and BA (0.5 mg l-1) was found optimum for inducing direct shoot regeneration. At this combination of
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BA and Kn Punjab Upma exhibited a better response in terms of shoot regeneration per cent (92.49) and average number of shoots per explant (4.78) in when compared to IPA-3. Chaturvedi et al. (2010) compared the regenerative potentiality of cotyledon explants of some Indigenous varieties of Cucurbits. The 7 day-old seedlings were used as explant source. They reported direct shoot regeneration for the first time from cotyledons of Cucurbita pepo under the influence of BA (5.0, 10.0 μM) and 3.0 μM each of BA and 2-iP. Indirect as well as direct regeneration was observed in Cucumis melo var. utilissimus; BA alone (5.0, 10.0 μM) supported shoot-bud differentiation indirectly via callusing while in combination with 2-iP at 1.0 μM each promoted direct regeneration of shoot-buds in cultures.
2.1.3.2 Effect of urea-based cytokinins on shoot regeneration
Fragmentary reports are available on TDZ mediated direct organogenesis from cotyledonary leaves. In most of the cases TDZ has been found to induce indirect organogenesis (Radhika et al. 2006). Murthy et al. (1996) achieved stimulation of direct organogenesis and somatic embryogenesis from cotyledons of Cicer artietinum when implanted on BA and TDZ amended MS medium. Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective as compared to BAP as an inductive signal of regeneration. The TDZ induced multiple shoot formation at all the concentrations tested (1.0 µM to 10.0 µM), although maximum morphogenic response was observed at 10.0 µM TDZ. Addition of NAA alone or in combination with BAP to the MS medium failed to invoke similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis (Murthy et al. 1996). High frequency of adventitious shoot regeneration (33.33%) and the highest number of shoots per explant (6.5) from cotyledons of Carthamus tinctorius was optimized at 0.5 mg l-1 TDZ and 0.25 mg l-1 IBA containing MS medium (Başalma et al. 2008).
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2.2 Indirect organogenesis
The undifferentiated mass of profusely dividing cells known as callus and callus mediated regeneration is termed as indirect organogenesis. In vitro callus can be induced from various parts of the plants like shoot tip, node, inetrnode, hypocotyl, cotyledon, root, leaf or floral organs and has multiple uses (Pande et al. 2002, Khurana et al. 2005, Sahai et al. 2010, Parveen and Shahzad 2011). The induction of callus growth and subsequent differentiation and organogenesis is accomplished with the differential application of growth regulators and the controlled environmental conditions in the culture medium (Tripathi and Tripathi 2003). Explants when cultured on the appropriate medium, usually with both an auxin and cytokinin, gave rise to an organized, growing and dividing mass of cells. In culture, callus proliferation can be maintained more or less indefinitely, provided that the callus is sub-cultured on the fresh medium periodically. During long-term culture, the culture may lose the requirement for auxin and/or cytokinins. This process is known as ‘habituation’. Gao and Bjork (2000) reported callus induction and plant regeneration in shoot tip explant of Valeriana officinalis with the manipulation of various combination and concentrations of auxins (IAA, IBA and NAA) and cytokinins (BA and Kn). Influence of different PGRs on high frequency plant regeneration via leaf callus was also reported in Coleus forskholii (Reddy et al. 2001). Rehman et al. (2003) reported regeneration via leaf derived callus of Cichorium intybus on modified MS medium containing 2.0 mM IAA, 5.0 mM Kn and 1000 mg l-1 casein hydrolysate (CH) with the production of at least five or more shoots from each callus. Efficient regeneration system has also been achieved from leaf derived callus in Solanum laciniatum (Okslar et al. 2002). Koroch et al. (2003) established a protocol for the induction of adventitious shoots from leaf calli of E. pallida. They reported optimum shoot regeneration frequency (63%) and number of shoots per explants (2.3 shoots per explants) on 26.6 µM BA and 0.11 µM NAA containing MS medium. Faisal and Anis (2003) reported callus formation from leaf explants of Tylophora indica with the application of dichlorophenoxy acetic acid (2,4-D) or trichlorophenoxy acetic acid (2,4,5-T) in which 100% cultures showed callus induction on MS medium supplemented with 2,4,5-T at high level of 10.0 µM. The characteristics of calli were also greatly influenced by the concentration of auxins and
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cytokinins. In general, maximum callus induction frequency was observed on a high level of auxin with low cytokinin level. Nodular callus was initiated from young leaf segments of Pluchea lanceolata, when cultures on Wood and Braun medium (1961) containing 2.0% sucrose and 5.0 mg l-1 Kn (Kumar et al. 2004). However, considerable amount of callus formation was observed with the combination NAA and BA in Leucaena leucocephala (Maity et al. 2005). The maximum callus induction frequency from stem callus of Ruta graveolens was observed in a combination of 2, 4- D and BA (Faisal et al. 2006b). Explants cultured on control medium (PGR-free MS medium) became necrotic and showed no sign of callus formation. Nandagopal and Ranjitha Kumari (2006) used ADS for high frequency shoot organogenesis from leaf- derived callus of Cichorium intybus. They observed highest percentage of callus
induction and multiple shoots proliferation was on MS plus B5 medium containing 6.66 μM BA, 2.85 μM IAA and 1.36 μM ADS. Auxins generally stimulate callus formation, but in some cases phenyl urea derivative i.e., TDZ was also found to possess callus induction properties. Phippen and Simon (2000) reported callus and shoot induction in Ocimum basilicum when leaf explants were placed on MS medium supplemented with 16.8 μM TDZ alone. The combination of TDZ with an auxin (NAA) greatly influences the callus formation frequency in leaf explants of Cimicifuga racemosa (Lata et al. 2002). Shahzad et al. (2006) documented maximum callus formation from mature green cotyledons on 0.6 µM TDZ supplemented MS medium in Acacia sinuata. In Hydrastis canadensis, high frequency of indirect organogenesis was achieved on 2.5 μM TDZ and 5.0 μM NAA, however sub-culturing of the parent tissue on BA (5.0 μM) containing medium maximized the production of shoots (He et al. 2007). Faisal et al. (2005) developed a protocol for high-frequency shoot regeneration and plant establishment from petiole- derived callus of Tylophora indica. In this plant, optimal callus was developed from petiole explants on MS medium supplemented with 10.0 µM 2, 4-D and 2.5 µM TDZ. Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5 µM TDZ. For Phyllanthus amarus, Nitnaware et al. (2011) reported maximum callus induction from leaf explant on 2.26 µM 2, 4-D and 2.32 µM Kn that exhibited higher shoot regeneration (32.4 shoots per culture) after transfer to MS medium containing TDZ.
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2.3 Other factors influencing regeneration
Several factors influence the efficiency of in vitro regeneration such as basal medium, growth regulators, types of additives, age of explants, age of culture, photoperiod which have been time-to-time reviewed by various workers (Batra 2001).
2.3.1 Effect of different culture media on shoot regeneration
One of the most important factors governing in vitro growth and morphogenesis of plant tissues is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants. Several media formulations are used for the majority of all cell and tissue culture work. These media formulation include those described by Murashige and Skoog (MS
1962), White (1963), Linsmaier and Skoog (1965), Gamborg et al. (B5 1968), Nitsch and Nitsch (NN 1969), Schenk and Hilderbrandt (SH 1972) and Lloyd and McCown (WPM 1980). The development of culture medium formulations was a result of systematic trials and experimentations. The MS medium was developed for tobacco, based primarily on the mineral analysis of tobacco tissue. Previously, it was referred as a ‘high salt’ medium due to its high content of potassium and nitrogen salts. The LS medium is basically MS medium with respect to its inorganic portion, but only inostiol and thiamine HCl are retained among the organic components. The B5 medium was devised for soyabean callus culture and has lesser amounts of nitrates + and especially NH4 than MS. Although, B5 was originally developed for the purpose of obtaining callus or for use with suspension culture but it also works well as a basal medium for whole plant regeneration. The SH medium was also formulated for callus culture of monocots and dicots while the White’s medium was designed for tissue culture of tomato roots. Whereas, the NN medium came in to existence for anther culture and contains a salt concentration intermediate to that of MS and White’s media (Beyl 1999). For most of the plant species MS medium was proved to be the best for micropropagation studies. Whereas, Faisal et al. (2007) examined the effect of different strengths of MS medium (¼ MS, 1/3 MS, ½ MS and MS), each comprising 2.5 µM BA, 0.5 µM NAA and 100 mg l-1 ascorbic acid for axillary shoot proliferation
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in Tylophora indica. A sharp decline in shoot proliferation efficiency of the explant was noticed on gradual reduction in salts’ concentration.
However, Chuenboonngarm et al. (2001) successfully used B5 medium for micropropagation of Gardenia jasminoides through shoo tip cultures. Similarly, Nandagopal and Ranjitha Kumari (2006) reported the highest percentage of callus
induction and multiplication shoot proliferation on MS and B5 medium supplemented with 6.66 µM BA and 2.852 µM IAA and 1.360 µM ADS. While, in nodal culture of Terminalia bellerica a maximum number of shoots per explant (10.6) was obtained on SH medium, but shoots were stunted and exhibiting yellow leaves which intensified on subsequent subcultures on the same fresh medium. However, growth of shoots was better on MS medium (Rathor et al. 2008). Woody plant medium (Lloyd and McCown 1980) has been reported to be more suitable medium for in vitro regeneration study of woody tree species. The pivot role of WPM for shoot proliferation has been reported in Cornus florida (Kaveriappa et al. 1997). The comparative influence of different culture media formulations on in vitro response has been assessed by various groups. In Gymnema sylvestre, Komalavalli and Rao (2000) found MS medium as the best basal medium for shoot sprouting
(62%), number (3.2) and length (2.2) with little callus formation followed by B5, SH, WPM and white’s medium. They noticed that shoot buds sprouted on White’s medium showed only limited development even if they were maintained for longer period. Similarly, in vitro propagation of various plants belonging to Asclepiadaceae has also been shown to have optimum growth in MS medium (Chi Won and John 1985, Pattnaik and Debata 1996, Komalavalli and Rao 1997). Husain et al. (2008) and Husain and Anis (2009) achieved the highest shoot multiplication as well as shoot length on MS basal medium over half-strength MS,
WPM and B5 basal media in Pterocarpus marsupium and Melia azedarach respectively. Similar results have also been reported on many woody plant species including Swartzia madagascariensis, Lagerstromia parviflora and Smilax china (Berger and Schaffner 1995, Tiwari et al. 2002, Song et al. 2010). Song et al. (2010) provided a comparative analysis of different culture media formulations (½ MS, MS,
2MS, WPM, B5 and SH) to achieve an efficient micropropagation protocol for Smilax china. As reported in most of the studied the MS medium exhibited higher growth than those of others. When three different strengths of MS medium were considered, ½ MS resulted in the highest shoot regeneration over MS and 2MS basal media. 41
On the other hand, Warakagoda and Subasinghe (2009) compared these media
(MS, WPM and B5) for in vitro seed germination of Jatropha curcas. In their study
among three media tested, B5 was the best for seed germination of J. curcus.
Therefore, for culture establishment only B5 was selected. Mhatre et al. (2000) used four different nutrient media with various modifications to determine their role in shoot regeneration in Vitis vinifera. The following media compositions were chosen for the micropropagation protocol; G16, initiation medium, comprised of NN major and minor salts, LS vitamins, Fe EDTA, 2% (w/v) sucrose, 10 mg l-1 thiamine HCl, 40.53 mg l-1 ADS, 218.4 mg l-1 monobasic sodium phosphate, 2.25 mg l-1 BAP and 0.09 mg l-1 NAA.
GM2, multiplication medium, comprised of WPM major and minor salts, B5 vitamins, Fe EDTA, 3% (w/v) sucrose, 2 mg l-1 calcium pantothenate, 168 mg l-1 monobasic sodium phosphate 0.5 mg l-1 IBA and 2.2 mg l-1 BAP MS2, shoot elongation medium, comprised of MS major and minor salts, MS vitamins, Fe EDTA, 2% (w/v) sucrose, 0.5 mg l-1 Bap and 0.2 mg l-1 IAA GR1, rooting medium (liquid), comprised of half-strength MS major and minor salts, full strength MS vitamins, Fe EDTA, 1% (w/v) sucrose and 0.1 mg l-1 IAA.
They reported that these modified media (G16 and GM2) resulted in healthy proliferation, none of the culture exhibited hyperhydricity. For this they suggested that both G16 and GM2 media contain monobasic sodium phosphate in addition to + BAP and this could be responsible for a synergistic effect of cytokinins and NH4 ions. In a report of 2004, Nas and Read hypothesized that the composition of minerals and organic substances in proportions similar to those found in the seed composition could provide an optimum tissue culture medium for micropropagation of higher plants. Their hypothesis would help to avoid factorial treatments, labour and explant requirement for obtaining defined tissue culture medium with optimum response. Using their hypothesis, they first developed a new tissue culture medium (Nas Medium, NM 2004) for hybrid hazelnuts (Corylus avellana). Threefold higher shoot length was observed on NM medium than any other media. Moreover, potential multiplication rates observed on NM (up to 107%) and WPM (up to 85%) were higher than those on other media. When the composition of NM was further improved (Nas
42 and Read medium, NRM) in accordance with their hypothesis, shoot length (up to three fold) and potential multiplication rate (up to 93%) were further enhanced.
2.3.2 Effect of different carbon sources on shoot regeneration
Carbohydrate compounds, normally found in the sieve-tube exudates of plants have been positively related with a suitable carbon source used in plant tissue culture medium (Welander et al. 1989). It is well documented that a specific carbohydrate may have different effects on morphogenesis in vitro. A carbohydrate, generally sucrose, is an indispensible ingredient of all culture media, as the photosynthetic ability of cultured tissue is limited because of low irradiance and limited gas exchange (Kozai 1991). It is also required as an osmotic agent (Thorpe 1985). Being easily translocable and resistant to enzymatic degradation due to non-reducing nature, sucrose is the most effective of choice among various carbohydrates for of plant tissue culture studies (Pontis 1978). But, now it is well established that carbohydrate requirements may show differences according to the species (Thompson and Thorpe 1987). A concentration of 20 to 40 g l-1 sucrose (a disaccharide made up of glucose and fructose) is the most often used carbon or energy source, since this sugar is also synthesized and transported naturally by the plant. Whereas, Murashige and Skoog (1962) stated that the use of 3% sucrose is better than 2 or 4%. The sugar concentration chosen is dependent on the type and age of the explant in culture. To justify this fact, Gürel and Gülsen (1998) investigated the requirement of sucrose concentration during three successive stages, namely initiation, transplantation and multiplication for Amygdalus communis cultures. Comparatively higher concentration of sucrose (5 and 6%) was required during initiation and transplantation stages as compared to multiplication phase (3 and 4%). Although sucrose is the most widely used carbohydrate in tissue culture, some reports indicate that it may cause hypoxia and ethanol accumulation due to fast metabolism and result in a significant decrease in osmotic potential of the medium (Neto and Otoni 2003). These conditions could in turn interfere with the nutrient uptake process. This interference would most likely result in the failure of absorption of diffusion or diffusion of some important elements. In such a critical situation, some reducing sugar like mono- or disaccharides and sugar alcohols such as glucose,
43 fructose, sorbitol and maltose may also be used to find an alternative carbon source (Nicoloso et al. 2003, Mosaleeyanon et al. 2004, Skrebsky et al. 2004, Pati et al. 2006, Rodrigues et al. 2006, Bandeira et al. 2007, Luo et al. 2009, Dobránszki and da Silva 2010, Mohamed and Alsadon 2010). The response of in vitro cultures to different carbon sources added to the medium has been compared for a number of plant species. Among three carbon sources (glucose, fructose and sucrose), sucrose proved to be the best for shoot regeneration in Pentanema indicum (Sivanesan and Jeong 2007). Similar result was obtained in Artemisia vulgaris (Sujatha and Ranjitha Kumari 2008). In fact, sucrose has been commonly used as a carbon source in tissue culture media (Fuentes et al. 2005). This is due to its efficient uptake across the plasma membrane (Borkowska and Szezebra 1991). Both sucrose and glucose gave a similar rate of proliferation in sour cherry (Borkowska and Szezebra 1991), Bixa orellana (Neto and Otoni 2003). While, Debnath (2005) reported the best response at 20 g l-1 sucrose in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well or better than sucrose depending upon cultivar type of Vaccinium vitis- idaea. But, carbohydrate concentration had a little effect on shoot vigour. Abou- Rayya et al. (2010) found glucose as the most effective carbon source for stimulating the production of shoots, fresh weight and shoot length followed by sucrose and fructose. In Solanum nigrum, Siridhar and Naidu (2011) also reported the highest number of shoots (24.0) on 4% fructose, but maximum shoot length (11.0 cm) was observed on 4% sucrose. The results obtained are in line with the earlier observation in Mulbery (Vijaya Chitra and Padmaja 2001), where addition of fructose instead of sucrose in the multiplication medium increases the shoot number and also growth of shoots. Sorbitol, a polyol that occurs abundantly in plant species, is a good carbon source for Malus species and Prunus persica tissue culture (Chong and Taper 1972, Coffin et al. 1976). The promotive effect of sorbitol on shoot multiplication rather than sucrose has also been reported for some plant species of Rosaceae (Pua and Chong 1984, Kadota et al. 2001, Ahmad et al. 2007). Energy source has also found to enhance the alkaloid content along with optimum morphogenesis. In Nepeta rtanjensis, Misic et al. (2005) noticed significant enhancement in shoot growth and nepetalactone accumulation on glucose. Besides, 44
they also determined the effect of different concentrations of carbohydrates in culture media on the internal carbohydrate status for the same plant species.
2.3.3 Effect of different pH on shoot regeneration
Scragg (1993) reported that at low pH, cells release H+ to the extracellular + environment affecting the absorption of nutrients, especially the NH4 while at high - - pH, cells release OH ions thus the absorption of NO3 is adversely affected. Based on the current report, it is hypothesized that the inhibitory effect of pH on shoot height is + - likely to be due to reduced uptake of NH4 and NO3 at low and high pH respectively. Every species requires an optimum pH for shoot regeneration and their subsequent proliferation. In most of the studies optimum regeneration was achieved at 5.8 pH (Sahai et al. 2010, Shahzad et al. 2011). However, Bhatia and Ashwath (2005) and Naik et al. (2010) suggested the requirement of acidic pH for maximum biomass production of Lycopersicon esculentum (5.5 pH) and Bacopa monniera (4.5 pH) respectively. Martins et al. (2011) studied the influence of low pH (4.5, 5.0 and 5.75) on in vitro growth and biochemical parameters ((lipid peroxidation, proline and carbohydrate content, antioxidant enzymes activities and total soluble protein) of Plantago almogravensis and P. algarbiensis. It was observed that medium pH did not affect in vitro proliferation and rooting. Interestingly, cultures of both species modify the initial pH value to the same final value. Results have shown that the lowest pH tested induced an increase in the level of lipid peroxidation in roots of both species and in shoots of P. algarbiensis, indicating plasma membrane damage. An accumulation of carbohydrates was observed in roots of P. almogravensis cultured at pH 4.5 and 5.0. Based on the results obtained it was concluded that Plantago species are apt to grow in vitro in medium with pH values much lower than the usually used in tissue culture, which is in agreement with the fact that both species colonize acid soils.
2.4 In vitro rooting of microshoots
Rooting is an important step in micropropagation studies (Moncousin 1991). Although, a number of plants root spontaneously in culture (some monocotyledons and other herbaceous species), shoots of most species multiplied in vitro lack a root
45 system (Yeoman 1987). Rooting can be achieved either by transferring the microshoots to medium lacking cytokinins with or without a rooting hormone or by treating the shoots as conventional cutting after removal from sterile culture medium. There is a great variation between species in the ease with which cultured shoots can be rooted and systematic trials are often needed to find the most effective conditions for rooting. All cytokinins inhibit rooting and BA (which is widely used for shoot multiplication) does so strongly, even after transfer to cytokinins-free medium (George and Sherrington 1984). The use of Kn or 2-iP in place of BA in the final stage of multiplication often improves subsequent rooting (e.g. in Pinus as reported by Webb and Street 1977). On the other hand, MS basal medium devoid of PGR has been found to induce in vitro rooting in some plant species (Cristina et al. 1990, Saxena et al. 1998, Faisal and Anis 2003, Ray and Bhattacharya 2008). The ease of root formation on auxin free medium may be due to the availability of endogenous auxin in the regenerated shoots (Minocha 1987). The concentration of rooting hormone (generally auxin) is often required to provide sufficient stimulus to initiate roots while preventing the excessive formation of callus. Root elongation may be inhibited by the levels of auxin required to initiate roots and the use of IAA which rapidly breaks down in cultured tissues is a useful way of overcoming this problem without having to provide a second rooting medium. The requirement of IAA for best rooting has also been reported in Artemisia vulgaris (Sujatha and Ranjitha Kumari 2007), Stevia rebaudiana (Ahmed et al. 2007, Anbazhagan et al. 2010), Gerbera jamesonii (Gantait et al. 2010) etc. Many species require the stronger axuin i.e., IBA or NAA to stimulate root fromation. Among the auxins tested, IBA was found to be the most suitable for in vitro rooting (Sreekumar et al. 2000, Faisal and Anis 2002, Liu et al. 2003, Sivaram and Mukundan 2003, Anis and Shahzad 2005, Feyissa et al. 2005, Faisal et al. 2005a, Faisal et al. 2006c, Faisal et al. 2007). Half-strength MS with IBA was proved to the best for in vitro root induction in Saussurea obvallata (Dhar and Joshi 2005), Centella asiatica (Mohapatra et al. 2008) and Spilanthes acmella (Saritha and Naidu 2008). However, NAA usually give rise to short and thick roots which may have the advantage of being better able to withstand accidental damage during planting out (Lane 1979). The stimulatory effect of NAA on root formation has been reported in many medicinal plants like Tagetes erecta (Misra and Datta 2001), Carthamus
46
tinctorious (Radhika et al. 2006), Trichosanthes dioica (Malek et al. 2007), Lysimachia species (Zheng et al. 2009) Sometimes combination of two different auxins or axuin with cytokinin found to induce optimum rooting response. The combination of IBA and IAA was found to induce best rooting in Ficus religiosa (Siwach and Gill 2011). Regenerated shoots of Anthurium andraeanum were best rooted on half-strength MS medium with 0.54 µM NAA and 0.93 µM Kn (Martin et al. 2003). Similarly, Beegum et al. (2007) reported best rooting in Ophiorrhiza prostrata when shoots were cultured on 10.74 µM NAA and 2.32 µM Kn containing half-strength MS medium. Different phenolic compounds like, phloroglucinol (PG), chlorengenic acid (CA) and salicyclic acid (SA) also facilitate in vitro rooting in recalcitrant species and PG was found to be a promotive phenolic compound for root induction in Pterocarpus marsupium (Anis et al. 2005, Husain et al. 2007a). However, Sakhanokho and Kelley (2009) observed that SA in combination with NaCl had a beneficial effect on root formation along with shoot multiplication and plant survival in Hibiscus moscheutos. Auxin promotes ethylene production that inhibits adventitious root formation in some species like pea cuttings (Nordstrom and Eliasson 1993) and Prunus avium shoot cultures (Biondi et al. 1990). Ma et al. (1998) demonstrated that the use of
ethylene inhibitors such as AgNO3 and CoCl2 may promote root formation in shoot
cultures of apple. Effect of AgNO3 on root fromation was also examined by Bais et al. (2000) for Decalepis hamiltonii. They reported best rooting response with the application of 40.0 µM AgNO3. However, Reddy et al. (2002) used triacontanol (TRIA) for in vitro rooting in D. hamiltonii.
2.5 Synseed production
Since the formulation of the concept of synseed by Murashige (1977), a number of studies have been undertaken in this area of plant biotechnology. The first report on synseed development was published by Kitto and Janick (1982) who produced desiccated carrot synseeds by coating the multiple somatic embryos in a water-soluble resin, polyoxyethyelene glycol (Polyox). Later on, Janick et al. (1993) extended this technology for encapsulating a mixture of carrot somatic embryos and embryogenic calli using Polyox. Redenbaugh et al. (1984) was the first to develop
47
hydrogel encapsulation technique for somatic embryos of alfalfa. Since then, encapsulation in hydrogel remains to be the most studied strategy of synseed production (Ara et al. 2000, Rai et al. 2009). A number of coating agents such as agar, sodium alginate, potassium alginate, sodium pectate carrageenan, sodium alginate with carboxymethyl cellulose, gelatin, gelrite, guargum, tragacanth gum etc. have been tested as hydrogels (Ara et al. 2000, Rai et al. 2009). Among these, sodium alginate has been frequently selected because of its moderate viscosity, low spin ability of solution, low toxicity, quick gellation, low cost and bio-compatibility characteristics (Swamy et al. 2009). Moreover, sodium alginate and calcium salt has been reported as the best combination since the ions are non-damaging, easy to use, have a low-price and provide an easy germination of encapsulated propagules. The encapsulation matrix composition is one of the important factors significantly affecting the re-growth performance of encapsulated tissue. For effective re-growth, the requirements of definite ingredients of hydrogel matrix (inorganic, organic, PGRs, carbohydrate etc.) are species specific. Generally, the addition of nutrients to the gel matrix results in improved re-growth performances (Chand and Singh 2004, Sundararaj et al. 2010, Ahmad and Anis 2010). Additionally, synseeds are reported to be highly susceptible to bacterial, fungal and other infections in the greenhouse (Vij and Kaur 1994). To reduce microbial contamination, various antimicrobial agents such as bavistin (Pattnaik et al. 1995), vitrofural G-1 (Nieves et al. 2003), plant preservative medium (PPM) (Micheli 2002) could be added to the gel matrix. However, such chemicals induce impairment in convertibility that could be successfully alleviated by adding PGRs in gel matrix. In agreement to this, additive effect of PPM and thidiazuron (TDZ) on overall plantlet development was observed by Lata et al. (2009) in Cannabis sativa synseeds. Previously, the synseed production was limited mostly to those plants in which somatic embryogenesis had been reported, but embryogenesis was not well documented for most of the plant species. In response to this, the possibility of using non-embryogenic vegetative propagules such as shoot tips, nodal segments, organogenic or embryogenic calli etc. has been explored as a suitable alternative to somatic embryos (Ara et al. 2000, Danso and Ford-Llyod 2003, Rai et al. 2008, Faisal and Anis 2007, Sharma et al. 2009a & b, West and Preece 2009, Ahmad and Anis 2010, Ozudogru et al. 2011). 48
With the use of such non-embryogenic plant propagules, synseed technology has been widely exploited to a range of plant species such as Morus spp. (Pattnaik et al. 1995, Pattnaik and Chand 2000), Eucalyptus grandis (Watt et al. 2000), Adhatoda vasica (Anand and Bansal 2002), Dalbergia sissoo (Chand and Singh 2004), Ananas comosus (Gangopadhyay et al. 2005), Chonemorpha grandiflora (Nishitha et al. 2006), Punica granatum (Naik and Chand 2006), Cannabis sativa (Lata et al. 2009), Spilanthes mauritiana (Sharma et al. 2009b), Zingiber officinale (Sundararaj et al. 2010), Vitex negundo (Ahmad and Anis 2010) etc. Amongst various vegetative propagules, nodal segments are most suitable for encapsulation studies as they posses pre-existing axillary meristem, however in vitro root induction is a major obstacle encountered in case of recalcitrant woody plant species. It is assumed that encapsulation inhibits the oxygen supply to explants and suppresses root induction (Piccioni 1997). Thus, to induce rooting different approaches have been exploited for root induction in synseeds. Piccioni (1997) suggested a method of incubating the explants in dark for root primordia induction and thereafter addition of PGRs in the gel matrix for higher conversion from synseeds. Pre-treatment of explants either with cytokinin or auxin was also suggested for improved synseed conversion frequency (Pattnaik et al. 1995, Soneji et al. 2002, Chand and Singh 2004, Germanà et al. 2011). Pinker and Abdel- Rahman (2005) emphasized that the addition of IAA to the gel matrix (prepared in modified MS) exhibited 100% root formation in encapsulated nodal segments of Dendraanthema × grandiflora. Nishitha et al. (2006) suggested the addition of silver
nitrate (AgNO3) along with IBA to enhance the conversion frequency in synseeds of Chonemorpha grandiflora. Addition of growth regulators to the germination medium eliminates the requirement of an additional in vitro root induction experiment prior to acclimatization. Ahmad and Anis (2010) found that the addition of Kn and NAA to MS basal medium improved the germination frequency of synseeds and induced a mean of 2.8 roots per synseed of Vitex negundo. Similar response has also been recorded previously for Pimpinella pruatjan (Roostika et al. 2006) and Tylophora indica (Faisal and Anis 2007). On the other hand, Gangopadhyay et al. (2005) devised a two step method to achieve maximum synseed conversion into complete plantlets in Ananus comosus; firstly, the microshoots were retrieved from synseeds and in the
49
second step, these microshoots were rooted in liquid medium (supplemented with IBA and Kn) supported with Luffa-sponge. Although a large number of plants can be produced in tissue culture through direct or indirect embryogenesis and organogenesis, but their delivery is cumbersome. Direct sowing of the synseeds in the soil or other type of substrates helps in escaping acclimatization procedure. The technique provides an ideal delivery system enabling easy flexibility in handling and transport of propagules as compared to large parcels of seedlings. Thus, germination of synseeds on nutrient free substrate is a prerequisite for sowing under non-sterile condition. In this context, Mandal et al. (2000) suggested that the successful conversion of synseeds into plantlets on simple planting substrate such as sand/soil/soilrite/vermi- compost is necessary for their use in commercial-scale propagation. Still successful germination of encapsulated tissues on various planting substrates has been reported only for a few plant species either in a controlled culture room environment or greenhouse conditions. The major limiting factor for reduced germination is the low- nutrient availability. Therefore, it is necessary to build up a nutrient reservoir for the encapsulated plant tissue, either endogenously or exogenously. Kavyashree et al. (2006) exogenously supplied half-strength LS nutrients in horticultural grade soilrite mix (peat: perlite: vermiculite 1:1:1) for ex vitro germination of mulberry synseeds with healthy shoot and root systems. Similarly, Lata et al. (2009) reported 100% conversion of synseeds on 1:1 potting mix-fertilome with coco natural growth medium, moistened with full-strength MS medium with 3% sucrose and 0.5% PPM in Cannabis sativa. Synseed technology also acts as a tool of germplasm exchange and short term conservation for rare and endangered plant species. For this purpose synseed storage is a critical factor which determines their successful germination after transportation abroad. Therefore, appropriate storage conditions and definite storage period are prerequisites to maintain viability during exchange of germplasm for successful commercialization of synseed technology. For short to medium-term storage, the aim is to increase the interval between subculture by reducing growth. In this respect, various strategies have been applied for slow growth maintenance of cultures. Low temperature and light intensity induce modifications in the physiology of stored explants, such as reduced respiration, water loss, wilting and ethylene production, thus allowing the storage of cultures from several months to years without 50
the necessity of transferring to fresh medium (Ozudogru et al. 2010). The temperature requirement for optimum viability varies from plant to plant. Generally, 4 ºC temperature is found to be most suitable for synseeds storage (Saiprasad and Polisetty 2003, Kavyashree et al. 2006, Faisal et al. 2007, Singh et al. 2007, Pintos et al. 2008, Sharma et al. 2009a & b, Ikhlaq et al. 2010, Tabassum et al. 2010). Gangopadhyay et al. (2005) stored the synseeds of Ananas comosus in different racks of a refrigerator with a range of temperature (4, 8, 12 and 16 ºC) for 60 days. Among the four temperature regimes, the beads stored at 8 ºC showed maximum germination frequency when allowed to re-grow again on nutrient media. Ray and Bhattacharya (2010) optimized best storage environment for Eclipta alba synseeds by changing in vitro physicochemical conditions. They extended storage duration up to 12 weeks by decreasing the sucrose concentration in the alginate matrix from 3 to 1 or 2%. Adriani et al. (2000) has also reported the pronounced effect of sucrose on re-growth ability of synseed and suggested that the sucrose availability can be a limiting factor in conversion ability of Actinidia synseeds.
2.6 Acclimatization of plantlets
The ultimate success of micropropagation depends on the ability to transfer and re-establish vigorously growing plants from in vitro to green house conditions. This involves acclimatization or hardening-off plantlets to conditions of significantly lower relative humidity and higher light intensity. Although, micropropagation has been extensively used for the rapid multiplication of various plant species and considerable efforts have been directed to optimize the conditions for in vitro stages of micropropagation, but the process of acclimatization of tissue culture raised plants to the natural environment has not been fully studied (Hazarika 2003). Micropropagation is often restricted due to high percentage of plantlets’ death during ex vitro transplantation (Pospisilova et al. 1999). The acclimatization of micropropagated plants remains a critical stage; in the first week after transfer to ex vitro conditions, plantlets cope with the different stresses and have to adapt to the external environmental conditions (Aragon et al. 2005). In fact, microparopagated plants are difficult to transplant due to following of two primary reasons:
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i) A heterotrophic mode of nutrition ii) Poor control of water loss (Kane 2000). A number of researches have been conducted to solve various problems related to acclimatization such as relative humidity and indicate that high humidity increase the survival percentage of micropropagated plants (Kozai 1991). A composite of anatomical and physiological features, characteristic of in vitro plant produced in 100% relative humidity, contributes to the limited capacity of microprapagted plants to regulate water loss immediately following transplanting. These features include no epicuticular wax formation, poor cuticle development, poorly differentiated mesophyll, poor connection between shoots and roots and improper function of stomata resulted in excessive water loss and poor photosynthetic capacity in ex vitro acclimatized plants (Ziv 1991, Kane 2000, Chen et al. 2006). Leaf surface covering agents, such as glycerol, paraffin and grease also promoted ex vitro survival of herbaceous species, but have not been evaluated over a long-term or examined on woody species (Selvapandiyan et al. 1988). Several growth retardants which reduce damage due to wilting without deleterious side effect have been suggested to improve ex vitro survival of regenerants. Absiccic acid (ABA) is considered as a growth retardant which may alleviate ‘transplantation shock’ and speed up acclimatization of tobacco plantlets under ex vitro conditions (Pospisilova et al. 1998). Ray and Bhattacharya (2008) established an efficient and simple protocol for in vitro propagataion of Eclipta alba including successful transplantation by priming the plantlets with a growth retardant, chlorocholine chloride (CCC) for the first time. Among various concentrations of CCC, 6.33 µM was found most effective for inducing certain beneficial changes. In 30 day-old treated shoots, they observed an increased number of roots, elevation in chlorophyll content and plant biomass. They reported that the arrested undesirable shoot elongation made the plants sturdy and more suitable for acclimatization. The primed plants exhibited 100% survival frequency as compared to control plants (84%). Priming of micropropagated propagules has already been recommended for obtaining better acclimatized plants (Nowak and Shulaev 2003, Hazarika 2003). The concept of priming the tissue culture raised plants to improve acclimatization is based on the fact that certain chemicals effectively pre-sensitize cellular metabolism of plants (Nowak and Shulaev 2003) and increase the adaptive ability of in vitro cultures (Conrath et al. 2002, Nowak and Pruski 2004). 52
Chapter 3 Materials & Methods
Chapter 3 MATERIALS AND METHODS
3.1 Plant material and explant source
The plant materials viz., nodal segments, shoot tips, leaves and cotyledons were excised from 3 week-old aseptic seedlings of S. acmella, S. mauritiana and D. hamiltonii.
3.2 Culture media
In vitro regeneration of whole plant greatly depends on the composition of media used. Several workers have used various formulations of culture media depending on their requirements. In the present study, the basal culture medium proposed by Murashige and Skoog (1962) was used for the in vitro cultivation of plant tissues. This medium is composed of three basic components; i. Essential elements (supplied as a complex mixture of salts) ii. An organic supplement (vitamins and amino acids) and iii. A fixed carbon source (generally supplied as sugar, sucrose). For practical purpose, the essential elements are further divided into the following two categories; a) Macro elements (major or macronutrients): Macro elements are classified as those elements which required in concentration greater than 0.5 mM l-1. They include nitrogen (N), phosphorous (P), potassium (K), calcium (Ca), magnesium + (Mg) and sulphur (S). Nitrogen is usually supplied in form of ammonium (NH4 ) - and nitrate (NO3 ) ions. Nitrate is superior to ammonium as the sole N source but use of ammomuim checks the increase of pH towards alkalinity. b) Micro elements (minor or micronutrients): Elements required less than 0.05 mM l-1. They are also termed as “trace elements”. These include manganese (Mn), zinc (Zn), boron (B), copper (Cu), molybdenum (Mo) and iron (Fe). Most critical of them being iron which is not available at low pH. Moreover, it has been observed that iron tartrate and citrate precipitate in the medium and pose difficulty in its utilization. Therefore, iron is used in a chelated form of EDTA (ethylene diamine tetra acetic acid) to make it available at wide range without
52
any precipitation. Na2EDTA usually used with FeSO4 which allows slow and continuous release of iron into the medium.
Besides, the SH, WPM and B5 media were used to compare the regeneration efficiency with MS medium. The different components of these nutrient media have been outlined in Table 2. Prepared SH, B5 and WPM media in powdered form were purchased from Duchefa, Netherlands. However, the MS medium was prepared in four separate stocks as described below.
3.3 Preparation of culture medium
3.3.1 Preparation of stock solutions
The constituents of MS medium given in Table 2 were prepared in four separate stocks (Table 3), consisting of I major salts (20x concentrated), II minor salts (200x concentrated), III iron salts (100x concentrated) and IV organic supplements (100x concentrated) by dissolving the required amounts in measured volume of double distilled water (DDW). These stock solutions were stored in dark bottles at 4°C in a refrigerator to prevent their photolysis and regularly checked for visible contamination. To prepare one liter of medium 50 ml of stock solution I, 5 ml of stock solution II and 10 ml each of stock solution III and IV were pipette out from the respective stock solutions using following formula;
S1V1 = S2V2 Where,
S1 = Strength of stock solution
V1 = Volume of stock solution required
S2 = Strength of desired solution
V2 = Volume of desired solution
3.3.2 Plant growth regulators (PGRs)
PGRs are the natural plant hormones and their synthetic analogous used for determining the developmental pathway of plant cells. In the present study, three main classes of PGRs namely cytokinins and auxins (adenine and urea derived) and gibberellins were used in different concentrations and combinations.
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Table 2. Nutritional composition of different culture media in mg l-1
Constituents MS medium B5 medium SH medium WPM (1962) (1968) (1972) (1980)
Macronutrients
MgSO4·7H2O 370 250 400 370 KH2PO4 170 - - 170 KNO3 1,900 2500 2500 - NH4NO3 1,650 - - 400 CaCl2·2H2O 440 150 151 96 NH4SO4 - 134 - - Ca(NO3)2·4H2O - - - 556 K2SO4 - - - 990 NaH2PO4 - 130.5 - - NH4 H2PO4 - - 300 -
Micronutrients
H3BO3 6.2 3.0 5.0 6.2 MnSO4·4H2O 22.3 10.0 10.0 22.3 ZnSO4·7H2O 8.6 2.0 1.0 8.6 Na2MoO4·2H2O 0.25 0.25 0.1 0.25 CuSO4·5H2O 0.025 0.025 0.2 0.25 CoCl2·6H2O 0.025 0.025 0.1 - KI 0.83 0.75 0.1 - FeSO4·7H2O 27.8 27.8 15.0 27.8 Na2EDTA·2H2O 37.3 37.3 20.1 37.3
Organic supplements
Vitamins Thiamine HCl 0.1 10.0 5.0 1.0 Pyridoxine HCl 0.5 0.1 0.5 0.5 Nicotinic acid 0.5 1.0 5.0 0.5 Myo-inositol 100 100 1000 100
Amino acid Glycine 2.0 - - 2.0
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Table 3. Nutritional composition of different stock solutions for MS medium Constituents (Strength) Amount (mg l-1) Stock solution I (20x)
MgSO4·7H2O 7,400
KH2PO4 3,400
KNO3 38,000
NH4NO3 33,000
CaCl2·2H2O 8,800 Stock solution II (200x)
H3BO3 1,240
MnSO4·4H2O 4,460
ZnSO4·7H2O 1,720
Na2MoO4·2H2O 50
CuSO4·5H2O 5
CoCl2·6H2O 5 Kl 166 Stock solution III (100x)
FeSO4·7H2O 2,780
Na2EDTA·2H2O 3,730 Stock solution IV (100x) Thiamine HCl 10 Pyridoxine HCl 50 Nicotinic acid 50 Myo-inositol 10,000 Glycine 200 pH 5.8
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• Auxins- induce cell division, cell elongation, apical dominance, adventitious root formation, somatic embryogenesis. When used in low concentration, auxins induce root initiation and in high, callus formation occurs. In the present study, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α- naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid (2,4-D) were used. • Cytokinins- promote cell division and stimulate initiation and growth of shoots in vitro. 6-Benzyladenine (BA), 6-furfurylaminopurine (Kn), 2- isopentanyladenine (2-iP) and thidiazuron (TDZ) were used in the present experimentation.
• Gibberellins- gibbrellic acid (GA3) is mostly used for internode elongation, seed germination and meristem growth. For each PGR, separate stock solutions were prepared by dissolving it in a small quantity of appropriate solvent (1N NaOH or absolute alcohol) and then the desired volume was adjusted with DDW to make an overall concentration of 1 mM. The different concentrations of growth regulators used in the present study were prepared from stock solutions by using foresaid formula;
S1V1 = S2V2 3.3.3 Carbon and energy sources
3% (w/v) sucrose was used throughout the experiment as a sole carbon and energy source. The effect of different energy sources like, sucrose, fructose, glucose and galactose on regeneration efficacy of the explants was also assessed with optimized combination of PGRs.
3.3.4 pH adjustment and gelling of the medium
The pH of the medium was adjusted to 5.8 by 1N NaOH using pH meter (L613, Elico Pvt. Ltd., India) before adding solidifying agent. Then medium was solidified with 0.8% (w/v) agar or 0.15% (w/v) phytagel and dissolved with the help of a microwave oven to make a homogenous solution. The effect of different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) on morphogenesis was also assessed using optimal combination of PGRs.
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3.3.5 Medium filling
The 15-20 ml nutrient medium was dispensed in 25 x 150 mm capacity culture tubes and 25 ml in 100 ml capacity wide mouth flasks. All the culture vessels containing media were plugged with non-absorbent cotton wrapped in a single layer of muslin cloth (cotton plug).
3.4 Sterilization
3.4.1 Sterilization of the medium
All the culture vessels containing media placed in plastic baskets which were wrapped with butter paper uniformly and autoclaved at 121 0C at 1.06 Kg cm-2 for 15- 20 min. After autoclaving culture tubes were tilted to prepare slant and leaved for overnight for solidification. These sterilized culture media were kept in the laminar air flow cabinet for further sterilization under ultraviolet (UV) radiations.
3.4.2 Sterilization of glass-wares, DDW and instruments
All the glass-wares, DDW and instruments like stainless steel forceps and scalpel were steam sterilized by autoclaving at 121 °C at 1.06 Kg cm-2 for 20 min after wrapping in aluminum foil or butter paper.
3.4.3 Sterilization of laminar airflow hood
Prior to inoculation of explants, the laminar air flow cabinet (horizontal type) was sterilized by exposing with UV rays (provided by 30 W UV tube, Phillips, India) at least for 15 min followed by switch on the air flow. The working bench of laminar airflow hood was disinfected by wiping with 70% ethanol before any experimentation.
3.4.4 Sterilization of seeds
The flower heads of S. acmella and S. mauritiana were procured from the Medicinal Plant Nursery of Tamnaar, Raigarh (Chhattisgarh) whereas the seeds of D. hamiltonii were collected from Central Food Technological Research Institute
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(CFTRI), Mysore (Karnataka). Similar procedure of seed sterilization was adopted for all the plant species. The healthy seeds were washed under running tap water for 30 min to remove any adherent particles. The seeds were kept in 1% (w/v) Bavistin (Carbendazim Powder), a broad spectrum fungicide, for 20 min and then washed in 5% (v/v) Teepol, a liquid detergent for 15 min. The treated seeds were agitated in sterilized DDW to remove the chemicals. The seeds were surface sterilized with 70% (v/v) ethanol and 2-3 drops (v/v) of Tween-20 for 30 s, followed by immersion in an aqueous solution
of 0.1% (w/v) HgCl2 for 3 min under the sterile conditions. Then seeds were washed 5-6 times with sterilized DDW to remove all traces of sterilants.
3.5 Inoculation of sterilized seeds and germination
The germination study was conducted through both in vivo and in vitro processes. In vivo experiment was carried out with uniform size pots containing garden soil and green manure (2:1) under field conditions. Watering was done on alternate days or as per required. The surface sterilized seeds were pre-incubated for 12 hrs in water (control) and in gibberellic acid (GA) at 0.5, 1.0 and 5.0 µM. Fifty and Twenty five per pot were used for Spilanthes species and D. hamiltonii respectively. For in vitro germination, the sterilized seeds were inoculated aseptically on to full- and half-strength MS medium amended with GA at 0.5, 1.0 and 5.0 µM. Ten and five seeds per culture tube were inoculated for both the Spilanthes species and D. hamiltonii respectively. The germination percentage and days to seedling formation were recorded periodically.
3.6 Culture establishment and shoot regeneration
Various plant parts like, nodal segments (1.0-1.5 cm), shoot tips (1.0 cm), leaves and cotyledons were excised from 3 week-old aseptic seedlings and used as the explants. Single explant was inoculated per culture tube containing regeneration medium. All these operations were performed under aseptic conditions of laminar airflow hood using sterilized instruments. The forceps and scalpel were time-to-time flame sterilized during inoculation by dipping them in rectified spirit followed by flaming and cooling. The MS basal medium with or without different cytokinins, BA,
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Kn, 2-iP and TDZ (1.0, 2.5, 5.0 and 7.5 µM) and auxins, NAA, IAA and IBA (0.1, 0.5 and 1.0 µM) either singly or in combination were tried to access the regenerative potentialities of explants. The data for shoot induction was recorded after 4 weeks of culture.
3.7 Sub-culturing and shoot proliferation
Regenerating cultures were cut into pieces and sub-cultured onto the optimized combination of PGRs dispensed in the flasks but the data were recorded as an aggregate of all the pieces. Sub-culturing was done up to sixth or eight passages with 3 or 4 weeks of interval as required.
3.8 In vitro rooting
For complete plantlets development in vitro rooting was performed. The in vitro raised microshoots (3.0-4.0 cm) with fully expanded leaves were harvested from shoot clusters and transferred to full- and half-strength MS media with or without auxins, NAA, IAA and IBA (1.0, 2.5 and 5.0 µM). The data for in vitro rooting was recorded after 4 weeks of culture.
3.9 Culture room conditions
All the culture vessels were placed in a culture room at 25 ± 2 °C under a 16-h photoperiod with 50 µmol m-2 s-1 photosynthetic photon flux density (PPFD) provided by cool white fluorescent tubes (40 W, Philips, India) with 55 ± 5% of relative humidity.
3.10 Synseed production
3.10.1 Plant material
The nodal segments (2-3 from the terminal bud, approximately 3-4 mm in size) were excised aseptically from 3 week-old in vitro raised seedlings of all the plant species and used as explants for synthetic seed production.
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3.10.2 Encapsulation matrix
Different concentrations 1, 2, 3, 4 and 5% (w/v) of sodium alginate were prepared using either liquid MS medium (with 3% sucrose) or DDW. For complexation 25, 50, 75, 100 and 200 mM calcium chloride solutions were prepared. Both, the gel matrix and complexing agent were sterilized by autoclaving at 1.06 Kg cm-2 for 15 min after adjusting the pH to 5.8.
3.10.3 Encapsulation and in vitro germination
Encapsulation was accomplished by mixing the nodal segments into sodium alginate solution and dropping them into calcium chloride solution. The droplets containing explants were held for at least 20-25 min to achieve polymerization of the sodium alginate. The alginate beads were then collected, rinsed with sterile liquid MS medium and transferred to sterilized filter paper placed in Petri-dishes for 5 min under the laminar airflow hood to absorb the excess of water and thereafter planted in wide mouth flask containing germination media. Synseeds having the gel matrix of MS medium were used for the optimization of germination medium. All the cultures were maintained at the same temperature, light and humidity as stated above for shoot induction and multiplication. The data for percent germination frequency of encapsulated beads was recorded after 6 weeks of culture to germination medium.
3.10.4 Low temperature storage
Three types of synseeds (encapsulated nodal segments having MS or DDW gel matrix and non-encapsulated nodal segments) were kept in sterilized beakers (moistened with DDW) sealed with two layers of Para Film and stored in a laboratory refrigerator at 4 °C. Five different exposure times (1, 2, 4, 6 and 8 weeks) were evaluated for synseed regeneration. After each storage period, encapsulated and non- encapsulated nodal segments were placed on to the optimized concentration of PGR for synseed conversion into plantlets. The complete plantlets (shoot and root) from synseeds were recovered after 6 weeks of culture.
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3.10.5 Direct or ex vitro sowing
Non-stored encapsulated nodal segments were also shown directly in soilrite moistened with tap water or quarter strength MS salts, soilrite and garden soil mixture (1: 1) moistened with tap water or quarter-strength MS salts for ex vitro conversion of into plantlets. The germination percentage of encapsulated nodal segments was recorded after 6 weeks of sowing.
3.11 Acclimatization of plantlets
Plantlets with well-developed roots were removed from the culture medium and after washing the roots gently under running tap water to remove the adhering medium; plantlets were immersed in 1% (w/v) Bavistin for half an hour, then transferred to thermocol cups (expanded polystyrene) containing autoclaved garden soil and green manure (2: 1), soilrite (75% Irish peat moss and 25% horticulture grade expanded perlite) (Keltech Energies Ltd., India) and vermicompost and irrigated with tap water as per requirement. The plantlets were covered with transparent polythene membrane to ensure high humidity for initial 2 weeks and then opened gradually in order to acclimatize plantlets to field conditions. After 4 weeks, successfully acclimatized plantlets were transferred to sterilized earthen pots filled with garden soil and green manure (2: 1). The potted plantlets were initially maintained inside the culture room conditions (4 weeks) and then transferred to greenhouse (4 weeks). Afterwards, the plantlets were transferred to field under full sun for further growth and development.
3.12 Physiological study
A set of in vitro regenerated plantlets with well developed shoot and roots were selected. Leaf samples were taken at transplantation day (day 0, control) and after 7, 14, 21, 28 days of acclimatization for physiological study.
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3.12.1 Chlorophyll (a, b and total) and carotenoids content estimation
The chlorophyll (a, b and total) and carotenoids contents from leaf tissue were estimated by using the method of MacKinney (1941) and MacLachan and Zalik (1963) respectively.
3.12.1.1 Procedure
About 100 mg fresh tissues of leaves were grinded in 5 ml acetone (80%) with the help of mortar and pestle. The suspension was filtered with Whatman filter paper number-1, if necessary the supernatant was re-grinded, washed and filtered, the total filtrate was taken in graduated test tubes and final volume was made up to 10 ml with 80% acetone.
3.12.1.2 Estimation
For chlorophyll contents, the optical density (O.D.) of above said solution was read at 645 and 663 nm and for carotenoids, the O.D. was read at 480 and 510 nm with the help of a spectrophotometer (UV-Pharma Spec 1700, Shimadzu, Japan). The chlorophyll and carotenoids contents were calculated according to the formula given below; Chlorophyll a (mg g-1 fresh tissue) 12.7(O.D.663)− 2.69(O.D.645) = ×V 1000×W Chlorophyll b (mg g-1 fresh tissue) 22.9(O.D.645)− 4.68(O.D.663) = ×V 1000×W Total Chlorophyll (mg g-1 fresh tissue) 20.2(O.D.645)+ 8.02(O.D.663) = ×V 1000×W Carotenoids (mg g-1 fresh tissue) 7.6(O.D.480)−1.49(O.D.510) = ×V D ×1000×W Where, O.D. = Optical density at given wavelength V = Final volume of chlorophyll extract in 80% acetone
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W = Fresh weight of leaf tissue D = Length of light path
3.12.2 Net photosynthetic rate (PN) estimation
The Net photosynthetic rate (PN) of in vitro regenerated plants were measured with the help of a portable photosynthetic system (LI-COR 6400, LI-COR Biosciences, Lincoln, USA) at 900 µmol m-2 s-1 photosynthetically active radiation
between 11:00 a.m. to 12:00 noon, on the basis of net exchange of CO2 between leaf and atmosphere by enclosing the leaf in the leaf chamber, and monitoring the rate at
which the CO2 concentration changed over a short time intervals (10-20 sec). The net -2 -1 photosynthetic rate was expressed as µmol CO2 m s .
3.13 Histological study
3.13.1 Fixation and storage
The differentiating explants were fixed in FAA solution consisting of formalin: glacial acetic acid: alcohol (70%) in the ratio of 4:6:90 (v/v). The fixed samples were stored in 70% alcohol.
3.13.2 Embedding and sectioning
Standard method of paraffin embedding (Johansen 1940) was followed for histological studies. Ethanol-xylol series was used for dehydration and infiltration. For complete infiltration of plant materials to be sectioned were kept in a vacuum oven at 60 °C for 15 min. Sections (longitudinal and transverse) of 10-12 µm thickness were cut from the wax block of samples using a Spencer 820 microtome (American Optical Corp. Buffalo, New York, US) and resulting paraffin ribbons were spread on clean slides, drick over night in an oven at 37 °C. The sections were de- waxed by immersion in xylene and passed through a series of deparafinizing solutions.
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3.13.3 Staining
Deparafized sections were immersed in coupling jar filled with 1% (w/v) ethanolic safranin for half an hour and washed with absolute alcohol. Permanent slides were made by mounting in Canada balsam. The sections were examined under a light microscope (Olympus CH 20i, Japan).
3.14 Chemicals and glass-wares used
Vitamins and amino acids (Thiamine HCl, Pyridoxine HCl, Nicotinic acid, Myo-inositol and Glycine) and PGRs (BA, Kn, 2-iP, TDZ, IAA, IBA, NAA) were obtained from Duchefa, Netherlands. The other chemicals like major and minor salts, sucrose, gelling agents (agar and phytagel), sterilants (Bavistin, Teepol and HgCl2), sodium alginate etc. were purchased from Qualigens, MERCK, SRL and /or Central Drug House. All chemicals used were of analytical grade. Glass-wares such as, test tubes (25 x 150 mm), Petri-dishes (17 x 100 mm), wide mouth flasks (100 ml), beakers (100, 250 and 500 ml) etc. used during the experiment were procured from Borosil, India.
3.15 Statistical analysis
The data was pooled from 3 separate experiments each with 20 replicates and analyzed statistically using SPSS version 16 (SPSS Inc., Chicago, IL, USA). The significance of differences among means was analyzed using Tukey’s test at 5% level of significance and data represented as mean ± standard error (SE).
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Chapter 4 Observations & Results
Chapter 4 OBSERVATIONS AND RESULTS
4.1 Spilanthes acmella
4.1.1 Seed germination and collection of explants
The seeds did not germinate in the field soil without any pre-treatment of GA. Therefore, the sterilized seeds were presoaked in GA at various concentrations (0.25, 0.5 and 1.0 µM). The lower most concentration of GA (0.25 µM) completely failed to stimulate any seed germination. While, the higher concentrations of GA (0.5 and 1.0 µM GA) triggered some germination but the frequency was very low (6.00 ± 0.63 and 12.40 ± 1.12% respectively) (Table 4). To improve the germination response sterilized seeds were inoculated aseptically on full and half-strength MS basal medium with or without GA (0.5, 1.0 and 5.0 µM). On the MS basal medium only 12.00 ± 2.00% seed germination was noticed which was slightly improved (42.00 ± 2.00%) on 1.0 µM GA wherein germination was started within 10.80 ± 0.63 days of incubation. However, the germination rate was significantly improved when the nutrient concentration was reduced to half (half-strength MS medium) along with the augmentation of GA. A maximum of 94.00 ± 2.44% seeds were germinated on medium comprised of half-strength MS nutrient plus 1.0 µM GA (Fig. 8 A & B). Radicals were first appeared just after 4.20 ± 0.37 days of inoculation on this optimal concentration of GA. On further increasing the concentration of GA (5.0 µM) germination delayed to 8.60 ± 0.40 days and only 70.00 ± 3.16% germination was noticed after 3 weeks of incubation (Table 5). Juvenile nodal segments (1-1.5 cm), shoot tips (0.8-1.2 cm with one pair of leaf primordia), leaves and cotyledons were excised from 3 week-old aseptic seedlings and used as the explants to determine their ability to induce multiple shoots.
4.1.2 Nodal segment culture
Nodal segments (each bearing two axillary buds) were excised from 3 week-old aseptic seedlings and cultured on three adenine-based cytokinins (BA, Kn and 2-iP) and one urea-based cytokinin (TDZ) with or without auxin (NAA, IAA and IBA). The recorded results are described as follows;
4.1.2.1 Effect of adenine-based cytokinins on shoot regeneration
The nodal segments failed to induce morphogenesis on control medium. They remained green up to 2 weeks, gradually turned yellowish-brown and died after 4 weeks of inoculation. Exogenous supplementation of cytokinins did not prove effective for axillary bud sprouting in multiple shoots. Among three adenine-based cytokinins, 1.0 µM BA supplemented MS basal medium induced a maximum of 2.00 ± 0.00 shoots per explant with 2.12 ± 0.11 cm length along with moderate callusing at the base of the explant in 89.00 ± 0.70% of cultures. On increasing or decreasing the BA concentrations percentage response and mean number of shoots per explant were further reduced due profuse callusing although shoot length was increased to 2.38 ± 0.07 cm on 2.5 µM BA. The Kn supplemented MS medium showed similar response on shoot regeneration as triggered by BA containing MS medium while 2-iP supplemented MS medium induced only high frequency callogenesis (Table 6).
4.1.2.2 Effect of cytokinin-auxin combinations on shoot regeneration
To see the synergistic effect of cyokinin-auxin combinations on multiple shoot induction, the nodal segments were also inoculated on BA (1.0 µM) with three auxins (NAA, IAA and IBA at 0.1, 0.5 and 1.0) supplemented to MS basal medium. Cytokinin- auxin combinations increased percent response and shoot length satisfactorily, but mean
number of shoots per explant did not improve effectively due to enhanced callogenesis. Initially explants enlarged in size within 1 week of incubation but axillary bud break was possible after 2 weeks of culture. Among the combinations tested, only a maximum of 2.40 ± 0.24 shoots per explant with 3.20 ± 0.08 cm shoot length were induced in 97.40 ± 1.24% cultures on 1.0 µM BA and 0.1 µM NAA combination. On the other hand, IAA and IBA were lesser effective (Table 7).
4.1.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Nodal segments were also inoculated on different concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) of TDZ supplemented to MS basal medium. Explants showed a little swelling on lower concentrations of TDZ (0.1-0.5 µM) after 2 weeks of culture, thereafter they remained as such up to 4 weeks of culture. While, higher concentrations (1.0-5.0 µM) induced huge callusing from the basal cut end of the explants. Since the nodal segments did not induce multiple shoots on any of the tried treatment of PGR, so the further experiments proceed with rest of the explants.
4.1.3 Shoot tip culture
The MS basal medium with or without different cytokinins, BA, Kn, 2-ip and TDZ (0.1, 0.5, 1.0, 2.5 and 5.0 µM) either singly or in combination with auxins, NAA, IAA and IBA (0.1, 0.5 and 1.0 µM) were tried to access the regenerative potentialities of shoot tip explants. The percent response, mean number of shoots per explant and mean shoot length varied according to the treatment used.
4.1.3.1 Effect of adenine-based cytokinins on shoot regeneration
The shoot tips cultured on PGR-free MS basal medium (control) failed to initiate multiple shoot buds even after 4 weeks of incubation. Each shoot tip elongated into 3.84 ± 0.19 cm single shoot (Fig. 9 A). The supplementation of cytokinins (BA, Kn or 2-iP) resulted in multiple shoot bud induction with the formation of new sets of leaves within 8-10 days of incubation (Fig. 9 B). Among adenine-based cytokinins tested, BA (1.0 µM) was found to be the optimal as inducing a mean number of 8.0 ± 0.31 shoots per explant with mean shoot length of 3.9 ± 0.30 cm in 66.0 ± 2.44% of cultures after 4 weeks of incubation (Fig. 9 C & D). On further increasing the concentration beyond the optimal level, the regeneration capacity was suppressed considerably due to intense basal callusing (white and friable). Addition of Kn or 2-iP to the medium did not found significant for shoot induction and multiplication. Among various concentrations tested, 1.0 µM Kn and 1.0 µM 2-iP induced mean number of 3.6 ± 0.40 (Fig. 9 E) and 1.6 ± 0.24 shoots per explant respectively. Prominent callusing was noticed on 5.0 µM 2-iP containing medium (Fig. 9 F) than similar treatment of BA and Kn. Thus, 1.0 µM BA was the most effective cytokinin for axillary bud initiation and subsequent proliferation than Kn and 2-iP (Table 8).
4.1.3.2 Effect of cytokinin-auxin combinations on shoot regeneration
The regeneration potential of the shoot tip explants considerably enhanced when the MS medium was supplied with the optimized BA (1.0 µM) in combination with various auxins (NAA, IAA and IBA) at different concentrations. The regeneration of shoot buds was noticed with the appearance of green bulged outgrowth at the compressed nodal region of the explants after 4-5 days of incubation followed by the emergence of shoot buds (Fig. 10 A). Thereafter, induced buds elongated and developed into cluster of multiple shoots. Among different treatments tried, 1.0 µM BA in combination with 0.1 µM NAA gave optimum result where a maximum of 96.0 ± 2.44% cultures responded
with a mean number of 33.0 ± 1.09 shoots per explant with mean shoot length of 5.2 ± 0.09 cm after 4 weeks of incubation (Fig. 10 B & C). While, higher concentration of NAA (0.5 and 1.0 µM) reduced the regeneration percentage because of simultaneous callus formation at the base of explant. Whereas, incorporation of IBA or IAA to the BA supplemented nutrient medium was lesser effective than NAA and reduced the regeneration efficiency significantly to 8.0 ± 0.31 shoots per explant on BA (1.0 µM) with IAA (0.1 µM) and 15.0 ± 0.31 shoots per explant on BA (1.0 µM) with IBA (0.5 µM) wherein the regeneration of shoot buds were initiated after 10 and 7 days of inoculation respectively (Fig. 10 D & E, Table 9).
4.1.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
The MS basal medium supplemented with different concentrations of TDZ (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) stimulated adventitious multiple shoot bud induction from the basal cut end of the explants. Apical bud sprouting in 2-3 shoot buds was also observed only in some replicates but it was not much evident, therefore only the data of adventitious shoots, induced from basal cut end, was recorded. Within 1 week of incubation, the explants swelled from their cut ends. First appearance of buds was observed from the swollen cut ends after 2 weeks of culture (Fig. 11 A). Thereafter, in the subsequent weeks the response of shoot bud induction increased significantly (Fig. 11 B & C). A maximum of 98.00 ± 2.00% regeneration was recorded at 0.25 µM TDZ wherein a mean number of 30.00 ± 0.30 shoots per explant with mean shoot length of 1.00 ± 0.10 cm was recorded, without any intervening callus formation, after 4 weeks of culture (Table 10). On this treatment, a thick rosette like clump of leaves was formed due to poor internodal elongation; hence each culture transformed into a dense mass of profusely regenerating stunted shoots except very few buds exhibited healthy growth (Fig. 11 D & E). Reduced concentration of TDZ (0.1 µM) adversely affected the mean number of shoots per explant from the basal cut end of the explants. While, increased
concentration of TDZ (0.5-2.5 µM) beyond the optimal level (0.25 µM) favored callusing (yellowish-white and lucid) that was associated with fasciated shoot differentiation (Fig. 11 F) and on 5.0 µM TDZ only 2.80 ± 0.40 shoots per explant were induced due to intense callogenesis. Since TDZ induced the stunted shoots even at the optimal level, therefore for further growth with enhanced multiplication, the shoots regenerated on 0.25 µM TDZ containing MS medium were transferred to the medium comprising MS nutrients plus BA (0.5, 1.0 and 2.5 µM) with or without NAA (0.5 µM).
4.1.3.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue
During this experiment, on BA supplemented medium multiplication was possible after 12 days of transfer while cytokinin-auxin combination improved the regeneration frequency just after 5-7 days of transfer. It was observed that BA alone did not found to be effective at all the concentrations tried for TDZ derived regenerating tissues as far as their growth was concerned. On the other hand, among different combinations of cytokinin and auxin, 1.0 µM BA with 0.5 µM NAA was found to be an effective combination for significant improvement in shoot proliferation and shoot growth wherein a maximum of 40.2 ± 0.70 shoots per explant having 3.8 ± 0.20 cm shoot length was induced after 4 weeks of culture (Table 11). However, 0.5 and 2.5 µM BA in combination with 0.5 reduced the regeneration efficiency of the regenerating explant due to callus formation. While, 5.0 µM BA and 0.5 µM IAA combination induced hyperhydricity in regenerating shoots exhibiting glassy appearance. Another experiment was designed to improve further growth in regenerated shoots. The regenerating tissues on 1.0 µM BA with 0.5 µM NAA were treated as “stock culture” for further sub-culturing (Fig. 12 A).
4.1.3.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture
The “stock culture” from each culture were divided into 4 pieces and further sub- cultured to half-strength MS medium, full-strength MS medium and full-strength MS basal medium augmented with BA (1.0 µM) and NAA (0.5 µM). It was observed that when “stock culture” was sub-cultured on to the same composition of BA and NAA while maximum of 76.00 ± 1.70 shoots per explant (2.5 fold increase than TDZ) with 5.30 ± 0.10 cm shoot length were regenerated, but all the shoots were highly hyperhydric (Fig. 12 B). Hyperhydricity caused translucent and brittle shoots having glossy appearance and adversely affected the survival rate. Therefore, for the prevention of hyperhydricity regenerating shoot clumps were sub-cultured to hormone-free MS basal medium. It was clearly observed that when the “stock culture” were sub-cultured to half- strength MS basal medium, growth in regenerated shoots was lesser (4.20 ± 0.10 cm) (Fig. 12 C). Although hyperhydricity was completely eliminated but drastic loss in shoot regeneration might be due to withdrawal of hormone and reduction in MS nutrients. However, on full-strength MS basal medium shoots were healthier with moderate elongation of shoots (4.70 ± 0.10 cm shoot length) as compared to half-strength MS medium (Fig. 12 D). No hyperhydricity was observed among the shoots however the shoot proliferation was very high (63.40 ± 1.10 shoots per explant) as compared to half- strength basal MS medium (42.80 ± 0.90 shoots per explant) (Table 12).
4.1.3.4 Effect of different pH on shoot regeneration
The pH of culture medium plays an important role in nutrients uptake and shoot proliferation. Therefore, the effect of different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) was tested on MS basal medium supplemented with an optimal combination of BA (1.0 µM) and NAA (0.1 µM). The optimum pH for shoot regeneration was found at 5.8 wherein
maximum 33.0 ± 1.09 shoots per explant with 5.20 ± 0.09 cm of shoot length were induced after 4 weeks of culture. Thereafter, a gradual decrease in shoot regeneration efficiency was observed with the increase in pH up to 6.6 wherein only 9.00 ± 0.44 shoots per explant having 2.26 ± 0.08 cm shoot length were induced after 4 weeks of inoculation. The shoot multiplication rate was severely affected with the decrease in pH below 5.8 where the acidic nature of medium inhibited shoots induction. Only 5.60 ± 0.50 hyperhydric shoots per explant with 1.78 ± 0.08 cm were induced at 5.4 pH whereas no shoot regeneration was noticed at 5.0 pH (Fig. 13).
4.1.3.5 Effect of different carbon sources on shoot regeneration
To study the effect of different carbon sources on shoots regeneration from shoot tip explants the MS basal medium containing an optimized combination of BA (1.0 µM) and NAA (0.1 µM) was used at 5.8 pH. Three carbohydrates i.e., sucrose, fructose and glucose were assessed at 3% (w/v) concentration. Among three carbon sources tested, sucrose induced a maximum of 33.0 ± 1.09 shoots per explant with 5.20 ± 0.09 cm shoot length after 4 weeks of culture. Whereas, fructose showed moderate response and induced an average of 23.60 ± 0.67 shoots per explant with 4.50 ± 0.10 cm shoot length. On the other hand, glucose was the least effective for shoot regeneration as regenerated only 9.60 ± 0.50 shoots per explant with 2.26 ± 0.08 cm shoot length (Fig. 14).
4.1.3.6 Effect of different culture media on shoot regeneration
Media proposed by various workers greatly affect the regeneration frequency of plant tissue due to their different formulations that is why the effect of three different basal media (B5, MS and SH) were also evaluated with an optimized combination of BA (1.0 µM) and NAA (0.1 µM) having 3% sucrose at 5.8 pH for shoot multiplication. The MS medium supplemented with an optimal PGR combination was found to be the most
suitable for maximum shoot proliferation (33.0 ± 1.09 shoot per explant) and shoot length (5.20 ± 0.09 cm shoot length). While, SH medium exerted satisfactory response with a
mean of 17.40 ± 0.87 shoots per explant and 3.36 ± 0.11 cm shoot length whereas B5 medium exhibited the poorest response as induced only 7.20 ± 0.73 shoots per explant and 2.68 ± 0.09 cm shoot length (Fig. 15).
4.1.4 Leaf culture
The leaf explants cultured on control treatment enlarged in size, but did not give any sign for organogenesis and indicated the requirement of PGR supply to the explants for adventitious or de novo organogenesis. The entire leaves along with their petioles were inoculated as such on MS medium supplemented with various cytokinins viz., BA, Kn, 2-iP and TDZ at varying concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) singly and in various combinations with different auxins, NAA, IAA and IBA (0.1, 0.5 and 1.0 µM). Leaves were placed with abaxial surface in contact with the culture media.
4.1.4.1 Effect of adenine-based cytokinins on shoot regeneration
The cultured leaves showed their initial response by enlargement in size along with a little bulging at their cut ends within 3-4 days of inoculation. Adventitious shoot buds appeared within 10 days of incubation from basal bulged petiolar end which were increased in number on further incubation while leaf lamina did not exhibit any sign of differentiation (Fig. 16 A). BA was found to be the best cytokinin for direct organogenesis from leaf explants followed by Kn and 2-iP. Among different treatments tested, when 2.5 µM BA was supplemented individually, a maximum of 6.20 ± 0.48 shoots per explant and 2.82 ± 0.10
cm shoot length were induced in 85.00 ± 1.84% cultures after 4 weeks of culture (Fig. 16 B, C & D). Kn and 2-iP both reduced all the three parameters i.e., percent response, mean number of shoots per explant and mean shoot length due to moderate to intense callus formation. On 2.5 µM Kn supplemented MS medium highest of 4.40 ± 0.50 shoots per explant with 3.30 ± 0.11 cm shoot length were induced after 4 weeks of incubation (Fig. 16 E). However, 5.0 µM 2-iP supplemented MS medium greatly delayed the organogenic behavior of explants due to intense callogenesis and only 2.60 ± 0.24 shoots per explant with 2.14 ± 0.07 cm of shoot length were induced in 23.00 ± 1.54% cultures (Fig. 16 F). The calli induced on lower and higher concentrations of BA, Kn and 2-iP were neither embryogenic nor organogenic. Thus, in the present investigation, a comparison of the relative effectiveness of different adenine-based cytokinins for adventitious shoot regeneration from leaf explants revealed the order of effectiveness as BA>Kn>2-iP (Table 13).
4.1.4.2 Effect of cytokinin-auxin combinations on shoot regeneration
In another set of experiment with the objective to formulate a better hormonal concentration for enhanced direct shoot induction, the effect of different cytokinin-auxin combinations was also studied. The synergism between cytokinin and auxin exhibited a positive role on the induction of shoots. On cytokinin-auxin combinations differentiation of shoot buds achieved earlier than only cytokinin containing medium. Cluster of shoot buds induced from the petiolar cut ends just after 1 week of culture (Fig. 17 A). An optimal concentration of BA (2.5 µM) was tried with various auxins viz., NAA, IAA and IBA (0.1, 0.5 and 1.0 µM) (Table 14). The MS medium with 2.5 µM BA and 0.5 µM NAA induced the highest number of shoots i.e., 27.80 ± 1.01 shoots per explant with mean shoot length of 4.50 ± 0.13 cm in 93.00 ± 2.00% of cultures (Fig. 17 B, C & D). The shoots regenerated on cytokinin-auxin combinations were found to be longer than those induced on MS medium augmented with BA (2.5 µM) individually. Increased
concentration of NAA (1.0 µM) favored white-friable lucid callus formation especially from the cut end of explants and nullified the caulogenic effectiveness of BA (Fig. 17D). All the combinations of BA and IAA or IBA were inferior for shoot induction as facilitated higher frequency of callogenesis (Fig. 17 E & F). After an incubation period of 4 weeks, mean number of 25.00 ± 1.00 and 16.80 ± 0.58 shoots per explant were noticed on 2.5 µM BA plus 0.5 µM IAA and 2.5 µM BA plus 0.5 µM IBA containing MS media respectively.
4.1.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
None of the leaves cultured on TDZ supplemented MS medium induce direct shoot formation. The leaf explants started to callus at all the concentrations of TDZ tested. The frequency of callogenesis varied from 40.00 ± 3.16 to 100.00 ± 0.00% according to the concentration (Table 15). Calli initiated from the cut end of explants after 5-7 days of culture and then spread over the leaf surface. Calli on TDZ containing media showed friable growth and characterized by soft (watery), white to greenish brown appearance. Three types of responses were observed on TDZ comprised nutrient media. The
first type of response (C1) corresponded to those explants which after initial swelling rapidly converted to calli without any sign of regeneration (Fig. 18 A). This response was
observed on lower concentrations of TDZ (0.1-1.0 µM). In the second type (C2) after initial swelling explants produced greenish-white lucid calli showing purplish-red pigmentation due to anthocyanin accumulation followed by the emergence of bud-like proturbances those were rapidly over crowed by profuse callusing (Fig. 18 B). Such calli were induced on 2.5 µM TDZ. While, on the highest level of TDZ (5.0 µM) exerted third
type of response (C3) which involved intense greenish-white to brown translucent callus formation with one or two bud like structures, but buds ceased to grow further and failed
to show any organogenic activity. Thus, only the second type of calli (C2) were used for further multiplication of shoots.
4.1.4.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue
This experiment was carried out with an aim of multiple shoot bud initiation from leaf derived calli followed by their growth in healthy shoots. To achieve greater success
in indirect organogenesis, the C2 calli were sub-cultured on various shoot regeneration media. Among the treatments tested, MS medium with 1.0 µM TDZ and 1.0 µM BA responded the best. On this treatment, sub-cultured calli started to grow vigorously after 1 week of sub-culturing and enhanced the pigmentation (Fig. 18 C). Regeneration was started from the marginal tissues of transferred calli. On the optimized combination of TDZ (1.0 µM) and BA (1.0 µM) a maximum of 16.00 ± 0.54 shoot buds per culture were initiated of which a mean of 14.00 ± 0.54 buds converted into shoots with 1.20 ± 0.11 cm shoot length after 4 weeks of incubation (Table 16). Initially an accurate quantitative determination of shoot buds was very difficult as they were arranged in a rosette fashion due to poor internodal growth. All the shoots were greenish-white in color and crumpled due to their vigorous production (Fig. 18 D). As none of the treatment improved shoot growth, therefore cultures grown on optimized combination of TDZ (1.0 µM) and BA (1.0 µM) were used as ‘stock culture’ for sub-culturing in order to further enhance the shoot growth.
4.1.4.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture
This experiment was carried out to manipulate the culture medium for further improvement in shoot growth (shoot length and leaf color) in TDZ and BA regenertaed shoots.
The shoot clumps from ‘stock culture’ was sub-cultured on fresh medium having similar combination of TDZ (1.0 µM) and BA (1.0 µM), half and full-strength MS medium with or without BA (1.0 µM). On TDZ and BA containing MS medium, shoot growth did not improve (1.20 ± 0.11 cm) moreover, the shoots (14.00 ± 0.54 shoots per explant) were hyperhydric and malformed (Fig. 18 E). A continued shoot multiplication was achieved through repeated sub- culturing of differentiating culture to 1.0 µM BA supplemented MS medium. A maximum of 20.40 ± 0.74 shoots per explant with 3.54 ± 0.08 cm of shoot length was induced on this treatment. Leaves were well expanded and greener as compared to the leaves of ‘stock culture’ (Fig. 18 F). Whereas, PGR-free MS medium (full and half-strength) did not improve shoot growth and still the leaves were whitish-green and crumpled (Table 17).
4.1.4.4 Effect of auxin (2, 4-D) on callus induction
Different concentrations of 2, 4-D (0.5, 1.0 and 2.5 µM) were also tested to induce callogenesis from leaf explants. Entire young leaves along with petioles were placed on 2, 4-D containing MS media. Callusing was observed in almost all the treatments although the morphology and growth of callus dependent on 2, 4-D concentration. Optimum callogenesis (97.60 ± 1.12%) was noticed on 1.0 µM 2, 4-D containing MS medium which became visible just after 5-7 days of inoculation (Fig. 19 A). On increasing (2.5 µM) or decreasing (0.5 µM) 2, 4-D concentration beyond an optimal level callogenesis was reduced to 78.60 ± 1.56 and 61.40 ± 0.97% respectively. The MS medium supplemented with 0.5 and 1.0 µM 2, 4-D induced yellow-lucid calli while on 2.5 µM 2, 4-D containing MS medium callus remained yellowish and lucid up to 3 weeks thereafter it turned to whitish-yellow and dried tissue (Fig. 19 B). With an aim to further increase the frequency of callogenesis, different concentrations of BA (0.5, 1.0 and 2.5 µM) were added to an optimal concentration of 2, 4-D (1.0 µM). Callusing was started within 3-5 days and the entire leaf explants were
covered with callus within 4 weeks of incubation. The texture of callus was considerably influenced by the concentration of 2, 4-D and BA. The calli induced on 2, 4-D and BA combinations were relatively more friable, lucid and yellow in color than 2, 4-D derived calli (Fig. 19 C). Among different combinations, 1.0 µM 2, 4-D and 1.0 µM BA exhibited maximum callus formation (100.00 ± 0.00%) and used for shoot regeneration (Table 18). When this optimal calli were transferred to regeneration medium comprising of 1.0 µM BA and 0.5 µM NAA only three or four small green buds were found but failed to elongate into healthy shoots even after 4 weeks of culture. Soon after callus tissue became necrotic and turned off-white (Fig. 19 D & E).
4.1.5 Cotyledon culture
The cotyledons cultured on control treatment enlarged in size, but did not give any sign for organogenesis, died after 4 weeks of culture and necessitated the PGR supplementation to the explants for de novo organogenesis. The entire cotyledons along with their petioles were inoculated on MS medium supplemented with various cytokinins viz., BA, Kn, 2-iP and TDZ at varying concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) singly and in various combinations with different auxins, NAA, IAA and IBA (0.1, 0.5 and 1.0 µM). The explants were inoculated with their abaxial surface in contact with the culture medium.
4.1.5.1 Effect of adenine-based cytokinins on shoot regeneration
The supplementation of different cytokinins enabled the cotyledons to show their morphogenetic potentiality differentially. Swelling was started form the proximal cut ends of the explant within 4-6 days of culture. Shoot buds were induced from swelled petiolar cut ends within 12-15 days of culture, but lamina did not involve in organogenesis.
A comparison of different cytokinins showed that the best response was achieved on 2.5 µM BA containing MS medium than Kn and 2-iP wherein shoot buds were induced after 12 days of incubation. On this concentration, no callogenesis was appeared at the site of organogenesis. However, a little friable green callus was appeared from the explants’ surface (Fig. 20 A). A maximum percentage response (66.0 ± 2.44) with the highest number of shoots per explant (7.2 ± 0.37) was observed on 2.5 µM BA after 4 weeks of culture (Fig. 20 B). Further increasing the BA concentration to 5.0 µM regeneration frequency was reduced to 52.00 ± 2.00% and 4.00 ± 0.31 shoots per explant. While, on 2.5 µM Kn and 2.5 µM 2-iP containing MS media maximum of 4.60 ± 0.24 and 2.00 ± 0.31 shoots per explant were induced respectively (Fig. 20 C). Meanwhile, the lowest concentration i.e., 0.5 µM of BA, Kn and 2-iP and the highest level of 2-iP (5.0 µM) did not exert any caulogenic response even after 4 weeks of incubation. The MS medium comprising 2-iP (5.0 µM) induced only callus that was whitish-green, lucid and compact (Fig. 20 D, Table 19).
4.1.5.2 Effect of cytokinin-auxin combinations on shoot regeneration
Addition of auxin (NAA, IAA and IBA) to an optimal cytokinin concentration improved regeneration potential of the cotyledons and showed a significant synergism between cytokinin and auxin. Among the various combinations, BA (2.5 µM) and NAA (0.5 µM) was found to be the most critical for direct organogenesis from the cotyledons wherein high frequency shoot bud regeneration was possible after 10 days of culture (Fig. 21 A). On this treatment a maximum of 17.40 ± 0.87 shoots per explant having mean shoot length of 4.50 ± 0.18 cm was induced in 78.0 ± 3.74% of cultures after 4 weeks of culture. White translucent callogenesis was also observed at the site of regeneration i.e., petiolar cut end but lamina remained entire (Fig. 21 B). Thus, for obtaining maximum shoot regeneration it was necessary to sub-culture the regenerating explants on fresh
medium of similar PGR treatment after removing the callus otherwise callus formation suppressed shoot regeneration (Fig. 21 C). On the other hand, BA (2.5 µM) and IAA (0.5 µM) combination induced only 11.80 ± 0.91 shoots per explant with 3.88 ± 0.08 cm of shoot length. While, BA (2.5 µM) and IBA (0.5 µM) containing MS medium delayed the response and found to be the least effective for shoot induction wherein only 2.60 ± 0.40 shoots per explant were induced due to intense callusing (Fig. 21 D). No regeneration was noticed on 2.5 µM BA and 1.0 µM IBA amended MS medium even after 4 weeks of culture (Table 20).
4.1.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Similar to the leaf explants, none of the tried concentrations of TDZ was able to exert direct caulogenic response through cotyledons. TDZ induced yellowish-white to whitish-green callus. A mean of 19.00 ± 4.00 to 67.60 ± 2.50% friable callogenesis was induced on the lower concentrations (0.1-0.5 µM) whereas higher concentrations (2.5-5.0 µM) induced comparatively compact callus in 77.40 ± 2.18 to 82.60 ± 1.66% cultures after 4 weeks of incubation (Table 21). Callus induced on 2.5 µM TDZ showed few meristemoids, but they failed to grow into shoot buds even transferred to the regeneration media as used for TDZ induced calli of leaf explants.
4.1.6 Effect of sub-culturing on shoot proliferation
As far as the literature is concerned, there is no report available on the effect of sub-culture passages on shoot proliferation for in vitro conservation of this highly medicinal plant species. Therefore, for further amplification and continued production of shoots, the regenerating tissues were sub-cultured periodically after every 3 weeks (up to 8th sub-culture passage) to the fresh optimized medium respective to the explant types. The MS medium with BA (1.0 µM) and NAA (0.1 µM) was used for shoot tip explants
while MS medium having BA (2.5 µM) and NAA (0.5 µM) combination was used for leaves and cotyledons. A high yield of shoot was achieved by sub-culturing the shoot clumps excised from primary cultures. The number of shoots developed was difficult to count and separate due to their vigorous production. During sub-culturing, elongated shoots were excised and simultaneously transferred to root induction medium and the remaining tissues were again sub-cultured on to the regeneration medium. From shoot tip explants, the frequency of shoot multiplication increased considerably from first sub- culture (42.20 ± 1.50 shoots per explant) to sixth sub-culture (77.20 ± 0.86 shoots per explant) and remained almost consistent at seventh sub-culture, but declined thereafter (Fig. 22 & 23). From leaves and cotyledons, regeneration efficiency continuously increased up to fifth sub-culture passage (73.00 ± 0.80 and 41.00 ± 1.18 shoots per leaf and cotyledon explant respectively). A remarkable increase in shoot number was observed from third (60.20 ± 0.60 and 33.60 ± 0.87 shoots per leaf and cotyledon respectively) to fifth sub-culture passage (73.00 ± 0.80 and 41.00 ± 1.18 shoots per leaf and cotyledon respectively). Nevertheless, a gradual increase in shoot length was observed after every passage of sub-culturing for all the explant types (Fig. 24, 25, 26 & 27).
4.1.7 In vitro rooting of microshoots
For in vitro rooting excised microshoots were transferred to half-strength MS medium with or without various auxins (NAA, IAA and IBA) at different concentrations of (1.0, 2.5 and 5.0 µM). The shoots were rooted in all the treatments with or without auxins within 3-7 days of transfer. Although, roots were induced on half-strength basal MS medium without auxins but they were very delicate and devoid of secondary branching due to which the survival rate of transplanted plantlets was adversely affected. Hence, it necessitated the supplementation of auxin to rooting medium for the induction
and development of healthy root system for the ease and maximum success during acclimatization. Among three auxins tested, NAA exhibited best rooting response than IAA and IBA. On lower concentration of NAA (1.0 µM) mean number of 20.20 ± 0.58 roots per shoot with 13.40 ± 0.21 cm root length were induced after 4 weeks of culture while 2.5 µM NAA induced highest of 30.40 ± 0.50 roots per shoot with 18.60 ± 0.18 cm root length. Although on both the aforesaid treatments of NAA roots induced directly from the basal cut end of microshoots but secondary rooting was possible only with 2.5 µM NAA that resulted in better survival after transplantation (Fig. 28 A & B). While, at higher concentration of NAA (5.0 µM) root induction was followed by a little callusing that inhibited not only further induction of roots but also inhibited their growth. The application of IBA and IAA were found inferior as the root induction was preceded by intense to moderate callus formation at the cut end of microshoots. Though in comparison to NAA, mean root number was quite higher (32.80 ± 1.15 roots per shoot) on IBA (2.5 µM) supplemented medium but the roots were stunted (3.20 ± 0.17 cm) and did not show any further elongation (Fig. 28 C, Table 22). Besides, on NAA supplemented nutrient medium few of the microshoots exhibited in vitro flowering during root induction (Fig. 28 D & E). When rooting was compared between agar and phytagel-solidified media, no significant difference was observed with respect to mean number of roots per shoot and root length (Fig. 28 F & G).
4.1.8 Acclimatization of plantlets
Since plantlets are grown in protective regime of culture conditions, it becomes imperative to make them autotrophic prior to their transplantation for better survival. Plantlets with 3-4 pairs of leaves were removed from the culture vessels, washed carefully with tap water, transferred to various planting substrates and hardened off,
adopting the procedure as described in materials and method. Among various type of planting substrates tested, maximum survival percentage of the plantlets was observed for soilrite (96.60%) followed by vermi-compost (81.60%) and mixture of garden soil with farmyard (2:1) (66.60%) (Fig. 29). After successful acclimatization, there was no detectable variation among hardened plantlets with respect to morphological and growth characteristics. All the micropropagated plantlets were free from external defects and flowered normally (Fig. 30 A, B & C).
4.1.9 Synseed production
The success of synseeds depends on various factors like the concentration of encapsulation gel matrix, complexing agent, germination medium and storage time at low temperature. The results on the respective experiments are as follows.
4.1.9.1 Effect of Na2-alginate concentration on synseed formation
Synseed formation was considerably influenced by the concentration of Na2-
alginate. Thus, in the first experiment of synseed, varying concentrations of Na2-alginate
(1, 2, 3, 4 and 5%) were tried with the combination of standard CaCl2·2H2O
concentration i.e. 100 mM. Among different concentration, 4% Na2-alginate produced firm, clear and isodiametric synseeds within 20 min of ion exchange (Fig. 31 A) and exhibited maximum conversion frequency into plantlets (74.80 ± 1.46%) after 6 weeks of
culture (Fig. 31 B). However, 3% Na2-alginate exhibited an average of 73.40 ± 1.88% germination, but synseeds were too soft to handle. On further lowering the concentration fragile synseeds were formed. On the other hand, highest level of Na2-alginate (5%) significantly reduced the germination frequency to 45.60 ± 1.69% after 6 weeks of culture (Table 23).
4.1.9.2 Effect of CaCl2·2H2O concentration on synseed formation
In the second experiment of synseed, different concentrations of CaCl2·2H2O (25,
20, 75, 100 and 200 mM) were tested with an optimal concentration of Na2-alginate i.e., 4% to obtain the synseeds of desired texture. Among different combinations of
CaCl2·2H2O and Na2-alginate, 100 mM CaCl2·2H2O with 4% Na2-alginate was proved to be the optimum for synseeds germination (74.80 ± 1.46%). Higher concentration of
CaCl2·2H2O (200 mM) was resulted in too hard synseeds that delayed the germination (39.00 ± 2.44%) while synseeds formed using the lower concentrations (25 and 50 mM) were fragile and burst during handling (Table 24).
4.1.9.3 In vitro plantlet regeneration from synseeds on culture medium
Synseeds were placed on different treatments of PGRs for complete plantlets (shoot and root development) recovery. The shoot re-growth was initiated after 2 weeks whereas rooting was started after 3 weeks of culture. Maximum conversion frequency of synseeds (87.80 ± 1.15%) was achieved on MS medium supplemented with BA (1.0 µM) and NAA (0.5 µM) after 6 weeks of culture (Fig. 31 C & D). The inclusion of IBA and IAA to the BA supplemented media reduced the germination frequency of synseeds to 73.40 ± 1.02 and 80.20 ± 2.83% respectively (Fig. 32 A & B, Table 25).
4.1.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage
In this experiment, the effect of different storage durations at 4°C was compared for shoot conversion ability of encapsulated and non-encapsulated nodal segments. After storage, they were placed onto the optimized combination of BA (1.0 µM) and NAA (0.5 µM). Maximum shoot regeneration capacity was obtained with non-encapsulated nodal
segments (99.00 ± 1.00%) as compared to encapsulate ones (87.80 ± 1.15%) without any storage. Up to 4 weeks of low temperature storage, viability of nodal segments was gradually decreased from 82.80 ± 1.15 to 73.60 ± 1.56% for the encapsulated nodal segments (with MS gel matrix) whereas a significant loss in the morphogenetic potential was observed for naked nodal segments (73.00 ± 2.00 to 28.00 ± 2.54%). After 4 weeks of storage, germination frequency was greatly reduced for synseed (having MS gel matrix) and naked nodal segments. On the other hand, encapsulated nodal-segments having DDW gel matrix did not survive after 2 weeks of storage (Table 26).
4.1.9.5 Acclimatization of plantlets
The plantlets with well developed shoot and root systems were removed from synseeds, washed carefully to remove remnant of encapsulating matrix and culture medium, transferred to thermocol cups containing sterile soilrite. These were covered with transparent polybags and acclimatized as described in materials and methods (Fig. 33 A-D). Plantlets were irrigated with normal tap water after every 4 day. Acclimatized plantlets showed more than 90% survival rate when transferred to field.
4.1.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
Among various planting substrates tested for ex vitro conversion of synseeds into complete plantlets, soilrite moistened with quarter-strength MS nutrient medium (63.00 ± 2.00% germination) was found to be the most suitable planting substrate followed by soilrite moistened with tap water (51.20 ± 0.96% germination) whereas no regeneration was noticed in soil having tap water supply even after 6 weeks of their inoculation (Table 27).
4.1.10 Physiological study
4.1.10.1 Chlorophyll a, b and total chlorophyll content during acclimatization
Chlorophyll a, b and total chlorophyll content showed similar trend during various phases of acclimatization. Plantlets showed decrease in chl a (0.75 ± 0.01 to 0.70 ± 0.02 mg g-1), chl b (0.36 ± 0.01 to 0.28 ± 0.01 mg g-1) and total chl (0.94 ±0.01 to 0.84 ±0.01 mg g-1) content during first 7 days of transfer. Thereafter, a linear increase was noticed with each passing week. After 14-28 days of transfer considerable increase in chl a (0.83 ± 0.01 to 1.12 ± 0.03 mg g-1), chl b (0.42 ±0.02 to 0.52 ±0.03 mg g-1) and total chl (1.03 ± 0.00 to 1.13 ± 0.01 mg g-1) content were seen, this further increased slightly after 28 days of acclimatization and stabilized to chl a 1.22 ±0.02 mg g-1, chl b 0.61 ±0.02 mg g-1 and total chl 1.23 ± 0.01 mg g-1 (Fig. 34).
4.1.10.2 Carotenoids content during acclimatization
Carotenoids content showed similar increasing trend like that of chlorophyll from 0 to 28 days of transfer of plants from in vitro to ex vitro conditions. It was slightly reduced (0.21 ± 0.03 to 0.18 ± 0.01 mg g-1) after 7 days of ex vitro transfer. However, a steep rise in carotenoids content (0.28 ± 0.01 mg g-1) was witnessed after 14 days of acclimatization. Finally, acclimatized plantlets showed slight increase and ultimately content was stabilized to 0.35 ± 0.01 mg g-1 after 28 days of transfer (Fig. 35).
4.1.10.3 Net photosynthetic rate (PN) during acclimatization
Net photosynthetic rate (PN) is an important indicator of the photosynthetic efficiency of plants. Plantlets transferred ex vitro were evaluated periodically for their net
photosynthetic rate for four weeks. Initially decrease in PN (1.76 ±0.02 to 1.69 ± 0.02 -2 -1 µmol CO2 m s ) was noticed when the plants were exposed to ex vitro conditions
-2 -1 during first week. However, significant increase (4.56 ±0.15 µmol CO2 m s ) in net
photosynthetic rate was observed in subsequent weeks when the PN value reached as high 2 -1 as 7.39 ± 0.16 µmol CO2 m s after 21 days of transfer. After 4 weeks of -2 -1 acclimatization, PN further slightly increased to 7.82 ± 0.1 µmol CO2 m s and remained almost consistent (Fig. 36).
4.1.11 Histological study
Histological studies were undertaken with an aim to establish the origin and mode of shoot regeneration from the responsive explants. In S. acmella, histological analysis was carried out for shoot tip and leaf explants as they were found to be more responsive for shoot regeneration than nodal segments and cotyledons. Fig. 37 A represents the section through compressed nodal region of 2 week-old shoot tip explants, cultured on BA (1.0 µM) and NAA (0.1 µM) and revealed the direct origin of shoot buds without any intermittent callus. This figure scrutinizes a well developed shoot bud with an apical meristem (AM), a pair of leaf primordia (LP) and pro-vascular strands (VS). While, Fig. 37 B showing direct organogenesis from the basal cut end of the 3 week-old shoot tip explant on 0.25 µM TDZ. Shoot bud initiation was started from the marginal cells that were followed by the inner cells. Formation of shoot buds directly from the swollen petiolar end of 2 week-old leaf (cultured on 2.5 µM BA and 0.5 µM NAA containing MS medium) is also evident in Fig. 37 C. The histological micrograph clearly reveals that the differentiation of shoot buds was direct from epidermal cells of cut end of petiole of young leaves without any associated callus. Leaves cultured on 2.5 µM TDZ supplemented MS medium for 2 and 3 weeks were also sectioned to show the indirect organogenesis. In case of indirect shoot organogenesis, whole leaf was converted into light green, friable callus. Meristematic zones were developed by rapidly dividing cells inside the callus. These meristematic zones were further expanded and acted as origin for shoot bud organogenesis. Fig. 38 A
shows adventitious shoot buds (ASB) embedded in callus while Fig. 38 B shows further development of shoot bud flanked by leaf primordial (LP) on both sides of apical dome (AD).
Table 4. Effect of pre-soaking of seeds in GA on in vivo seed germination in S. acmella after 3 weeks of sowing GA (µM) Mean no. of days to Germination frequency (%) germination Control 0.00 ± 0.00c 0.00 ± 0.00c GA (0.25) 0.00 ± 0.00c 0.00 ± 0.00c GA (0.5) 19.20 ± 0.73a 6.00 ± 0.63b GA (1.0) 12.00 ± 0.83b 12.40 ± 1.12a Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 5. Effect of different GA concentrations on in vitro seed germination in S. acmella after 3 weeks of culture Treatment (µM) Mean no. of days to Germination frequency (%) germination MS 17.40 ± 0.67a 12.00 ± 2.00e MS + GA (0.5) 11.80 ± 0.66cd 26.00 ± 2.44d MS + GA (1.0) 10.80 ± 0.63de 42.00 ± 2.00c MS + GA (5.0) 15.80 ± 0.73ab 22.00 ± 2.00de ½ MS 14.40 ± 0.67bc 30.00 ± 3.16cd ½ MS + GA (0.5) 7.40 ± 0.24e 62.00 ± 4.89b ½ MS + GA (1.0) 4.20 ± 0.37f 94.00 ± 2.44a ½ MS + GA (5.0) 8.60 ± 0.40e 70.00 ± 3.16b Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 6. Effect of different cytokinins on shoot multiplication through nodal segments of S. acmella after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 0.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00d - BA (0.5) 84.80 ± 1.31b 1.40 ± 0.24ab 1.68 ± 0.09bc + BA (1.0) 89.00 ± 0.70a 2.00 ± 0.00a 2.12 ± 0.11a + BA (2.5) 70.00 ± 0.63d 2.00 ± 0.00a 2.38 ± 0.07a + + BA (5.0) 62.40 ± 1.12e 1.60 ± 0.24ab 1.52 ± 0.09bc + + + Kn (0.5) 78.20 ± 1.28c 1.20 ± 0.20b 1.46 ± 0.10bc + Kn (1.0) 83.00 ± 1.04b 2.00 ± 0.20a 1.74 ± 0.08b + + Kn (2.5) 67.40 ± 0.87d 2.00 ± 0.20a 2.18 ± 0.08a + + Kn (5.0) 62.00 ± 1.37e 1.60 ± 0.24ab 1.38 ± 0.05c + + + 2-iP (0.5) 0.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00d + + 2-iP (1.0) 0.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00d + + + 2-iP (2.5) 0.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00d + + + 2-iP (5.0) 0.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00d + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 7. Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA o n shoot multiplication through nodal segments of S. acmella after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 97.40 ± 1.24a 2.40 ± 0.24a 3.20 ± 0.08a - NAA (0.5) 88.40 ± 0.74b 2.20 ± 0.20ab 2.60 ± 0.10ab + NAA (1.0) 77.00 ± 1.26c 2.00 ± 0.00ab 2.36 ± 0.10bc + + + IBA (0.1) 84.80 ± 0.96b 2.00 ± 0.73ab 2.62 ± 0.12ab + + IBA (0.5) 77.00 ± 0.94c 2.00 ± 0.31ab 2.52 ± 0.08abc + + + IBA (1.0) 72.80 ± 0.96c 1.80 ± 0.50abc 1.84 ± 0.16cd + + + IAA (0.1) 57.80 ± 0.80d 1.60 ± 0.31bc 1.46 ± 0.11de + + IAA (0.5) 50.60 ± 0.74e 1.20 ± 0.40c 0.92 ± 0.37e + + + IAA (1.0) 0.00 ± 0.00f 0.00 ± 0.31d 0.00 ± 0.00f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 8. Effect of different concentration of growth regulators on shoot number and shoot length through shoot tip explants after 4 weeks of culture PGR % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 100.0 ± 0.00a 1.00 ± 0.00f 3.84 ± 0.19bcd - BA (0.5) 38.0 ± 2.00de 4.4 ± 0.40bc 2.7 ± 0.50def - BA (1.0) 66.0 ± 2.44b 8.0 ± 0.31a 3.9 ± 0.30bcd - BA (2.5) 60.0 ± 3.16b 5.8 ± 0.37b 4.7 ± 0.28ab + BA (5.0) 60.0 ± 3.16b 4.0 ± 0.31cd 4.2 ± 0.15abc + + Kn (0.5) 46.0 ± 2.44cd 2.6 ± 0.40de 4.0 ± 0.17abcd - Kn (1.0) 64.0 ± 2.44c 3.6 ± 0.40cd 4.5 ± 0.29ab + Kn (2.5) 62.0 ± 2.00c 1.6 ± 0.24e 5.3 ± 0.27a + + Kn (5.0) 56.0 ± 4.00bc 1.6 ± 0.24e 4.4 ± 0.20ab + + ef e 2-iP (0.5) 32.0 ± 2.00 1.4 ± 0.24 2.1 ± 0.12efg + 2-iP (1.0) 44.0 ± 4.00cde 1.6 ± 0.24e 2.3 ± 0.18efg + + 2-iP (2.5) 54.0 ± 2.44bc 1.4 ± 0.24e 1.6 ± 0.13fg + + 2-iP (5.0) 44.0 ± 2.44cde 1.2 ± 0.20e 1.2 ± 0.10g + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 9. Effect of different combination of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot number and shoot length through shoot tip explants after 4 weeks of culture PGR (µM) % Response Mean no. of Mean shoot Frequency of shoots/explant length (cm) callogenesis NAA (0.1) 96.0 ± 2.44a 33.0 ± 1.09a 5.2 ± 0.09a - NAA (0.5) 92.0 ± 2.00ab 28.2 ± 0.66b 5.5 ± 0.17a + NAA (1.0) 82.0 ± 2.00bc 23.2 ± 0.86c 5.6 ± 0.22a + + + IBA (0.1) 92.0 ± 3.74ab 12.8 ± 0.73d 6.1 ± 0.11a + + IBA (0.5) 72.0 ± 3.74cd 15.0 ± 0.31d 5.6 ± 0.28a + + + IBA (1.0) 62.0 ± 3.74de 13.6 ± 0.50d 5.9 ± 0.13a + + + IAA (0.1) 76.0 ± 2.44c 8.0 ± 0.31e 5.5 ± 0.41a + + IAA (0.5) 56.0 ± 2.44ef 2.6 ± 0.40f 2.1 ± 0.10b + + + IAA (1.0) 48.0 ± 2.00f 1.0 ± 0.31f 1.4 ± 0.13b + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 10. Effect of different concentrations of TDZ on multiple shoot formation through shoot tip explants of S. acmella after 4 week of culture TDZ % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis 0.1 84.00 ± 2.40b 6.80 ± 0.60cd 1.40 ± 0.10a - 0.25 98.00 ± 2.00a 30.00 ± 0.30a 1.00 ± 0.10ab - 0.5 82.00 ± 3.70b 11.80 ± 0.90b 1.00 ± 0.10ab - 1.0 70.00 ± 3.10c 8.80 ± 0.60c 1.00 ± 0.10ab + 2.5 48.00 ± 2.00d 5.60 ± 0.40d 0.80 ± 0.00b + 5.0 46.00 ± 2.40d 2.80 ± 0.40e 1.10 ± 0.10ab + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + + indicate no, slight, moderate callusing respectively.
Table 11. Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated shoot tip explants of S. acmella after 4 weeks of transfer BA NAA Mean number of Mean shoot (µM) (µM) shoots/explant length (cm) - - 30.0 ± 0.3c 1.5 ± 0.5e 0.5 - 31.0 ± 0.4c 2.1 ± 0.1d 1.0 - 36.4 ± 0.6b 2.4 ± 0.1c 2.5 - 34.2 ± 0.5b 2.2 ± 0.1d 0.5 0.5 35.2 ± 0.4b 2.8 ± 0.1b 1.0 0.5 40.2 ± 0.7a 3.8 ± 0.2a 2.5 0.5 35.6 ± 0.7b 3.1 ± 0.2b 5.0 0.5 30.2 ± 0.7c 2.9 ± 0.2b Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 12. Effect of PGRs and PGR-free nutrient media on growth and hyperhydricity of TDZ regenerated shoots through shoot tip explants of S. acmella after 4 weeks of transfer Treatment Mean number of shoots/explant Mean shoot length (cm) BA (1.0 µM) + NAA (0.5 µM) 76.0 ± 1.7a (Hyperhydric shoots) 5.3 ± 0.1a Full-strength MS 63.4 ± 1.1b 4.7 ± 0.1b Half-strength MS 42.8 ± 0.9c 4.2 ± 0.1c Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 13. Efficacy of cytokinins on direct shoot organogenesis through leaf explants of S. acmella after 4 weeks of culture PGR % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 00.00 ± 0.00i 0.00 ± 0.00f 0.00 ± 0.00g - BA (0.5) 28.20 ± 1.28f 2.20 ± 0.20de 2.64 ± 0.11de + BA (1.0) 55.60 ± 1.69c 4.60 ± 0.40ab 3.14 ± 0.07abc + BA (2.5) 85.00 ± 1.84a 6.20 ± 0.48a 2.82 ± 0.10bcd ++ BA (5.0) 72.60 ± 1.53b 4.20 ± 0.37bc 2.48 ± 0.13def - Kn (0.5) 9.40 ± 0.40h 1.40 ± 0.40ef 2.82 ± 0.10bcd + Kn (1.0) 44.20 ± 1.24d 3.00 ± 0.31bcde 3.26 ± 0.08ab ++ Kn (2.5) 72.80 ± 1.15b 4.40 ± 0.50b 3.30 ± 0.11a +++ Kn (5.0) 54.20 ± 1.42c 3.40 ± 0.40bcd 2.78 ± 0.10cd + 2-iP (0.5) 00.00 ± 0.00i 0.00 ± 0.00f 0.00 ± 0.00g + 2-iP (1.0) 18.80 ± 1.06g 1.80 ± 0.37de 2.22 ± 0.10ef + + 2-iP (2.5) 35.00 ± 1.84e 3.40 ± 0.50bcd 2.50 ± 0.08def + + 2-iP (5.0) 23.00 ± 1.54fg 2.60 ± 0.24cde 2.14 ± 0.07f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 14. Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on shoot multiplication through leaf explants of S. acmella after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 86.60 ± 1.07ab 23.80 ± 1.35ab 4.06 ± 0.13abc - NAA (0.5) 93.00 ± 2.00a 27.80 ± 1.01a 4.50 ± 0.13a + NAA (1.0) 76.40 ± 1.72c 16.40 ± 0.67cd 3.70 ± 0.08c + + + IBA (0.1) 81.00 ± 1.00bc 19.60 ± 1.63bc 3.70 ± 0.17c + + IBA (0.5) 85.40 ± 1.72b 25.00 ± 1.00c 4.32 ± 0.14ab + + + IBA (1.0) 65.80 ± 1.68d 12.80 ± 0.96de 3.92 ± 0.12bc + + + IAA (0.1) 21.40 ± 0.97e 11.00 ± 0.44e 2.58 ± 0.05e + + IAA (0.5) 25.60 ± 1.69e 16.80 ± 0.58cd 3.16 ± 0.09d + + + IAA (1.0) 0.00 ± 0.00f 0.00 ± 0.00f 0.00 ± 0.00f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 15. Effect of different concentrations of TDZ on callus induction through leaf explants of S. acmella after 4 weeks of culture TDZ % Response Frequency of Texture of callus (µM) callogenesis 0.00 0.00 ± 0.00f - - e 0.10 40.00 ± 3.16 + Yellowish-white friable C1 callus d 0.25 54.00 ± 2.44 + Yellowish-white friable C1 callus c 0.50 72.00 ± 3.74 + Yellowish-white friable C1 callus b 1.00 84.00 ± 2.44 + + Yellowish-white friable C1 callus b 2.50 92.00 ± 2.00 + + Off-white friable pigmented C2 callus a 5.00 100.00 ± 0.00 + + + Off-white slight compact C3 callus Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively. C1: non-organogenic callus. C2: organogenic callus with red pigmentation. C3: less organogenic callus.
Table 16. Effect of different combinations of TDZ with BA on shoot induction through TDZ derived leaf calli of S. acmella after 4 weeks of culture Treatment (µM) % Response Mean no. of shoot Mean no. of Mean shoot length(cm) buds/culture ± SE shoots/culture ± SE TDZ (1.0) 62.00 ± 3.74b 6.80 ± 0.86bc 4.20 ± 0.37bc 1.00 ± 0.07a TDZ (2.5) 38.00 ± 3.74c 5.80 ± 0.37cd 2.80 ± 0.37c 0.98 ± 0.06a TDZ (1.0) + BA (1.0) 84.00 ± 2.44a 16.00 ± 0.54a 14.00 ± 0.54a 1.20 ± 0.11a TDZ (1.0) + BA (2.5) 46.00 ± 4.00c 8.60 ± 0.60b 5.4 ± 0.50b 1.12 ± 0.05a TDZ (2.5) + BA (1.0) 24.00 ± 2.44d 4.00 ± 0.44d 2.60 ± 0.40c 0.62 ± 0.05b TDZ (2.5) + BA (2.5) 0.00 ± 0.00e 0.00 ± 0.00e 0.00 ± 0.00d 0.00 ± 0.00c Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 17. Shoot proliferation through TDZ derived leaf calli of S. acmella after 4 weeks of transfer Treatment (µM) Mean no. of Mean shoot Remark shoots/explant length (cm) TDZ (1.0) + BA (1.0) 14.00 ± 0.54b 1.20 ± 0.11d Shoots green or white but hyperhydric and malformed leaves BA (1.0) 20.40 ± 0.74a 3.54 ± 0.08a Shoots with green and well expanded leaves MS 17.60 ± 1.12ab 2.94 ± 0.08b Whitish-green and crumpled shoots ½ MS 14.80 ± 1.24b 2.26 ± 0.10c Whitish-green and crumpled shoot Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 18. Effect of different concentrations of 2,4-D and BA on callus induction through leaf explants of S. acmella after 4 weeks of incubation 2, 4-D (µM) BA (µM) % Callogenesis Texture of callus 0.5 - 61.40 ± 0.97d Yellow, compact lucid 1.0 - 97.60 ± 1.12ab Yellow, less compact lucid 2.5 - 78.60 ± 1.56c Whitish-yellow, compact and dried 1.0 0.5 98.80 ± 0.48a Yellow, friable and lucid 1.0 1.0 100.00 ± 0.00a Yellow, friable and lucid 1.0 2.5 93.60 ± 1.56b White, compact and dry Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 19. Efficacy of cytokinins on direct shoot organogenesis through cotyledons of S. acmella after 4 weeks of culture PGR % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis BA (0.5) 00.00 ± 0.00e 0.00 ± 0.00f 0.00 ± 0.00e - BA (1.0) 00.00 ± 0.00e 0.00 ± 0.00f 0.00 ± 0.00e - BA (2.5) 66.00 ± 2.44a 7.20 ± 0.37a 2.80 ± 0.09c - BA (5.0) 52.00 ± 2.00bc 4.00 ± 0.31bc 2.38 ± 0.09d - Kn (0.5) 00.00 ± 0.00e 0.00 ± 0.00f 0.00 ± 0.00e + Kn (1.0) 40.00 ± 3.16c 2.80 ± 0.20de 2.12 ± 0.08d - Kn (2.5) 58.00 ± 3.74ab 4.60 ± 0.24b 3.90 ± 0.11a - Kn (5.0) 44.00 ± 4.00c 3.20 ± 0.37cd 2.42 ± 0.11d + 2-iP (0.5) 00.00 ± 0.00e 0.00 ± 0.00f 0.00 ± 0.00e + + 2-iP (1.0) 20.00 ± 3.16d 0.00 ± 0.00f 0.00 ± 0.00e - 2-iP (2.5) 22.00 ± 3.74d 2.00 ± 0.31e 3.28 ± 0.08b + 2-iP (5.0) 00.00 ± 0.00e 0.00 ± 0.00f 0.00 ± 0.00e + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 20. Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on shoot multiplication through cotyledons of S. acmella after 4 weeks of culture
Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 62.00 ± 3.74ab 13.60 ± 0.67b 3.94 ± 0.11ab +
NAA (0.5) 78.00 ± 3.74a 17.40 ± 0.87a 4.50 ± 0.18a +
NAA (1.0) 46.00 ± 5.09bc 9.80 ± 0.66cd 3.22 ± 0.18c + +
IBA (0.1) 52.00 ± 3.74bc 8.20 ± 0.48d 2.96 ± 0.16cd +
IBA (0.5) 38.00 ± 3.74cd 11.80 ± 0.91bc 3.88 ± 0.08b + +
IBA (1.0) 38.00 ± 3.74cd 7.20 ± 0.37d 2.56 ± 0.06de + +
IAA (0.1) 24.00 ± 4.44de 4.20 ± 0.37e 2.24 ± 0.12ef +
IAA (0.5) 14.00 ± 2.44ef 2.60 ± 0.40ef 1.88 ± 0.10f + +
IAA (1.0) 00.00 ± 2.00f 0.00 ± 0.00f 0.00 ± 0.13g + + +
Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. +, + +, + + +, indicate slight, moderate, intense callusing respectively.
Table 21. Effect of different concentrations of TDZ on callus formation through cotyledons of S. acmella after 4 weeks of culture TDZ (µM) % Response Frequency of Callus texture callogenesis 0.1 19.00 ± 4.00d + Yellowish-white, friable 0.25 31.60 ± 2.24c + Yellowish-white, friable 0.5 67.60 ± 2.50b + Yellowish- white, friable 1.0 82.60 ± 1.66a + + Yellowish- white, compact 2.5 77.40 ± 2.18ab + + + Dark-green, compact 5.0 81.00 ± 3.31a + + Dark-green, compact Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callus formation respectively.
Table 22. Effect of different auxins augmented to half-strength basal MS media on in vitro rooting in S. acmella after 4 weeks of transfer Auxin Mean number Mean root Frequency (µM) of roots/shoot length (cm) of callogenesis Control 8.60 ± 0.40fg 8.80 ± 0.26c - NAA (1.0) 20.20 ± 0.58bc 13.40 ± 0.21b - NAA (2.5) 30.40 ± 0.50a 18.60 ± 0.18a - NAA (5.0) 20.40 ± 0.50bc 12.80 ± 0.41b + IBA (1.0) 23.20 ± 0.96b 5.50 ± 0.18d - IBA (2.5) 32.80 ± 1.15a 3.20 ± 0.17ef + IBA (5.0) 16.80 ± 0.96cd 2.20 ± 0.19g + + IAA (1.0) 11.60 ± 1.02ef 4.10 ± 0.13e + IAA (2.5) 14.40 ± 0.67de 5.30 ± 0.09d + + IAA (5.0) 7.60 ± 0.67g 2.90 ± 0.11fg + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 23. Effect of sodium alginate concentration on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium Sodium alginate (% w/v) Conversion response (%) into plantlets 1.0 Fragile beads 2.0 Fragile beads 3.0 73.40 ± 1.88a (but soft to handle) 4.0 74.80 ± 1.46a 5.0 45.60 ± 1.69b Different concentrations of sodium alginate and 100 mM CaCl2·2 H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 24. Effect of calcium chloride concentration on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium Calcium chloride (mM) Conversion response (%) into plantlets 25 Fragile beads 50 Fragile beads 75 65.40 ± 2.03b(but soft to handle) 100 74.80 ± 1.46a 200 39.00 ± 2.44c Different concentrations of sodium alginate and 100 mM CaCl2·2 H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 25. Effect of different treatments on conversion of encapsulated nodal segments of S. acmella after 6 weeks of culture on MS medium Treatment (µM) Conversion response (%) into plantlets MS 74.80 ± 1.46b MS + BA (1.0) + NAA (0.5) 87.80 ± 1.15a MS + BA (1.0) + IAA (0.5) 80.20 ± 2.83b MS + BA (1.0) + IBA (0.5) 73.40 ± 1.02b Different concentrations of CaCl2·2H2O and 4% (w/v) sodium alginate were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 26. Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of S. acmella into plantlets Storage Conversion of Conversion of Conversion of duration encapsulated nodal encapsulated nodal non-encapsulated (weeks) segments into plantlets segments into plantlets nodal segments (%) (encapsulation (%) (encapsulation into plantlets (%) matrix prepared in MS matrix prepared in basal medium) distilled water) 0 87.80 ± 1.15a 27.00 ± 1.22a 99.00 ± 1.00a 1 82.80 ± 1.15ab 15.00 ± 1.58b 73.00 ± 2.00b 2 76.60 ± 1.88bc 04.00 ± 0.31c 51.00 ± 3.31c 4 73.60 ± 1.56b 00.00 ± 0.00d 28.00 ± 2.54d 6 58.00 ± 2.00d 00.00 ± 0.00d 16.00 ± 1.87e 8 46.00 ± 1.87e 00.00 ± 0.00d 09.20 ± 0.37e Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 27. Effect of different planting substrates on ex vitro conversion of encapsulated nodal segments of S. acmella into plantlet after 6 weeks of sowing Treatment (µM) Conversion response (%) into plantlets Soilrite + ¼ MS salts 63.00 ± 2.00a Soilrite + tap water 51.20 ± 0.96b Soilrite + soil + ¼ MS salts 46.60 ± 1.88b Soilrite + soil + tap water 26.80 ± 1.93c Soil + ¼ MS Salts 15.60 ± 2.31d Soil + tap water 00.00 ± 0.00e Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Explanation of Figure 8 Seed germination in S. acmella
A. Seeds of S. acmella obtained from dried flower heads B. Aseptic seedlings grown on half-strength MS medium supplemented with 1.0 µM GA, 3 week-old culture
Figure 9
A B C
D E F
Explanation of Figure 9 Shoot regeneration through shoot tip explants
A. Elongated shoot tips on PGR-free MS medium (control), 4 week-old culture B. Shoot bud sprouting through shoot tip explant on MS medium containing 1.0 µM BA, 1 week-old culture C. Multiple shoot regeneration on MS medium containing 1.0 µM BA, 2 week-old culture D. –do-, 3 week-old culture E. Elongated shoots on MS medium containing 1.0 µM BA, 4 week-old culture F. Shoot regeneration on 1.0 µM Kn supplemented MS medium, 3 week-old culture G. Culture showing high frequency callogenesis on 5.0 µM 2-iP supplemented MS medium, 4 week-old culture
Figure 10
A
C
B D E
Explanation of Figure 10 Shoot regeneration through shoot tip explants
A. Regeneration of multiple shoots through compressed nodal region of shoot tip explant on MS medium containing 1.0 µM BA and 0.1 µM NAA, 1 week-old culture B. High frequency regeneration on above mentioned medium, 2 week-old culture C. Proliferation and elongation of microshoots on MS medium containing 1.0 µM BA and 0.1 µM NAA, 4 week-old culture D. Shoot tip culture showing multiple shoot regeneration with simultaneous callusing through basal cut end of the explant on MS medium containing 1.0 µM BA and 0.5 µM IAA, 2 week-old culture E. Shoot regeneration through shoot tip explant on MS medium containing 1.0 µM BA and 0.5 µM IBA, 2 week-old culture
Figure 11
A B C
D E F
Explanation of Figure 11 Shoot regeneration through shoot tip explants
A. Adventitious shoot regeneration through the basal cut end of shoot tip on 0.25 µM TDZ containing MS medium, 2 week-old culture B. Sprouting of induced buds 0.5 µM TDZ containing MS medium, 2 week-old culture C. Culture transferred on phyatgel-solidified MS medium amended 0.25 µM TDZ containing MS medium, 2 week-old culture D. Cluster of multiple shoots induced adventitiously on 0.25 µM TDZ containing MS medium, 3 week-old culture E. –do-, 4 week-old culture F. Fasciated shoot regeneration with simultaneous callusing on 1.0 µM TDZ, 4 week-old culture
Figure 12
A B
C D
Explanation of Figure 12 Shoot regeneration through shoot tip explants
A. TDZ derived “stock culture” of shoot tip explant maintained on BA (1.0 µM) with NAA (0.5 µM), 4 week-old culture B. Vitrified and glossy shoots on the above mentioned treatment, 4 weeks after sub-culture
C. TDZ derived shoots on half-strength MS medium, 4 weeks after sub-culture D. Healthy shoots with proper leaf expansion on full-strength MS medium, 4 week after sub-culture
50 Mean shoot no./explant 6 Mean shoot length (cm) a 40 b 5 a
30 4
3 20 c b d 2 Mean shoot no./explant shoot Mean Mean shoot length shoot Mean (cm) c 10 d 1
e 0 0 5.0 5.4 5.8 6.2 6.6
pH of the culture medium
Figure 13. Effect of different pH of the culture medium on shoot regeneration through shoot tip explants of S. acmella supplemented with BA (1.0 µM) in combination with NAA (0.1 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
50
Mean shoot no./explant 6 a Mean shoot length (cm) 40 b 5 a
30 4 b 3 20 c
2 Mean shoot no./explant shoot Mean Mean shoot lengthshoot Mean (cm) c 10 1
0 0 Sucrose Fructose Glucose
Carbon source
Figure 14. Effect of different carbon sources on shoot regeneration through shoot tip explants of S. acmella supplemented with BA (1.0 µM) in combination with NAA (0.1 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
50 Mean shoot no./explant 6 a Mean shoot length (cm) 40 5 a
4 30 b
3 20 b b
2 Mean shoot no./explant shoot Mean Mean shoot lengthshoot Mean (cm)
10 c 1
0 0 MS SH B5 Culture medium
Figure 15. Effect of different culture media on shoot regeneration through shoot tip explants of S. acmella supplemented with BA (1.0 µM) in combination with NAA (0.1 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 16
A B C
D E F
Explanation of Figure 16 Shoot regeneration through leaf explants
A-D. Adventitious shoot bud differentiation through basal bulged petiolar end of leaf on MS medium having 2.5 µM BA, 10 day , 2, 3 and 4 week-old cultures A. Leaf culture showing reduced regeneration due to callus through on 2.5 µM Kn supplemented MS medium, 3 week-old culture B. Shoot regeneration on 2.5 µM 2-iP supplemented MS medium, 3 week-old culture
Figure 17
A B C
E
D F
Explanation of Figure 17 Shoot regeneration through leaf explants
A. Direct multiple shoot bud induction on MS medium with 2.5 µM BA and 0.1 µM NAA through the basal petiolar end of leaf explant, 1 week-old culture B. High frequency shoot bud differentiation on 2.5 µM BA along with 0.5 µM NAA, 1 week-old culture C. Elongation of regenerated shoots on above combination, 4 week-old culture. D. Shoot proliferation on MS medium with 2.5 µM BA and 0.1 µM NAA, 4 week-old culture E. Poor regeneration of direct shoot buds on MS medium supplemented with 2.5 µM BA and 1.0 µM NAA showing callogenesis, 1 week-old culture F. Shoot bud regeneration on 2.5 µM BA and 0.1 µM IBA showing callogenesis, 2 week-old culture
Figure 18
A B C
D E F
Explanation of Figure 18 Shoot regeneration through leaf explants
A. Callus induced through leaf explant on 0.5 µM TDZ supplemented MS medium, 4 week-old culture B. Leaf-derived callus on 2.5 µM TDZ showing purplish-red pigmentation, 3 week-old culture C. Shoot buds induced through callus tissue on 2.5 µM TDZ, 4 week-old culture D. Greenish-white and crumpled shoots regenerated through leaf derived callus on TDZ (1.0 µM) and BA (1.0 µM) combination, 4 week-old culture E. Hyperhydric and malformed shoots on TDZ (1.0 µM) and BA (1.0 µM), 4 weeks after sub-culture from BA (1.0 µM) containing MS medium F. Culture showing well expanded and green leaves on full-strength MS medium, 2 weeks after sub-culture from BA (1.0 µM) containing MS medium
Figure 19
A B C
D E
Explanation of Figure 19 Callus induction through leaf explants
A. Yellow-lucid callus initiation through leaf on 1.0 µM 2, 4-D containing MS medium, 2 week-old culture B. Whitish-yellow and dried callus regenerated through leaf explant on 2.5 µM 2, 4-D containing MS medium, 4 week-old culture C. Yellow, friable and lucid callus induced on 1.0 µM 2, 4-D and 1.0 µM BA containing MS medium, 4 week-old culture D. Callus exhibiting few green meristemoids on 1.0 µM BA and 0.5 µM NAA, 2 week-old culture E. Culture showing necrotic tissue on 1.0 µM BA and 0.5 µM NAA, 4 week-old culture
Figure 20
A B
C D
Explanation of Figure 20 Shoot regeneration through cotyledons
A. Direct shoot bud induction through petiolar cut end of the cotyledon on 2.5 µM BA containing MS medium, 2 week- old culture B. Elongation of regenerated shoot buds on above medium, 3 week-old culture C. Shoot regeneration through cotyledon on 2.5 µM 2-iP containing MS medium, 3 week-old culture D. Callus obtained through cotyledons on 5.0 µM 2-iP containing MS medium, 3 week-old culture
Figure 21
A B
C D
Explanation of Figure 21 Shoot regeneration through cotyledons
A. Shoot regeneration through petiolar cut end of the cotyledon on 2.5 µM BA and 0.5 µM NAA containing MS medium, 2 week-old culture B. Further regeneration of shoot buds with simultaneous callogenesis on above medium, 3 week-old culture C. Proliferating culture placed on fresh MS medium supplemented with 2.5 µM BA and 0.5 µM NAA after removing callus tissue, 4 week-old culture D. Cotyledon showing poor regeneration on BA (2.5 µM) and IBA (0.5 µM) containing MS medium
8 a Mean shoot no./explant 100 Mean shoot length (cm) b bc c cd 7 a a 80 de a a
ef b b 6 f 60 c
e 5 40 Mean shoot no./explant shoot Mean Mean shoot length shoot Mean (cm)
20 4
0 3 1 2 3 4 5 6 7 8 Sub-culture passage
Figure 22. Effect of sub-culture passage on shoot proliferation efficiency of shoot tip explants. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 23
A B
C D E
Explanation of Figure 23 Sub-culturing of shoot tip derived tissues
A. Shoot proliferation through mother tissue of shoot tip explant, after 4th sub-culture. B. Group of maintained cultures C. Regenerating tissue through mother tissue of shoo tip explant, after 5th sub-culture passage D. Regenerating tissue through mother tissue of shoo tip explants, after 6th sub-culture passage E. Proliferated culture showing elongated shoots, after 8th sub-culture passage
a Mean shoot no./explant ab 100 Mean shoot length (cm) bc cd 6 de ef 80 f a a a
b g c b 4 60 d
e 40 Mean shoot no./explant shoot Mean Mean shoot length shoot Mean (cm) 2
20
0 0 1 2 3 4 5 6 7 8
Sub-culture passage
Figure 24. Effect of sub-culture passage on shoot proliferation efficiency of leaf explants. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 25
A
B
Explanation of Figure 25 Sub-culturing of leaf derived tissues
A. Shoot proliferation through mother tissue of leaf explant after 7th sub-culture passage B. Group of maintained cultures of leaf explant
8 Mean shoot no./explant Mean shoot length (cm) a a a 60 7 b
c bc c a ab abc 6 40 bc bc d cd d e 5 Mean shoot no./explant shoot Mean 20 lengthshoot Mean (cm) 4
0 3 1 2 3 4 5 6 7 8 Sub-culture passage
Figure 26. Effect of sub-culture passage on shoot proliferation efficiency of cotyledons. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 27
A B
C D
Explanation of Figure 27 Sub-culturing of cotyledon derived tissues
A. Shoot proliferation through mother tissue of cotyledons, after 4th sub-culture passage B. Shoot proliferation through mother tissue of cotyledons, after 5th sub-culture passage C. Shoot elongation after 6th sub-culture passage D. Shoot elongation after 7th sub-culture passage
Figure 28
A B C D
E F G
Explanation of Figure 28 In vitro root induction of microshoots
A. Microshoot showing rooting on half-strength MS medium supplemented with 1.0 µM NAA, 3 week-old culture B. Microshoot showing best rooting on half-strength MS medium supplemented with 2.5 µM NAA, 3 week-old culture C. Stunted and profuse rooting on half-strength MS medium supplemented with 2.5 µM IBA, 4 week-old culture D. Microshoot showing rooting and in vitro bud induction on half-strength MS medium supplemented with 2.5 µM NAA, 2 week-old culture E. An enlarged view of flower bud F. An enlarged view of rooting on agar-solidified nutrient medium with an optimal concentration of NAA (2.5 µM), 4 week-old culture G. An enlarged view of rooting on phytagel-solidified nutrient medium with an optimal concentration of NAA (2.5 µM), 4 week-old culture
120
Soilrite Vermi-compost 100 Garden soil + farmyard (2:1)
80
60 % Survival%
40
20
0 Soilrite Vermi-compost Garden soil + farmyard (2:1)
Types of planting substrate
Figure 29. Effect of different planting substrates on survival percentage of regenerated plantlets after 2 months of field transfer. The bars represent the results of 300 plantlets for each substrate.
Figure 30
A B
C
Explanation of Figure 30 Successfully hardened micropropagted plantlets
A. Group of successfully acclimatized plantlets of S. acmella in thermocol cup containing soilrite, 4 week-old B. Potted plantlets in soil mix with farmyard (2:1) showing flowering after 2 month of field transfer C. Closed view of plantlets
Figure 31
A B
C D
Explanation of Figure 31
Synseeds production and germination
A. Encapsulated nodal segments with 4% Na 2-alginate and 100 mM CaCl 2·2H2O placed on MS basal medium, 1 day- old culture B. Germination of synseeds on MS basal medium, 3 week-old culture C. Shoot emergence on MS medium supplemented with 1.0 µM BA and 0.5 µM NAA, 2 week-old culture D. –do- Root induction, 3 week-old culture
Figure 32
A B
Explanation of Figure 32 Synseed germination
A. Culture showing complete plantlets (elongated shoots with roots) recovery through synseeds on MS medium supplemented with 1.0 µM BA and 0.5 µM NAA, 6 week-old culture B. An isolated synseed showing shoot and root system
Figure 33
A
B C D
Explanation of Figure 33 Successfully hardened plantlets obtained from synseeds
A. An acclimatized plantlet recovered from synseed in soilrite, 4 week-old B. An over view of potted plantlet in soil C. Plantlet with flower head D. A twig showing flower heads
Chl a (mg g-1) Chl b (mg g-1) Total Chl (mg g-1) 1.4
a 1.2 b a
) c a -1 1.0 e
d 0.8 c b bc
a 0.6 a
Chlorophyll content (mg g (mg content Chlorophyll b 0.4 bc c
0.2 0 7 14 21 28 Acclimatization period (days)
Figure 34. Change in chlorophyll contents (a, b & Total) (mg g-1) during acclimatization. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
) Carotenoids (mg g-1) 0.40
a
) 0.35 -1
ab 0.30 abc
0.25 bc
Carotenoids content (mg g (mg content Carotenoids 0.20 c
0.15 0 7 14 21 28 Acclimatization period (days)
Figure 35. Change in carotenoids content (mg g-1) during acclimatization. The bars represents mean ± S. E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
-2 -1 Net photosynthetic rate (PN) (µmol CO2 m s ) 9 )
1 a 8 s- a -2 m 2 7
6 ) (µmol CO N 5 b
4
3
2 c c Netphotosynthetic rate (P
1 0 7 14 21 28 Acclimatization period (days)
-2 -1 Figure 36. Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization. The bars represents mean ± S. E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 37
LP
AM VS
A
SB
B
C
Explanation of Figure 37 Histological study of regenerating tissues
A. An adventitious bud with apical meristem (AM), leaf primordial (LP) and pro-vascular strands (VS) regenerated from shoot tip explant on BA (1.0 µM) and NAA (0.1 µM) B. Section of 2 week-old regenerating shoot tip explant of S. acmella on TDZ (0.25 µM) showing multiple shoot bud formation (SB) from the peripheral cells of basal cut end of the explant along with vascularization C. Shoot bud originating directly from the swollen petiolar cut end of 2 week-old leaf explant of S. acmella on MS medium containing BA (2.5 µM)
Figure 38
LP
ASB AD LP
V ASB
A B
Explanation of Figure 38 Histological study of regenerating tissues
A. Two well differentiated adventitious shoot buds (ASB) embedded in callus tissue induced from leaf explant on TDZ (2.5 µM) B. Apical dome (AD) of shoot bud with a pair of leaf primordia (LP) along with vascularization (V) in the callus.
4.2 Spilanthes mauritiana
4.2.1 Seed germination and collection of explants
The sterilized seeds without pre-soaking in GA (control) failed to germinate in vivo. The pre-soaking of seeds in GA (0.25, 0.5 and 1.0 µM) slightly increased in vivo seed germination from 6.20 ± 0.58 (0.25 µM) to 14.0 ± 1.37% (1.0 µM) which was possible after 19.80 ± 0.80 and 11.60 ± 0.92 days of sowing respectively (Table 28). For further improvement in the germination frequency, in vitro seed germination was performed. The sterilized seeds were inoculated aseptically on full and half-strength MS basal medium with or without varying concentrations of GA (0.5, 1.0 and 5.0 µM). On MS basal medium devoid of GA (control), seeds did not germinate satisfactorily and resulted in only 8.80 ± 0.58% germination after 3 weeks of incubation. Incorporation of GA enhanced the germination percentage. Among different treatments tested, highest seed germination (86.00 ± 1.87%) was observed on half-strength MS supplemented with 1.0 µM GA after 3 weeks of incubation (Fig. 39 A & B). On this treatment, seeds were started to germinate within a mean of 5.80 ± 0.58 days of incubation which was the earliest among all the treatments tested. Full-strength MS medium amended with similar concentration of GA (1.0 µM) delayed their germination to 14.20 ± 0.58 days and only 31.40 ± 0.97% seedlings were developed. Further, decreasing or increasing the concentration of GA did not improve the germination on both the strength of MS medium (full and half-strength) (Table 29).
The nodal segments (1-1.5 cm), shoot tips (0.8-1.2 cm with one pair of leaf primordia), leaves and cotyledon were excised from 3 week-old aseptic seedlings and used as the explants to induce multiple shoots.
4.2.2 Nodal segment culture
Nodal segments, each bearing two axillary buds were cultured on three adenine- based cytokinins (BA, Kn and 2-iP) and one urea-based cytokinin (TDZ) with or without auxin (NAA, IAA and IBA).
4.2.2.1 Effect of adenine-based cytokinins on shoot regeneration
The nodal segments failed to respond morphogentically on control medium. They remained green up to 2 weeks, gradually turned yellowish-brown and died after 4 weeks of inoculation. Exogenous supplementation of cytokinins did not exhibit any beneficial role on axillary bud multiplication. Only 1.40 ± 0.24 to 2.00 ± 0.00 shoots per explant along with moderate to intense basal callusing were recorded on BA or Kn supplemented MS medium while on 2-iP supplemented MS medium all the nodal segments failed to induce any shoot and only high frequency callogenesis was noticed after 4 weeks of culture. Among the treatments tested, maximum regeneration response (99.60 ± 0.40) was noticed on 1.0 µM BA supplemented MS medium while maximum elongation (1.50 ± 0.07 cm) was noticed on 2.5 µM Kn supplemented MS medium (Fig. 40 A & B, Table 30).
4.2.2.2 Effect of cytokinin-auxin combinations on shoot regeneration
Excised nodal segments also inoculated on cytokinin-auxin combinations to improve multiple shoot induction as compared to cytokinin alone. Different concentrations (0.1, 0.5 and 1.0 µM) of three auxins (NAA, IAA and IBA) were added to 1.0 µM BA containing MS medium. Surprisingly, these combinations also did not improve axillary bud sprouting due to high frequency callogenesis. Initially nodal
segments increased their size within 5-7 days of inoculation; axillary buds became prominent during elongation and started to sprout after an incubation period of 10-12 days. Callogenesis increased consistently from 0.1 to 1.0 µM NAA and IAA. Only 1.80 ± 0.20 to 2.20 ± 0.20 shoots per explant were induced on 1.0 µM BA plus 1.0 µM NAA and 1.0 µM BA plus 0.5 µM IAA containing MS medium respectively. However, these shoots were longer than induced on individual cytokinins. Among all the combinations, longest shoots (3.20 ± 0.09 cm) were achieved on MS medium supplemented with 1.0 µM BA and 0.1 µM IAA (Fig. 40 C). On the other hand, only intense callusing was noticed on IBA supplemented MS medium (Table 31).
4.2.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
TDZ supplemented (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) MS medium adversely affected the totipotency of nodal segments. All the treatments of TDZ failed to induce shoot. Explants remained fresh and green on 0.1 to 1.0 µM TDZ up to 3 weeks of culture, thereafter degenerated. On further increasing the concentration (2.5 and 5.0 µM), the nodal segments increased their girth, became translucent and remained as such even after 4 weeks of culture.
4.2.3 Shoot tip culture
The objective of the first experiment with shoot tip explants was to identify the most effective growth regulator type and its concentration for multiple shoot induction. Different concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) of various cytokinins (BA, Kn, 2-iP and TDZ) were tested individually and in combination with three concentrations (0.1, 0.5 and 1.0 µM) of different auxins (NAA, IAA and IBA).
4.2.3.1 Effect of adenine-based cytokinins on shoot regeneration
The shoot tip explants remained green and fresh but failed to induce multiple shoot buds on control medium. On control treatment, they elongated into an average of 4.90 ± 0.11cm long shoots after 4 weeks of culture (Fig. 41 A). However, different
concentrations of cytokinins (0.5, 1.0, 2.5 and 5.0 µM) triggered the multiple shoot bud formation when supplied to MS basal medium. Among three adenine-based cytokinins (BA, Kn and 2-iP) tested, BA (1.0 µM) was proved to be optimal with 59.80 ± 1.56% response. Initial response was noticed within 8-10 days of inoculation in terms of shoot elongation and apical bud break (Fig. 41 B). Thereafter induced buds elongated and developed into cluster of multiple shoots (Fig. 41 C). On 1.0 µM BA supplemented MS medium a maximum of 6.00 ± 0.44 shoots per explant measuring 2.36 ± 0.14 cm was recorded after 4 weeks of inoculation (Fig. 41 D). However, on increasing or decreasing the BA concentration beyond an optimal level gradual loss in shoot regeneration efficiency was noticed. On MS medium supplemented with 2.5 µM BA response was decreased to 52.60 ± 1.02% with mean number of 5.00 ± 0.31 shoots per explant and mean shoot length of 2.90 ± 0.11 cm followed by mean number of 3.60 ± 0.50 shoots per explant and mean shoot length of 3.44 ± 0.12 cm with 44.20 ± 2.28% response on MS basal medium with 5.0 µM BA (Fig. 41 E). On replacing BA with the similar concentrations of Kn (0.5, 1.0, 2.5 and 5.0 µM), shoot regeneration efficiency of shoot tip explants was reduced and regeneration was initiated after 10-12 days of inoculation. A maximum of 4.40 ± 0.50 shoots per explant with mean shoot length of 3.32 ± 0.15 cm was induced on MS augmented with 1.0 µM Kn (Fig. 41 F). On further increasing or decreasing Kn concentration beyond the optimal level, in vitro response of shoot tip explants was further delayed and lesser number of shoots were obtained as compared to similar concentrations of BA. On 2.5 µM Kn amended MS medium, only 3.80 ± 0.58 shoots per explant were observed in 43.20 ± 1.46% cultures while on 5.0 µM Kn containing MS medium only 2.80 ± 0.37 shoots per explant were differentiated in 37.00 ± 1.54% cultures after 4 weeks of inoculation. The shoot tip explants cultured on MS medium supplemented with similar concentrations of 2-iP showed the weakest response for shoot regeneration as compared to BA and Kn. Explants showed shoot emergence after 14-15 days of incubation on 2-iP supplemented medium. Among all the concentrations of 2-iP tested, MS medium supplemented with 2.5 µM 2-iP induced only a maximum of 2.60 ± 0.24 shoots per explant with shoot length of 2.42 ± 0.15 cm in 25.60 ± 1.69% of cultures after same
incubation period of 4 weeks whereas 5.0 µM 2-iP induced high frequency of callogenesis (Table 32).
4.2.3.2 Effect of cytokinin-auxin combinations on shoot regeneration
The effect of various auxins (NAA, IAA and IBA) was also studied with the optimized concentration of most effective cytokinin (1.0 µM BA). As a result of synergism between cytokinin and auxin, frequency of multiple shoot induction was improved than cytokinin alone. Among three auxins tested, IAA with BA was found to be most effective for multiple shoot regeneration. Regeneration was being earliest and multiple shoot induction was noticed just after 5-7 days of inoculation. Out of three combinations of BA and IAA, the highest shoot regeneration frequency (98.0 ± 2.00%) along with a maximum number (18.80 ± 0.48) of shoots per explant was recorded without an intervening callus on MS medium supplemented with 1.0 µM BA and 0.5 µM IAA ((Fig. 42 A-D). On this combination not only the vigor of shoots, proper leaf expansion and shoot elongation were also observed. However, a slight decrease in mean number of shoots was observed with further increase in IAA concentration to 1.0 µM and resulted in greenish-white friable and fleshy callus at the base of shoots. The callus was removed continuously in order to check its adverse effect on shoot multiplication efficiency of explants. Similar pattern of regeneration was observed with BA and NAA combination, although the regeneration frequency was lesser than BA and IAA combination due to callusing. On BA and NAA supplemented MS medium regeneration of shoot buds was initiated after 8-10 days of incubation. A maximum of 13.20 ± 0.48 shoot per explant and 3.72 ± 0.13 cm shoot length was noticed on MS medium supplemented with 1.0 µM BA and 0.5 µM NAA after 4 weeks of incubation (Fig. 42 E & F). On the other hand, shoot tip explants cultured on MS medium augmented with BA (1.0 µM) and IBA (0.1, 0.5 and 1.0 µM) showed the weakest response for direct shoot multiplication wherein regeneration was noticed after 12-14 days of inoculation. The MS medium containing 1.0 µM BA and 0.5 µM IBA was found to be critical for inducing a maximum of only 10.80 ± 0.37 shoots per explant with mean shoot length of
3.36 ± 0.15 cm in 54.0 ± 4.00% of cultures. However, on increased concentration of IBA (1.0 µM) response was slightly reduced due to intense callusing (greenish-white) and only 6.60 ± 0.40 shoots per explant were recorded after same incubation period of 4 weeks. Thus, the affectivity order of auxins added to an optimal level of BA for multiple shoot regeneration through this explant was IAA>NAA>IBA (Table 33).
4.2.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
The exogenous augmentation of TDZ to the MS basal medium significantly improved shoot multiplication efficiency of shoot tip explants as compared to individual adenine-based cytokinins, however the response was significantly varied according to the concentration of TDZ used (Table 34). Similar to S. acmella, adventitious shoot buds induced directly from the basal cut ends without any intervening callus. Apical bud of shoot tip explants did not sprout in any of the culture. All the shoot tip explants elongated in to single shoot with simultaneous swelling in the basal cut end on MS basal medium with TDZ (0.1-1.0 µM) after 1 week of culture. Cluster of shoot buds was firstly appeared on 0.25 µM TDZ after 2 weeks of culture (Fig. 43 A). Elongated shoot was cut and only the basal regenerating tissue was transferred to the fresh medium of similar concentration of TDZ (0.25 µM) for further growth of shoot buds. A maximum of 26.20 ± 1.35 shoots per explants with 93.60 ± 3.86% response was achieved on MS medium supplemented with 0.25 µM TDZ after 4 weeks of inoculation (Fig. 43 B). In contrast, 2.5 µM TDZ drastically reduced the shoot formation and only 3.80 ± 0.58 shoots per explant were recorded during the same incubation period of 4 weeks. While, further increasing the TDZ concentration to 5.0 µM, shoot tip explants failed to respond any morphogenesis and only callus was induced after 4 weeks of incubation (Fig. 43 C). Although the mean number of shoots per explant was quite higher on TDZ containing MS medium than individual adenine-based cytokinin but shoot elongation was not evident even after 4 weeks of culture, indicating the inhibitory effect of TDZ on shoot elongation. The mean length of regenerated shoots was found to be inversely proportional to the concentration of TDZ (up to 2.5 µM). The use of 2.5 µM TDZ resulted in the shortest shoots (1.10 ± 0.08 cm) and the longest shoots (1.76 ± 0.11 cm) were induced on
0.1 µM TDZ containing MS medium. The observed abnormal effect of TDZ like poor intermodal elongation was not a permanent phenomenon and could be overcome by transferring the explants to other PGR containing or PGR-free medium (control). The shoots regenerating on optimal concentration of TDZ (0.25 µM) were only selected for further transfer.
4.2.3.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue
BA (0.5, 1.0 and 2.5 µM) alone did not improve the growth of regenerated shoots satisfactorily when transferred from optimal TDZ (0.25 µM), thus necessitated the supplementation of auxin to BA containing MS basal medium. Among different combinations, 1.0 µM BA and 0.5 µM IAA significantly improved the shoot regeneration efficiency of the mother tissue and a maximum of 41.00 ± 1.18 shoots per explant with 2.11 ± 0.06 cm shoot length was induced after 4 weeks of transfer ((Fig. 43 D, Table 35). While, the MS medium supplemented with 2.5 and 5.0 µM BA in combination with 0.5 µM IAA induced high frequency of callogenesis and reduced the regeneration efficiency of explant (Fig. 43 E). Moreover, on this treatment shoots had a glossy appearance and hyperhydric texture which made them brittle. On further culture of such shoots on the same concentration, most of the shoots turned yellowish and died after 4 weeks of transfer. Still none of the combination improved the shoot length successfully. Thus, for further improvement in shoot elongation, the regenerated shoots were further sub- cultured on to full and half-strength MS basal medium with or without 1.0 µM BA and 0.5 µM IAA. The cultures multiplied on MS supplemented with 1.0 µM BA and 0.5 µM IAA was treated as ‘stock culture”.
4.2.3.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture
In this experiment, the ‘stock culture’, from each replicate was divided into 4 pieces and each piece was sub-cultured to the above mentioned regeneration media.
Among the treatments tested, BA and IAA amended MS medium considerably enhanced the potential of regenerating stock tissue and a maximum of 72.00 ± 1.92 shoots per explant (2.74 fold increase) and with 4.00 ± 0.10 cm of shoot length was recorded after 4 weeks of sub-culturing. Although the shoot proliferation efficiency and shoot growth of the stock culture was maximum but most of the regenerated shoots were hyperhydric which adversely affected the survivability of shoots. Hyperhydricity was started to first appear after 2 weeks of sub-culture which increased thereafter (Fig. 44 A). On the other hand, PGR-free full-strength MS basal medium satisfactory improved overall growth pattern (mean shoot number, shoot length and heath of shoot) of stock tissue and a maximum of 64.00 ± 1.37 shoots per explant with mean shoot length of 3.72 ± 0.08 cm was achieved after 4 weeks of sub-culture. On this medium, all the regenerated shoots were healthier than shoots sub-cultured on PGR supplemented nutrient medium. On full-strength MS medium none of the shoot exhibited hyperhydricity and glassy appearance (Fig. 44 B). While, half-strength MS basal medium significantly reduced the potential of mother tissue and induced lesser number of shoots per explant (50.00 ± 1.70) with 3.42 ± 0.07 cm of shoot length than on full-strength MS medium might be due to lesser nutrient supply. All the shoots were healthy as found on full-strength MS medium (Table 36).
4.2.3.4 Effect of different pH on shoot regeneration
The effect of different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) was tested on MS basal medium supplemented with an optimal combination of BA (1.0 µM) and IAA (0.5 µM) (Fig. 3). Among different pH levels tested, optimum shoot proliferation and elongation was achieved at 5.8 wherein a maximum 18.80 ± 0.48 shoots per explant and 4.26 ± 0.10 cm of shoot length were induced after 4 weeks of culture. On further increasing the pH to 6.6, a gradual loss in shoot proliferation efficiency (6.80 ± 0.80 shoots per explant) was observed. But the shoot multiplication rate was severely affected with a slight reduction in pH to 5.4 and only 4.00 ± 0.54 shoots per explant were noticed. At this pH, all the regenerated shoots were highly hyperhydric that caused the poorest growth in regenerants. Further lowering the pH to 5.0, all the shoot tip explants turned
brown after 2 weeks, thereafter died might be due to the acidic nature of medium (Fig. 45).
4.2.3.5 Effect of different carbon sources on shoot regeneration
The response of shoot tip explants to three carbon sources i.e., sucrose, fructose and glucose (each tested at 3% concentration) was also tested with MS basal medium augmented with BA (1.0 µM) and IAA (0.5 µM) at 5.8 pH. Among three carbon sources, sucrose proved to be the best for shoot regeneration as induced a maximum of 18.80 ± 0.48 shoots per explant and 4.26 ± 0.10 cm shoot length after 4 weeks of culture. The MS basal medium supplemented with fructose showed moderate response and induced 16.00 ± 1.37 shoots per explant and 2.88 ± 0.17 cm shoot length. Whereas glucose was the least effective for shoot regeneration as regenerated only 4.60 ± 0.50 shoots per explant with 2.02 ± 0.06 cm shoot length (Fig. 46).
4.2.3.6 Effect of different culture media on shoot regeneration
The effect of three different basal media (B5, MS and SH) was also evaluated with the optimum treatment of BA (1.0 µM) and IAA (0.5 µM) comprising 3% sucrose at 5.8 pH for improvement in shoot multiplication and elongation. The MS medium was found to be the most suitable for maximum shoot proliferation (18.80 ± 0.48 shoots per
explant and 4.26 ± 0.10 cm shoot length). B5 exhibited poor response for all the evaluated parameters and induced only 5.60 ± 0.67 shoots per explant and 2.94 ± 0.06 cm shoot length, while SH showed the satisfactory result with a mean of 10.60 ± 1.32 shoots per explant and 3.10 ± 0.11 cm shoot length (Fig. 47).
4.2.4 Leaf culture
As the effect of different cytokinins and auxins on shoot regeneration was observed for shoot tip explants likewise the cumulative effect of cytokinins and auxins was also observed for leaf explants.
4.2.4.1 Effect of adenine-based cytokinins on shoot regeneration
Leaf explants failed to induce shoot bud differentiation on control treatment, only enlargement in their size was noticed even up to 4 weeks of incubation. However, augmentation of cytokinins (BA, Kn and 2-iP) at 0.5, 1.0, 2.5 and 5.0 µM to the MS basal medium favored multiple shoot from the leaf explants differentially. Initially, enhancement in leaf size was observed with simultaneous swelling at petiolar cut ends within 4-5 days of incubation. Thereafter, green bulging was noticed from the swelled cut end followed by differentiation of multiple adventitious buds after 2 weeks of incubation while distal end of the leaf did not differentiate even after 4 weeks of inoculation (Fig. 48 A). Differentiated buds increased in number on further culture on same regeneration medium.
Frequency of shoot organogenesis increased linearly on BA up to 2.5 µM concentration afterward a gradual reduction was noticed. Among all the concentrations, 2.5 µM BA was proved to be the optimum concentration as induced a maximum of 5.60 ± 0.40 shoot per explant and 3.44 ± 0.16 cm shoot length with 87.00 ± 2.00% response frequency after 4 weeks of incubation (Fig. 48 B & C). However, on increasing the concentration of BA to 5.0 µM the frequency of callogenesis was increased that negatively affected shoot organogenesis as only 4.40 ± 0.50 shoots per explant were observed in 75.60 ± 1.69% cultures during similar incubation period of 4 weeks. Similarly, leaf explants induced multiple shoot formation on MS medium supplemented with different concentrations of Kn (0.5, 1.0, 2.5 and 5.0 µM). However, the shoot regeneration efficiency was weaker than BA and only 4.20 ± 0.37 shoots per explant with mean shoot length of 3.76 ± 0.11 cm were observed in 75.60 ± 1.69% cultures on 2.5 µM Kn after 4 weeks of incubation. The frequency of callogenesis was more pronounced on 2.5 µM Kn as compared to the similar concentration of BA (Fig. 48 D). That obstructed the direct shoot organogenesis pathway. The effect of another cytokinin (2-iP) was also observed on direct shoot organogenesis. However, 2.5 µM 2-iP supplemented MS medium induced higher
frequency of callogenesis with a maximum of only 3.20 ± 0.37 shoots per explant and mean shoot length of 2.74 ± 0.11 cm (Fig. 48 E, Table 37).
4.2.4.2 Effect of cytokinin-auxin combinations on shoot regeneration
The combined effect of various auxins (IAA, IBA and NAA) at different concentrations (0.1, 0.5 and 1.0 μM) with optimal concentration of BA (2.5 μM) on multiple shoot induction form leaf explants was also evaluated. As a result of interaction between cytokinin and auxin, higher frequency of shoot regeneration was observed on combination treatments as compared to cytokinin alone. Cytokinin-auxin combinations also enhanced the frequency of callogenesis as compared to individual cytokinin. Among the combinations tested, BA along with IAA exhibited a synergetic effect on multiple shoot formation after 1 week of incubation. The MS medium supplemented with 2.5 µM BA and 0.5 µM IAA was found to be the most critical combination as resulted the production of a maximum of 15.00 ± 0.31 shoots per explant with 4.22 ± 0.11 cm of shoot length in 98.00 ± 2.00% cultures after 4 weeks of culture (Fig. 49 A-E). On replacement of IAA by NAA (0.1, 0.5 and 1.0 µM) to BA supplemented nutrient medium, a significant decrease in shoot induction frequency was observed. The MS medium with 2.5 µM BA and 0.5 µM NAA induced a maximum of 12.20 ± 0.48 shoots per explant and 3.52 ± 0.09 cm of shoot length in 94.00 ± 1.87% culture after 4 weeks of incubation . On this combination a heavy callogenesis was occurred leading to reduced shoot regeneration. The frequency of callogenesis increased on further increasing the concentration of NAA to 1.0 µM. The regeneration of shoots further lowered with intense callogenesis when BA and IBA combination was tried. It was observed that the lowest concentration of IBA (0.1 µM) in combination with BA (2.5 µM) was effective to induce shoot regeneration with a maximum of only 5.80 ± 0.37 shoots per explant with mean shoot length of 2.30 ± 0.15 cm in only 17.00 ± 2.00% of cultures after same 4 weeks of incubation ((Fig. 49 F, Table 38).
4.2.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Various concentrations of TDZ (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) were also tested to explore the caulogenic potential of young leaf explants. A wide range of variation in regeneration response was observed on TDZ supplemented MS medium. On 0.1 µM TDZ leaf explants failed to respond morphogenetically, only a little callusing was noticed at wounded end of the explants. Whereas, the leaf explants implanted on MS basal medium having 0.25 and 0.5 µM TDZ induced calli first then started differentiation directly. Callusing was started at the excised petiolar end after 4-7 days of inoculation and subsequently covered the entire leaf surface in next 7 days. The callus was whitish- green and fleshy in appearance. Incidentally, callus did not differentiate any shoot bud and ceased to grow further after 12 days of initiation. After scarping the callus tissue, leaf explants were further transferred to fresh culture medium of similar concentrations of TDZ. Reddish-green pigmentation was noticed on leaf surface after 1 week of transference. The leafy structures were emerged directly from the pigmented area adjacent to mid rib region and their subsequent differentiation into shoots with dark green leaves and stunted growth was observed after 4 weeks of transference (Fig. 50 A). Among different concentrations, 0.5 µM TDZ proved to be the optimal concentration as induced a maximum of 8.40 ± 0.50 mean number of shoots per explant and 1.10 ± 0.04 cm shoot length in 81.00 ± 1.18% cultures. However, on slight increasing the TDZ concentration to 1.0 µM, leaf surface expanded due to callusing after 1 week of culture. After scraping the callus tissue jointed shoots were directly induced from the expanded leaf after 2 week of culture and a maximum of 4.20 ± 0.37 shoots were induced in 36.80 ± 0.96% cultures on 1.0 µM TDZ containing MS medium after 4 week of culture (Fig. 50 B & C). On further increasing the TDZ concentration to 2.5 and 5.0 µM, only fast growing greenish-white compact and dry calli were noticed respectively. None of the concentration (2.5 and 5.0 µM) exhibited shoot regeneration even after 4 weeks of culture (Table 39). The shoots induced on optimal concentration of TDZ i.e., 0.5 µM did not grow further on the same culture medium. Therefore, the entire leaf explants bearing the stunted shoots (regenerated on 0.5 µM TDZ) were transferred to cytokinin and auxin supplemented MS medium for further shoot growth.
4.2.4.3.1 Effect of cytokinin-auxin combinations on shoot regeneration from TDZ derived tissue
To study the synergistic effect (if any) of auxin and cytokinins, different concentrations of BA (0.5, 1.0 and 2.5 µM) were tested with or without NAA and IAA (1.0, 0.5 and 1.0 µM) for further shoot proliferation in TDZ exposed leaf explants. None of the individual concentration of BA was found significant. However, cytokinin-auxin combination considerably enhanced the shoot proliferation of mother tissue. Of the various combinations tested, 1.0 µM BA in conjunction with 0.5 µM IAA proved to be the best shoot proliferation medium, giving a maximum of 12.40 ± 0.74 shoots per explant with 2.20 ± 0.07 cm shoot length after 4 weeks of transfer (Fig. D) and so was selected as the optimal shoot multiplication medium for TDZ treated leaf explants. In fact, addition of 0.5 µM IAA to 1.0 µM BA supplemented medium resulted in nearly 1.2 fold increase in shoot proliferation as compared to that on 1.0 µM BA alone. The shoots obtained on 1.0 µM BA and 0.5 µM IAA supplemented medium were healthy. On the other hand, shoots obtained on 1.0 µM BA and 0.5 µM NAA supplemented medium were malformed as most of them were jointed and overcrowded by white friable dry callus (Table 40). For further enhancing the regeneration potential of leaf explants, shoots on 1.0 µM BA and 0.5 µM IAA supplemented MS medium were treated as ‘stock culture’ those were sub-cultured to similar combination of BA and IAA, full and half-strength basal MS medium.
4.2.4.3.2 Effect of different nutrient strengths on shoot proliferation from stock culture
Among different treatments tested, PGR-free MS basal medium was found most effective for further shoot proliferation and overall shoot growth. A maximum of 15.00 ± 0.31shoots per explant with mean shoot length of 2.62 ± 0.04 cm was achieved after 4 weeks of sub-culturing. All the shoots were healthy and exhibited fully expanded leaves (Fig. 51 A). Whereas half-strength MS basal medium slightly reduced both the parameters i.e., mean shoot number (13.40 ± 0.60) and shoot length (2.50 ± 0.03 cm) might be due to reduced nutrients.
On the other hand, when sub-culturing was done on to the similar combination of BA and IAA shoots were hyperhydric might be due to continuous exposure of PRGs (Fig. 51 B), but the shoot proliferation was almost similar to full-strength MS medium (16.20 ± 0.58) (Table 41).
4.2.4.4 Effect of auxin (2, 4-D) on callus induction
Leaf explants were used to obtain regeneration through callus. Explants were cultured on 2, 4-D (1.0, 2.5 and 5.0 µM) containing MS medium to induce callogenesis. Optimum callusing (96.60 ± 1.02%) was noticed on 2.5 µM 2, 4-D that was started within 5-7 days of incubation. Leaf explants induced only 63.00 ± 1.54% callusing at lower most concentration i.e., 1.0 µM while higher concentration (5.0 µM) induced 87.60 ± 1.93% callogenesis. Thus, 1.0 µM 2, 4-D was tested in combination with varying concentrations (0.5, 1.0 and 2.5 µM) of BA to achieve high frequency callogenesis. BA and 2, 4-D combinations slightly improved callus formation. Among the combinations of BA and 2, 4-D, 1.0 µM BA and 2.5 µM 2, 4-D yielded maximum callogenesis (99.60 ± 0.40%) after 4 weeks of culture. Whereas 98.20 ± 0.91 and 92.40 ± 1.12% callusing was noticed on 0.5 and 2.5 µM BA when added to 2.5 µM 2, 4-D containing MS medium (Table 42). Thus, only the calli induced on 1.0 µM BA with 2.5 µM 2, 4-D combination were transferred to shoot regeneration medium (1.0 µM BA and 0.5 µM IAA) wherein few meristemoids were noticed after 2 weeks of transference but ceased to grow further and died after 4 weeks of culture (Fig. 52 A-D).
4.2.5 Cotyledon culture
Similar to the other explants, cotyledons were also tested on different hormonal regimes to explore their caulogenic potential. The entire explants along with their petioles were inoculated as such on MS medium supplemented with various cytokinins viz., BA, Kn, 2-iP and TDZ at varying concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) singly or with various combinations of auxins viz., NAA, IAA and IBA (0.1, 0.5 and 1.0 µM).
4.2.5.1 Effect of adenine-based cytokinins on shoot regeneration
The regeneration response of cotyledons after 4 weeks of culture on different cytokinins has been represented in Table. No shoot regeneration was recorded on basal MS medium. Exogenous augmentation of cytokinins to the MS basal medium induced multiple shoots. All the explants exerted their initial response by swelling and enlargement in size after 5-7 days of culture. Callusing (greenish-white and lucid) was noticed at the petiolar cut end while lamina surface remained entire (Fig. 53 A). Among the cytokinins tested, BA was found to be the best at 2.5 µM to induce a maximum of 4.80 ± 0.37 shoots per explant having 3.16 ± 0.09 cm of shoot length directly from the petiolar cut end in 83.00 ± 2.54% cultures (Fig. 53 B & C). On increasing the BA concentration beyond the critical level and only 1.80 ± 0.37 shoots per explant with 2.90 ± 0.11cm shoot length was noticed on 5.0 µM BA whereas no regeneration was noticed on reduced concentrations of BA (0.5 and 1.0 µM). The Kn supplemented MS basal medium considerably suppressed the regeneration potential of the explants because of pronounced white, friable and fleshy callus as compared to the similar concentrations of BA (Fig. 53 D). On the other hand, 2-iP at all the concentrations did not exhibit any caulogenic response due to intense callogenesis (Table 43).
4.2.5.2 Effect of cytokinin-auxin combinations on shoot regeneration
Additive effect of various auxins (NAA, IAA and IBA) on caulogenesis was also assessed with MS medium comprising the optimized level of BA. Similar to other explants types, cytokinin-auxin combinations exerted their synergistic effect on direct organogenesis from cotyledons. All the explants induced callusing first then initiated organogenesis. Callusing was initiated from the incised end that subsequently covered the entire surface of cotyledon after 1 week of culture. The callus was greenish-white, compact and watery that gave fleshy appearance to the explant. After 2 weeks of culture dark green shoot primordials were noticed from petiolar cut end and its adjacent area (Fig. 54 A). Callus formation was also observed at the site of regeneration. Thus, for maximized shoot regeneration it was necessary to sub-culture the explants on fresh medium after 15-20 days otherwise callus formation suppressed the shoot regeneration (Fig. 54 B). Among the various combinations, BA (2.5 µM) and IAA (0.5 µM) was
found to be the critical for direct organogenesis from cotyledons. A maximum of 14.0 ± 1.09 shoots having the mean shoot length of 4.44 ± 0.22 cm was induced in 82.00 ± 2.09% of cultures after 4 weeks of incubation (Fig. 54 C). The BA and NAA combinations showed similar response like that of BA and IAA combinations; however regeneration frequency was lesser (Table 44). On the other hand, 2.5 µM BA with 0.1 µM IBA containing MS medium induced only 5.80 ± 0.58 shoots per explant in 13.00 ± 2.00% cultures along with fast growing callus that covered the regenerating shoot bud and hampered their further growth (Fig. 54 D). Whereas, higher concentrations of IBA (0.5 and 1.0 µM) failed to induce a single shoot bud even after 4 weeks of incubation.
4.2.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
None of the tried concentration of TDZ showed caulogenic response through cotyledons; only callusing was started within 7-12 days of culture. Calli were yellowish- white to dark green in color and fleshy in texture. Calli induced on the lower concentrations (0.1-0.5 µM) were friable whereas higher concentration (1.0-5.0 µM) induced comparatively compact calli (Table 45). None of callus tissues induced a single shoot even after their sub-culturing to the regeneration medium.
4.2.6 Effect of sub-culturing on shoot proliferation
For further proliferation, the regenerating tissues were sub-cultured continuously (up to 8th sub-culture passage) to the fresh medium supplemented with an optimized treatment of PGRs respective to the explant types. The MS medium with BA (1.0 µM) and IAA (0.5 µM) was used for shoot tip explants while MS having BA (2.5 µM) and IAA (0.5 µM) combination was used for leaves and cotyledons. Sub-culturing gradually increased the regeneration efficiencies’ of all the explant types. The shoots grew vigorously that made their processing (during sub-culturing) difficult. During sub- culturing, the excised shoots were simultaneously transferred to root induction medium while the remaining tissues were again sub-cultured on to the fresh regeneration medium. For shoot tip explants, the frequency of direct shoot multiplication increased considerably from first sub-culture (34.00 ± 1.37 shoots per explant) to sixth sub-culture (73.20 ± 1.82
shoots per explant) and remained almost consistent at seventh sub-culture passage (72.20 ± 0.83 shoots per explant), but declined thereafter (64.00 ± 1.37 shoots per explant). For the leaf explants, regeneration efficiency continuously increased up to fifth sub-culture passage (70.20 ± 0.80 shoots per explants), remained same at sixth passage. Thereafter, a gradual loss in shoot number was observed at seventh (67.40 ± 0.87) and eight sub- culture passage (65.60 ± 0.67). On the other hand, through cotyledons maximum number of shoots (39.80 ± 0.80) was obtained after fifth sub-culture passage. Irrespective to the explant types, a continuous increase in shoot length was noticed after every sub-culture passage (Fig. 55, 56, 57 & 58).
4.2.7 In vitro rooting of microshoots
Although roots induced simultaneously on proliferation medium in all the explant types, but those roots did not help in successful establishment of plantlets in the field conditions as they were thin and delicate. Therefore, the regenerated shoots were excised from proliferating culture medium and transferred to half-strength MS medium supplemented with or without three auxins i.e., IAA, IBA or NAA at different levels (1.0, 2.5 and 5.0 µM). The half-strength MS basal medium devoid of auxin (control) induced fibrous roots without any secondary branching. Auxin supplementation significantly improved rooting in excised microshoots. Rooting was noticed within 1 one of culture in all the treatments of auxin. The half-strength MS basal medium augmented with NAA was found the best for healthy root induction as compared to IAA and IBA. A maximum of 26.40 ± 1.12 roots per microshoot with 8.02 ± 0.17 cm root length was achieved without an intervening callogenesis on half-strength MS medium augmented with 2.5 µM NAA (Fig. 59 A & B). Further increasing the NAA concentration to 5.0 µM callogenesis was noticed that adversely affected the root induction and a mean of 20.20 ± 0.66 roots per microshoot induced after 4 weeks of transfer. IBA showed similar pattern of in vitro root induction as shown by NAA supplemented medium, although mean number of roots per microshoot and their length were lesser than NAA containing medium. However, IAA was found to be the least effective in terms of in vitro root induction as induced huge callus associated with the stunted roots (Table 46).
4.2.8 Acclimatization of plantlets
Fully developed plantlets (with shoot and root) were removed from the culture medium, washed carefully in order to remove remnant of culture medium, transferred to thermocol cups containing sterile soilrite, vermin-compost and garden soil mixed with farmyard. These were covered with transparent polybags and acclimatized as described in materials s and methods (Fig. 59 C). Plantlets were irrigated after every 4 day with normal tap water. Among three planting substrates tested, maximum survival percentage of the regenerated plantlets was observed for soilrite (92.60%) followed by vermi- compost (83.30%) and garden soil (34.00%) (Fig. 60). After acclimatization plantlets were transferred to field where they grew well and flowered normally (Fig. 61).
4.2.9 Synseed production
The optimal conditions are very important for the preparation of ideal synseeds and their subsequent regeneration. The results on the respective experiments performed for the synseed formation followed by their successful germination are as follows.
4.2.9.1 Effect of Na2-alginate concentration on synseed formation
The morphology of syneeds in respect to shape, texture and transparency varied
with different concentrations of Na2-alginate (1, 2, 3, 4 and 5%) with CaCl2·2H2O (100
mM). The 4% Na2-alginate produced clear and uniform beads within ion exchange
duration of 20 min (Fig. 62 A & B). Further increasing the concentration of Na2-alginate solution resulted in the production of hard beads and cause considerable delay in germination. However, Na2-alginate concentration beyond the optimum level was also not suitable for encapsulation because resulting beads were fragile and difficult to handle (Table 47).
4.2.9.2 Effect of CaCl2·2H2O concentration on synseed formation
Various concentrations of CaCl2·2H2O (25, 50, 75, 100 and 200 mM) were tested
with 4% Na2-alginate solution to obtain beads with desired texture. The 100 mM was found to be optimum for the production of uniform synseeds (Table 48). Lower
concentrations of CaCl2·2H2O resulted in either non-formation or very soft bead formation which may burst during handling. Higher concentration of CaCl2·2H2O than the optimal level (100 mM) resulted in the production of hard beads.
4.2.9.3 In vitro plantlet regeneration from synseeds on culture medium
Synseeds prepared with 4% Na2-alginate and 100 mM CaCl2·2H2O exhibited re- growth within 2-3 weeks of incubation on MS medium augmented with 1.0 µM BA and 0.5 µM NAA, IAA or IBA and complete plantlets with well developed root and shoot systems were obtained after 6 weeks of culture (Fig. 62 C & D). The maximum conversion response (83.0 ± 2.09%) was observed on BA (1.0 µM) and IAA (0.5 µM) combination followed by 79.00 ± 1.97 and 64.00 ± 1.37% germination on BA (1.0 µM) with NAA (0.5 µM) and BA (1.0 µM) with IBA (0.5 µM) respectively (Table 49).
4.2.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage
Storage time (0, 1, 2, 4, 6 and 8 weeks) was also found to influence the regeneration frequency. For encapsulated nodal segments (with MS gel matrix), a gradual decrease in regeneration frequency was observed up to 4 weeks of storage (75.40 ± 1.43% germination), thereafter the percent conversion frequency decreased considerably (Fig. 63 A-C). While, non-encapsulated nodal segments showed 42.60 ± 1.66% germination frequency just after 2 weeks of culture. After that germination frequency was greatly reduced. On the other hand, none of the encapsulated nodal segments having DDW gel matrix survived after 2 weeks of storage (Table 50).
4.2.9.5 Acclimatization of plantlets
The plantlets with well developed shoot and root systems were removed from culture medium, washed carefully to remove remnant of culture medium and gel-matrix, transferred to thermocol cups containing sterile soilrite. These were covered with transparent polybags and acclimatized successfully (Fig. 64 A & B). Acclimatized plantlets showed more than 90% survival rate when transferred to field and grew well in the same manner as the non-encapsulated plantlets.
4.2.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
Various planting substrates were also assessed for the direct conversion of synseeds into complete plantlets under ex vitro conditions. Among different planting substrates, soilrite moistened with quarter-strength MS nutrient medium was found to be the most suitable for direct conversion of synseeds (63.40 ± 1.02% germination), followed by soilrite moistened with tap water (34.60 ± 2.03) whereas no regeneration was noticed in soil having tap water supply even after 6 weeks of sowing (Table 51).
4.2.10 Physiological study
4.2.10.1 Chlorophyll a, b and total chlorophyll content during acclimatization
Chlorophyll a and b content along with total chlorophyll content was estimated after 0, 7, 14, 21 and 28 days of acclimatization. Initial transfer of plants from in vitro to ex vitro conditions negatively affected the chlorophyll content by showing decrease in Chl a (0.76 ± 0.02 to 0.74 ± 0.04 mg g-1), Chl b (0.37 ± 0.00 to 0.32 ±0.01 mg g-1) and Total chl (1.06 ±0.02 to 0.98 ±0.03 mg g-1) content during first week of acclimatization. Thereafter a linear increase was noticed during subsequent weeks in all the parameters. A sharp rise in Chl a (0.80 ± 0.02 mg g-1), Chl b (0.44 ±0.01 mg g-1) and Total chl (1.15 ± 0.01 mg g-1) was recorded after 2 weeks of acclimatization with a comparatively constant rise thereafter. During late phase of acclimatization (4th week) the Chl a (1.25 ±0.03 mg g-1), Chl b (0.65 ±0.01 mg g-1) and Total chl (1.63 ± 0.03 mg g-1) contents almost stabilized with a very gradual increase (Fig. 65).
4.2.10.2 Carotenoids content during acclimatization
Carotenoids content was also evaluated along with other photosynthetic pigments during first 4 weeks of acclimatization. Like the chlorophylls, carotenoids slightly after first week of transfer to ex vitro conditions. However, a significant increase in carotenoids content was observed during second (0.26 ± 0.01 mg g-1) and third week (0.33 ±0.01 mg g-1) of acclimatization. During the fourth week of acclimatization carotenoids content was recorded 0.36 ± 0.02 mg g-1 (Fig. 66).
4.2.10.3 Net photosynthetic rate (PN) during acclimatization
Net photosynthetic rate (PN) reflects the photosynthetic competence of plantlets
during variable conditions. Net photosynthetic rate is the estimation of CO2 absorbed by the unit area of plant per unit per second. After an initial slight decrease (2.00 ±0.03 to -2 -1 1.87 ± 0.03 µmol CO2 m s ) in PN on transfer of plantlets from in vitro to ex vitro conditions, a steady increase was noticed during subsequent weeks of acclimatization. A
steep rise in net photosynthetic rate was found during 14 days of transfer where PN value -2 -1 increased up to 3.76 ±0.16 µmol CO2 m s . Further enhancement in net photosynthetic -2 -1 rate was found after 21 days of transfer (5.88 ± 0.08 µmol CO2 m s ). Thereafter, a -2 -1 slight increase in net photosynthetic rate (6.88 ± 0.07 µmol CO2 m s ) was noticed during the late phase of acclimatization (Fig. 67).
4.2.11 Histological study
Regenerating shoot tip explants placed on BA (1.0 µM) + IAA (0.5 µM) were fixed after 7 days of incubation. The fixed tissues analyzed histologically, showed clearly the induction of shoot bud in the form of prominent bulging at the axillary region of the explant (Fig. 68 A & B). Adventitious shoot formation from leaf explants was also studied. One week-old section of regenerating leaf tissue incubated on BA (2.5 µM) and IAA (0.5 µM) containing MS medium showed distinctly well developed shoot buds originating directly from the leaf tissue without any intermittent callus phase (Fig. 68 C). In basal petiolar end of leaf blade, the abaxial (dorsal) epidermal cells that touched the
medium, increased in size and simultaneously with hypodermal cells, divided in several planes. Some of these epidermal cells exhibited dense cytoplasm and conspicuous nucleus and nucleolus indicating exogenous origin of meristemoids.
Table 28. Effect of pre-soaking of seeds in GA on in vivo seed germination in S. mauritiana after 3 weeks of sowing GA (µM) Mean no. of days to Germination frequency (%) germination Control 0.00 ± 0.00d 0.00 ± 0.00d GA (0.25) 19.80 ± 0.80a 6.20 ± 0.58c GA (0.5) 16.60 ± 0.92b 10.00 ± 0.63b GA (1.0) 11.60 ± 0.92c 14.00 ± 1.37a Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 29. Effect of different GA concentrations on in vitro seed germination of S. mauritiana after 3 weeks of culture Treatment (µM) Mean no. of days to Germination frequency germination (%) MS 19.80 ± 0.48a 8.80 ± 0.58g MS + GA (0.5) 16.60 ± 0.50bc 16.00 ± 1.30f MS + GA (1.0) 14.20 ± 0.58c 31.40 ± 0.97d MS + GA (5.0) 17.20 ± 0.58ab 22.60 ± 1.02e ½ MS 15.40 ± 0.40bc 20.80 ± 1.15ef ½ MS + GA (0.5) 10.40 ± 0.74d 60.00 ± 1.70c ½ MS + GA (1.0) 5.80 ± 0.58e 86.00 ± 1.87a ½ MS + GA (5.0) 9.20 ± 0.80d 76.60 ± 1.88b Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 30. Effect of different cytokinins on shoot multiplication through nodal segments of S. mauritiana after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 00.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00e - BA (0.5) 90.00 ± 0.00bcd 1.60 ± 0.24ab 1.28 ± 0.07cd + BA (1.0) 99.60 ± 0.40a 2.00 ± 0.00a 1.50 ± 0.07abc + + BA (2.5) 93.20 ± 0.96b 2.00 ± 0.00a 1.76 ± 0.11a + + BA (5.0) 99.00 ± 1.00a 2.00 ± 0.00a 1.38 ± 0.12bcd + + + Kn (0.5) 86.20 ± 1.24d 1.40 ± 0.24b 1.14 ± 0.04d + Kn (1.0) 91.00 ± 1.18bc 2.00 ± 0.00a 1.28 ± 0.05cd + + Kn (2.5) 87.00 ± 0.94cd 2.00 ± 0.00a 1.66 ± 0.06ab + + + Kn (5.0) 75.80 ± 1.68e 1.80 ± 0.20ab 1.32 ± 0.05cd + + + 2-iP (0.5) 00.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00e + + + 2-iP (1.0) 00.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00e + + + 2-iP (2.5) 00.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00e + + + 2-iP (5.0) 00.00 ± 0.00f 0.00 ± 0.00c 0.00 ± 0.00e + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 31. Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through nodal segments of S. mauritiana after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 95.20 ± 0.96b 2.00 ± 0.00a 2.60 ± 0.08b + + NAA (0.5) 89.00 ± 1.18c 2.00 ± 0.00a 2.26 ± 0.10cd + + + NAA (1.0) 81.80 ± 0.91d 1.80 ± 0.20a 1.92 ± 0.05e + + + IAA (0.1) 99.80 ± 0.20a 2.20 ± 0.20a 3.20 ± 0.09a + + + IAA (0.5) 92.60 ± 1.24b 2.00 ± 0.00a 2.50 ± 0.08bc + + IAA (1.0) 80.40 ± 0.40d 2.00 ± 0.00a 2.10 ± 0.04de + + + IBA (0.1) 00.00 ± 0.00e 0.00 ± 0.00b 0.00 ± 0.00f + + + IBA (0.5) 00.00 ± 0.00e 0.00 ± 0.00b 0.00 ± 0.00f + + + IBA (1.0) 00.00 ± 0.00e 0.00 ± 0.00b 0.00 ± 0.00f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 32. Effect of different cytokinins on shoot multiplication through shoot tip explants of S. mauritiana after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 52.00 ± 0.00a 1.00 ± 0.00f 4.90 ± 0.11a - BA (0.5) 44.20 ± 1.42b 4.00 ± 0.31bcd 2.08 ± 0.11e - BA (1.0) 59.80 ± 1.56a 6.00 ± 0.44a 2.36 ± 0.14de - BA (2.5) 52.60 ± 1.02a 5.00 ± 0.31ab 2.90 ± 0.11cd - BA (5.0) 44.20 ± 2.28b 3.60 ± 0.50bcde 3.44 ± 0.12bc + Kn (0.5) 36.40 ± 1.80bc 3.20 ± 0.37bcde 2.32 ± 0.10e - Kn (1.0) 57.00 ± 2.09a 4.40 ± 0.50abc 3.32 ± 0.15bc - Kn (2.5) 43.20 ± 1.46bc 3.80 ± 0.58bcd 3.72 ± 0.09b - Kn (5.0) 37.00 ± 1.54bc 2.80 ± 0.37cdef 3.30 ± 0.12bc + + 2-iP (0.5) 24.60 ± 2.03d 1.80 ± 0.20ef 1.98 ± 0.06e - 2-iP (1.0) 36.00 ± 1.87c 2.20 ± 0.37def 2.52 ± 0.09de + 2-iP (2.5) 25.60 ± 1.69d 2.60 ± 0.24cdef 2.42 ± 0.15de + + 2-iP (5.0) 12.40 ± 1.12e 1.80 ± 0.37ef 1.96 ± 0.05e + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 33. Effect of different combinations of auxin (NAA, IBA and IAA) with 1.0 µM BA on shoot multiplication through shoot tip explants of S. mauritiana after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 86.0 ± 5.09ab 8.80 ± 0.37de 2.68 ± 0.17e - NAA (0.5) 89.0 ± 3.78ab 13.20 ± 0.48b 3.72 ± 0.13bc + NAA (1.0) 67.0 ± 3.74c 7.80 ± 0.20ef 3.22 ± 0.08cde + IAA (0.1) 90.0 ± 3.16ab 14.20 ± 0.66b 4.82 ± 0.08de - IAA (0.5) 98.0 ± 2.00a 18.80 ± 0.48a 4.26 ± 0.10cd - IAA (1.0) 72.0 ± 3.74bc 12.40 ± 0.50bc 3.50 ± 0.11de + IBA (0.1) 58.0 ± 5.83cd 6.80 ± 0.37ef 3.02± 0.06a + + IBA (0.5) 54.0 ± 4.00cd 10.80 ± 0.37cd 3.36 ± 0.15ab + + IBA (1.0) 46.0 ± 2.44d 6.60 ± 0.40f 2.96 ± 0.16cd + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 34. Effect of different concentrations of TDZ on multiple shoot formation through shoot tip explants of S. mauritiana after 4 weeks of culture TDZ % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis 0.1 72.00 ± 3.74bc 3.40 ± 0.81c 1.76 ± 0.11a - 0.25 93.60 ± 3.86a 26.20 ± 1.35a 1.46 ± 0.05ab - 0.5 82.00 ± 2.54ab 12.20 ± 1.01b 1.42 ± 0.13abc - 1.0 60.00 ± 4.28c 8.80 ± 0.58b 1.38 ± 0.10bc + 2.5 39.00 ± 3.31d 3.80 ± 0.58c 1.10 ± 0.08b + 5.0 00.00 ± 0.00e 1.00 ± 0.00c 1.68 ± 0.09a + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + + indicate no, slight, moderate callusing respectively.
Table 35. Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated shoot tip explants of S. mauritiana after 4 weeks of transfer BA IAA Mean number of Mean shoot (µM) (µM) shoots/explant length (cm) - - 28.00 ± 0.83cd 1.54 ± 0.01f 0.5 - 29.40 ± 0.40cd 1.71 ± 0.01de 1.0 - 33.40 ± 1.66bc 1.83 ± 0.01cd 2.5 - 26.60 ± 0.96d 1.66 ± 0.01ef 0.5 0.5 29.00 ± 1.73cd 1.92 ± 0.01bc 1.0 0.5 41.00 ± 1.18a 2.11 ± 0.06a 2.5 0.5 36.00 ± 1.22ab 2.14 ± 0.06a 5.0 0.5 27.00 ± 0.94d 2.01 ± 0.02ab Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 36. Effect of PGR combination and PGR free nutrient media on growth and hyperhydricity of TDZ treated shoot tip explants of S. mauritiana after 4 weeks of transfer Treatment (µM) Mean no. of Mean shoot Frequency of shoots/explant length (cm) hyperhydricity BA (1.0) + IAA (0.5) 72.00 ± 1.92a 4.00 ± 0.10a + + MS 64.00 ± 1.37b 3.72 ± 0.08ab - ½ MS 50.00 ± 1.70c 3.42 ± 0.07b - Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. – indicates no hyperhydricity and + + indicates moderate hyperhydricity.
Table 37. Effect of different cytokinins on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 0.00 ± 0.00j 0.00 ± 0.00f 0.00 ± 0.00e + BA (0.5) 48.00 ± 2.54ef 1.40 ± 0.24ef 2.60 ± 0.15cd - BA (1.0) 67.6 0 ± 1.12bc 2.60 ± 0.24cde 3.06 ± 0.08bc - BA (2.5) 87.00 ± 2.00a 5.60 ± 0.40a 3.44 ± 0.16ab - BA (5.0) 75.60 ± 1.69b 4.40 ± 0.50bc 2.38 ± 0.16cd + Kn (0.5) 37.60 ± 2.50g 1.20 ± 0.20ef 3.06 ± 0.16bc - Kn (1.0) 57.80 ± 1.01cde 2.00 ± 0.31e 3.44± 0.15ab - Kn (2.5) 75.60 ± 1.69b 4.20 ± 0.37ab 3.76 ± 0.11a - Kn (5.0) 60.00 ± 3.16cd 1.80 ± 0.37de 2.62 ± 0.26cd + + 2-iP (0.5) 15.00 ± 2.23e 1.20 ± 0.20ef 2.14 ± 0.09d - 2-iP (1.0) 40.00 ± 3.18fg 1.40 ± 0.24ef 2.44 ± 0.11cd + 2-iP (2.5) 54.00 ± 1.87de 3.20 ± 0.37bcd 2.74 ± 0.11cd + + 2-iP (5.0) 27.00 ± 2.00h 1.60 ± 0.24de 2.24 ± 0.09d + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 38. Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 83.00 ± 2.00c 9.00 ± 0.44c 3.30 ± 0.15c + NAA (0.5) 94.00 ± 1.87ab 12.20 ± 0.48b 3.52 ± 0.09bc + NAA (1.0) 68.00 ± 3.74d 8.40 ± 0.24c 3.10 ± 0.11c + + IAA (0.1) 90.60 ± 1.16abc 12.80 ± 0.37b 4.02 ± 0.06ab + IAA (0.5) 98.00 ± 2.00a 15.00 ± 0.31a 4.22 ± 0.11a + IAA (1.0) 87.00 ± 3.00bc 11.40 ± 0.40b 3.62 ± 0.17abc + + IBA (0.1) 17.00 ± 2.00e 5.80 ± 0.37d 2.30 ± 0.15d + + IBA (0.5) 8.00 ± 1.22ef 4.60 ± 0.40d 1.88 ± 0.17d + + + IBA (1.0) 0.00 ± 0.00f 0.00 ± 0.00e 0.00 ± 0.00e + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 39. Effect of different concentrations of TDZ on direct shoot organogenesis through leaf explants of S. mauritiana after 4 weeks of culture TDZ (µM) % Response Mean no. of Mean shoot Frequency of shoots/explant length (cm) callogenesis 0.10 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c + 0.25 17.80 ± 1.01c 2.80 ± 0.37c 1.24 ± 0.08a + + 0.50 81.00 ± 1.18a 8.40 ± 0.50a 1.10 ± 0.04a + + 1.00 36.80 ± 0.96b 4.20 ± 0.37b 0.80 ± 0.08b + + 2.50 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c + + + 5.00 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. +, + +, + + +, indicate slight, moderate, intense callusing respectively.
Table 40. Effect of different cytokinin-auxin combinations on shoot proliferation through TDZ treated leaf explants of S. mauritiana after 4 weeks of transfer Treatment (µM) Mean no. of Mean shoot length Frequency of shoots/explant (cm) callogenesis BA (0.5) 8.60 ± 0.40c 1.56 ± 0.06d - BA (1.0) 10.20 ± 0.48abc 1.98 ± 0.08abc - BA(2.5) 9.00 ± 0.44bc 1.62 ± 0.03d + + BA (1.0) + NAA (0.1) 9.80 ± 0.58bc 1.82 ± 0.08bcd + BA (1.0) + NAA (0.5) 10.00 ± 0.70abc 1.74 ± 0.08cd + BA (1.0) + NAA (1.0) 9.00 ± 0.31bc 1.80 ± 0.07bcd + + BA (1.0) + IAA (0.1) 12.40 ± 0.74a 2.20 ± 0.07a + BA (1.0) + IAA (0.5) 11.20 ± 0.37ab 2.08 ± 0.05ab + + BA (1.0) + IAA (1.0) 10.20 ± 0.48abc 1.80 ± 0.03bcd + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + + indicate no, slight, moderate callusing respectively.
Table 41. Effect of PGR combination and PGR-free nutrient medium on growth and hyperhydricity of TDZ derived tissue through leaf explants of S. mauritiana after 4 weeks of transfer Treatment (µM) Mean no. of Mean shoot Frequency of shoots/explant length (cm) hyperhydricity BA (1.0) + IAA (0.5) 16.20 ± 0.58a 2.78 ± 0.06a + + MS 15.00 ± 0.31ab 2.62 ± 0.04ab - ½ MS 13.40 ± 0.60b 2.50 ± 0.03b - Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. – indicates no hyperhydricity and + indicates hyperhydricity.
Table 42. Effect of different concentrations of 2,4-D and BA on callus induction through leaf explants of S. mauritiana after 4 weeks of incubation 2, 4-D (µM) BA (µM) % Callogenesis Texture of callus 0.5 - 63.00 ± 1.54d Yellow, compact lucid 1.0 - 96.60 ± 1.02ab Yellow, less compact lucid 2.5 - 87.60 ± 1.93c Whitish-yellow, compact and lees lucid 1.0 0.5 98.20 ± 0.91a Greenish-white, compact and less lucid 1.0 1.0 99.60 ± 0.40a Greenish-white, compact and less lucid 1.0 2.5 92.40 ± 1.12bc White, compact and dry Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 43. Effect of different cytokinins on direct shoot organogenesis through cotyledons of S. mauritiana after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c - BA (0.5) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + BA (1.0) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + BA (2.5) 83.00 ± 2.54a 4.80 ± 0.37a 3.16 ± 0.09b + BA (5.0) 57.00 ± 3.00b 1.80 ± 0.37b 2.90 ± 0.11c + Kn (0.5) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + Kn (1.0) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + Kn (2.5) 54.00 ± 4.00b 2.60 ± 0.50b 3.22 ± 0.18a + Kn (5.0) 42.00 ± 3.74c 1.60 ± 0.24b 3.14 ± 0.12a + + 2-iP (0.5) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + + 2-iP (1.0) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + + + 2-iP (2.5) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + ++ 2-iP (5.0) 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 44. Effect of different combinations of auxin (NAA, IBA and IAA) with 2.5 µM BA on direct shoot organogenesis through cotyledons of S. mauritiana after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 67.00 ± 2.00c 10.60 ± 0.50cd 3.24 ± 0.09b + NAA (0.5) 79.60 ± 2.37a 14.00 ± 0.54b 3.70 ± 0.08b + + NAA (1.0) 52.00 ± 3.39d 8.60 ± 0.40d 2.60 ± 0.13c + + IAA (0.1) 78.60 ± 2.99ab 15.00 ± 0.44b 3.70 ± 0.09b + IAA (0.5) 82.00 ± 2.09a 17.80 ± 1.01a 4.44 ± 0.22a + + IAA (1.0) 69.40 ± 1.16bc 12.40 ± 0.74bc 3.32 ± 0.09b + + IBA (0.1) 13.00 ± 2.00e 5.80 ± 0.58e 2.56 ± 0.16c + + IBA (0.5) 0.00 ± 0.00f 0.00 ± 0.00f 0.00 ± 0.00d + + + IBA (1.0) 0.00 ± 0.00f 0.00 ± 0.00f 0.00 ± 0.00d + + +
Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 45. Effect of different concentrations of TDZ on callus formation through cotyledons of S. mauritiana after 4 weeks of culture TDZ (µM) % Response Frequency of Callus texture callogenesis 0.1 22.00 ± 0.94d + Yellowish-white, friable 0.25 26.60 ± 1.88d + Yellowish-white, friable 0.5 66.60 ± 1.88c + + Yellowish- white, friable 1.0 77.00 ± 1.54b + + + Yellowish- white, compact 2.5 81.40 ± 0.97b + + + Dark-green, compact 5.0 91.40 ± 1.00a + + + Dark-green, compact Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments.
Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. +, + +, + + +, indicate slight, moderate, intense callusing respectively.
Table 46. Effect of different auxins augmented to half-strength basal MS medium on in vitro rooting in S. mauritiana after 4 weeks of transfer Auxin Mean number Mean root Frequency (µM) of roots/shoot length (cm) of callogenesis Control 6.20 ± 0.73f 5.18 ± 0.15e - NAA (1.0) 15.60 ± 0.67c 7.00 ± 0.17b -
NAA (2.5) 26.40 ± 1.12a 8.02 ± 0.17a - NAA (5.0) 20.20 ± 0.66b 6.38 ± 0.13c + IBA (1.0) 9.40 ± 0.60ef 4.34 ± 0.15f - IBA (2.5) 13.40 ± 0.87cd 4.94 ± 0.06ef + IBA (5.0) 8.40 ± 0.50ef 3.56 ± 0.06g + + IAA (1.0) 13.20 ± 0.58cd 5.42 ± 0.14de + IAA (2.5) 19.20 ± 0.86b 5.94 ± 0.06cd + + IAA (5.0) 10.20 ± 0.66de 5.18 ± 0.08e + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 47. Effect of sodium alginate concentration on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium Sodium alginate (% w/v) Conversion response (%) into plantlets
1.0 Fragile beads 2.0 Fragile beads 3.0 73.00 ± 2.00a (but soft to handle) 4.0 74.40 ± 1.93a 5.0 46.60 ± 1.88b
Different concentrations of sodium alginate and 100 mM CaCl2·2H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 48. Effect of calcium chloride concentration on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium Calcium chloride (mM) Conversion response (%) into plantlets 25 Fragile beads 50 Fragile beads 75 63.40 ± 1.88b(but soft to handle) 100 74.40 ± 1.93a 200 33.60 ± 1.56c
Different concentrations of sodium alginate and 100 mM CaCl2·2H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 49. Effect of different treatments on conversion of encapsulated nodal segments of S. mauritiana after 6 weeks of culture on MS medium Treatment (µM) Conversion response (%) into plantlets MS 74.40 ± 1.93b MS + BA (1.0) + NAA (0.5) 79.00 ± 1.97ab MS + BA (1.0) + IAA (0.5) 83.00 ± 2.09a MS + BA (1.0) + IBA (0.5) 64.00 ± 1.37c
Different concentrations of CaCl2·2H2O and 4% (w/v) sodium alginate were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 50. Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of S. mauritiana into plantlets Storage Conversion of Conversion of Conversion of duration encapsulated nodal encapsulated nodal non-encapsulated (weeks) segments into plantlets segments into plantlets nodal segments (%) (encapsulation (%) (encapsulation into plantlets (%) matrix prepared in MS matrix prepared in basal medium) distilled water) 0 83.00 ± 2.09a 17.20 ± 0.96a 97.20 ± 0.96a 1 82.00 ± 2.07ab 10.60 ± 0.60b 70.20 ± 3.55b 2 77.40 ± 1.77ab 04.00 ± 0.63c 42.60 ± 1.66c 4 75.40 ± 1.43b 00.00 ± 0.00d 16.00 ± 1.37d 6 57.40 ± 1.12c 00.00 ± 0.00d 08.80± 0.58de 8 41.00 ± 1.18d 00.00 ± 0.00d 04.20 ± 0.58e Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 51. Effect of different planting substrates on ex vitro germination of encapsulated nodal segments of S. mauritiana after 6 weeks of sowing Planting substrate Conversion response (%) into plantlets Soilrite + ¼ MS salts 63.40 ± 1.02a Soilrite + tap water 34.60 ± 2.03b Soilrite + soil + ¼ MS salts 28.60 ± 0.97c Soilrite + soil + tap water 13.00 ± 1.54d Soil + ¼ MS Salts 6.80 ± 0.96e Soil + tap water 0.00 ± 0.00f Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Figure 39
A B
Explanation of Figure 39 Seed germination in S. mauritiana
A. Seeds obtained through dried flower heads B. Aseptic seedlings grown on half-strength MS medium supplemented with 1.0 µM GA, 2 week-old culture
Figure 40
A
B C
Explanation of Figure 40 Shoot regeneration through nodal segments
A. Axillary bud sprouting on MS medium supplemented with 0.5 µM BA, 2 week-old culture B. Shoot elongation along with basal callusing through the nodal segments on MS medium supplemented with 1.0 µM BA, 3 week-old culture C. Shoot regeneration through nodal segment on MS medium supplemented with 1.0 µM BA and 0.1 µM IAA, 4 week-old culture
Figure 41
A B C
D E F
Explanation of Figure 41 Shoot regeneration through shoot tip explants
A. Elongated shoot tip showing basal callusing on PGR-free MS medium (control), 4 week-old culture B. Shoot bud sprouting through shoot tip explant on MS medium containing 1.0 µM BA, 1 week-old culture C. Multiple shoot regeneration on MS medium containing 1.0 µM BA, 2 week-old culture D. Elongated shoots on MS medium containing 1.0 µM BA, 4 week-old culture E. Shoot regeneration on 5.0 µM BA supplemented MS medium, 3 week-old culture F. Shoot regeneration on 1.0 µM Kn supplemented MS medium, 3 week-old culture
Figure 42
A B C
D E F
Explanation of Figure 42 Shoot regeneration through shoot tip explants
A. Regeneration of multiple shoots through compressed nodal region of shoot tip explant on MS medium containing 1.0 µM BA and 0.5 µM IAA, 1 week-old culture B. High frequency regeneration on above mentioned medium, 2 week-old culture. C. Shoot elongation of microshoots of above culture, 3 week-old culture D. Shoot proliferation in flask containing MS medium augmented with 1.0 µM BA and 0.5 µM IAA, 4 week-old culture E. Shoot bud sprouting on MS medium containing 1.0 µM BA and 0.5 µM NAA, 2 week-old culture F. Shoot bud sprouting on MS medium containing 1.0 µM BA and 0.5 µM NAA, 3 week-old culture
Figure 43
A B C
D E
Explanation of Figure 43 Shoot regeneration through shoot tip explants
A. Adventitious shoot bud induction through the basal cut end of shoot tip on 0.25 µM TDZ containing MS medium, 2 week-old culture B. Clump of multiple shoots on 0.25 µM TDZ containing MS medium, 4 week-old culture C. Shoot tip showing huge basal callusing on 5.0 µM TDZ containing MS medium, 4 week-old culture D. Multiple shoot regeneration through TDZ exposed shoot tip when transferred to 1.0 µM BA and 0.5 µM IAA, 3 week- old culture E. Fasciated and distorted shoots regenerated through TDZ exposed shoot tip when transferred to 2.5 µM BA and 0.5 µM IAA, 4 week-old culture
Figure 44
A B
Explanation of Figure 44 Shoot regeneration through shoot tip explants
A. Vitrified and glossy shoots induced through TDZ exposed shoot tip on 1.0 µM BA and 0.5 µM IAA when sub- cultured through the same treatment after 4 weeks of sub-culturing B. Healthy shoots with proper leaf expansion on full-strength MS medium after 4 week of sub-culturing
25 6 Mean shoot no./explant a Mean shoot length (cm) a 5 20 b
4 b 15
3 b 10 c 2 Mean shoot no./explant shoot Mean c length shoot Mean (cm)
c 5 1
d 0 5.0 5.4 5.8 6.2 6.6 pH of the culture medium
Figure 45. Effect of different pH of the culture medium on shoot regeneration through shoot tip explants of S. mauritiana supplemented with BA (1.0 µM) in combination with IAA (0.5 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
30 5 Mean shoot no./explant a Mean shoot length (cm) 25 4
20 a b 3 b 15 c 2 10 Mean shoot no./explant shoot Mean Mean shoot lengthshoot Mean (cm)
c 1 5
0 0 Sucrose Fructose Glucose
Figure 46. Effect of different carbon sources on shoot regeneration through shoot tip explants of S. mauritiana after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
25 5 Mean shoot no./explant a Mean shoot length (cm)
20 a 4
b b 15 3
b
10 2 Mean shoot no./explant shoot Mean c lengthshoot Mean (cm)
5 1
0 0 MS SH B5 Culture medium
Figure 47. Effect of different culture media on shoot regeneration through shoot tip explants of S. mauritiana supplemented with BA (1.0 µM) in combination with IAA (0.5 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 48
A D
B C E
Explanation of Figure 48 Shoot regeneration through leaf explants
A. Direct bud induction through basal petiolar cut end of the leaf explant on MS medium amended 2.5 µM BA, 2 week-old culture B. Shoot regeneration through leaf explant on MS medium amended 2.5 µM BA, 3 week-old culture C. Elongation of regenerated shoots on above treatment, 4 week-old culture D. Shoot regeneration through leaf explant on 2.5 µM Kn supplemented MS medium, 2 week-old culture E. Poor shoot regeneration on 2.5 µM 2-iP supplemented MS medium, 2 week-old culture
Figure 49
A B C
D E F
Explanation of Figure 49 Shoot regeneration through leaf explants
A. Swelling in the basal petiolar cut end of the leaf explant on MS medium augmented with 2.5 µM BA and 0.5 µM IAA, 5 day-old culture B. Adventitious shoot bud differentiation through basal bulged petiolar end of leaf on MS medium augmented with 2.5 µM BA and 0.5 µM IAA, 1 week-old culture C. High frequency shoot regeneration through leaf explant on MS medium augmented with 2.5 µM BA and 0.5 µM IAA, 2 week-old culture D. Shoot elongation of the above culture on the same PGR combination, 3 week-old culture E. Shoot proliferation of the above culture in flask containing similar PGR combination, 4 week-old culture F. Shoot regeneration through leaf explant on 2.5 µM BA and 0.1 µM IBA, 4 week-old culture
Figure 50
A B
C D
Explanation of Figure 50 Shoot regeneration through leaf explants
A. Shoot regeneration through expanded leaf (after scraping the surface callus) on 0.5 µM TDZ, 4 week-old culture B. Joint shoots regenerated through leaf surface on 1.0 µM TDZ, 2 week-old culture C. Elongation of jointed shoots on 1.0 µM TDZ, 4 week-old culture D. Shoot multiplication through TDZ (0.5 µM) treated leaf explant on 1.0 µM BA in conjunction with 0.5 µM IAA, after 3 weeks of transfer
Figure 51
A B
Explanation of Figure 51 Shoot regeneration through leaf explants
A. Healthy shoot proliferation on PGR-free full strength MS medium, 4 week-old culture B. Culture showing hyperhydric shoots on BA (1.0) + IAA (0.5), 4 week-old culture
Figure 52
A B
C D
Explanation of Figure 52 Callus induction through leaf explants
A. Callus initiation through leaf explant on 1.0 µM 2,4-D containing MS medium, 1 week-old culture B. Callus initiation through leaf explant on 2.5 µM 2,4-D containing MS medium, 4 week-old culture C. Callus initiation through leaf explant on 1.0 µM 2,4-D and 1.0 µM BA containing MS medium, 3 week-old culture D. Leaf-derived callus on 1.0 µM BA and 0.5 µM IAA containing MS medium, after 2 weeks of transfer
Figure 53
A B
C D
Explanation of Figure 53 Shoot regeneration through cotyledons
A. Cotyledons cultured on MS medium supplemented with 2.5 µM BA, 1 week-old culture B. Multiple shoot regeneration through cotyledon on MS medium supplemented with 2.5 µM BA, 2 week-old culture C. Shoot elongation on cotyledon derived shoots on MS medium supplemented with 2.5 µM BA, 4 week-old culture D. Shoot regeneration through cotyledon cultured on MS medium supplemented with 2.5 µM Kn, 3 week-old culture
Figure 54
A B
C D
Explanation of Figure 54 Shoot regeneration through cotyledons
A. Cotyledon shoot bud initiation overcrowded by huge callusing on 2.5 µM BA and 0.5 µM IAA, 2 week-old culture B. Shoot regeneration 2.5 µM BA and 0.5 µM IAA through cotyledon after scraping the callus, 16 day-old culture C. Shoot elongation of above culture on similar PGR combination, 4 week-old culture D. Shoot regeneration with simultaneous callusing 2.5 µM BA and 0.1 µM IBA through cotyledon after scraping the callus, 4 week-old culture
8 a Mean shoot no./explant ab 100 Mean shoot length (cm) bc c cd 80 6 de a c ab ef bc cd d 60 f
e 4
40 f Mean shoot no./explant shoot Mean Mean shoot length shoot Mean (cm)
20 2
0 1 2 3 4 5 6 7 8 Sub-culture passage
Figure 55. Effect of sub-culture passage on shoot proliferation efficiency of shoot tip explants. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
8 a Mean shoot no./explant 100 Mean shoot length (cm) b c d 80 e 6 a a ab f bc f c g 60 d
e 4 40 f Mean shoot no./explant shoot Mean Mean shoot length shoot Mean (cm)
20 2
0 1 2 3 4 5 6 7 8
Sub-culture passage
Figure 56. Effect of sub-culture passage on shoot proliferation efficiency of leaf explants. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
8 a Mean shoot no./explant ab bc 50 Mean shoot length (cm) c cd de ab ef bc 6 40 a cd
f a d
30 e 4
f Mean shoot no./explant shoot Mean 20 length shoot Mean (cm)
2 10
1 2 3 4 5 6 7 8
Sub-culture passage
Figure 57. Effect of sub-culture passage on shoot proliferation efficiency of cotyledons. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s
Figure 58
A B C
D E
Explanation of Figure 58 Sub-culturing of shoot tip, leaf and cotyledon derived tissues
A. Shoot proliferation through shoot tip mother tissue on 1.0 µM BA and 0.5 µM IAA, after 5th sub-culture passage B. Shoot proliferation through mother tissue of leaf explant on 2.5 µM BA and 0.5 µM IAA, after 5th sub-culture passage C. Shoot proliferation through mother tissue of cotyledon on 2.5 µM BA and 0.5 µM IAA, after 3rd sub-culture passage D. Elongated shoots regenerated through cotyledon, after 8th sub-culture passage E. A group of maintained cultures
Figure 59
A B C
Explanation of Figure 59 In vitro root induction of microshoots
A. Microshoot showing in vitro root induction on half-strength MS medium supplemented with 2.5 µM NAA, 2 week-old culture B. Closed view of above culture, 3 week-old C. An acclimatized plantlet of S. mauritiana in soilrite, 4 week-old
Soilrite 100 Vermi-compost Garden soil + farmyard (2:1)
80
60 % Survival%
40
20
Types of planting substrate
Figure 60. Effect of different planting substrates on survival percentage of regenerated plantlets after 2 months of field transfer. The columns represent the results of 300 plantlets for each substrate.
Figure 61
Explanation of Figure 61 Plantlets transferred in field soil mixed with farmyard showing flowering
Figure 62
B
A
C D
Explanation of Figure 62 Synseed production and germination
A. Encapsulated nodal segments placed on MS basal medium, 1 day-old culture. B. Shoot emergence through synseeds on MS basal medium, 1 week-old culture C. Complete plantlet recovery through synseeds on MS medium supplemented with 1.0 µM BA and 0.5 µM IAA, 4 week-old culture D. Elongated plantlets on MS medium supplemented with 1.0 µM BA and 0.5 µM IAA, 5 week-old culture
Figure 63
A B
C
Explanation of Figure 63 Synseed germination
A. Synseeds (having MS gel matrix) germination after 4 week of storage, 3 week-old culture B. –do-, after 6 weeks of incubation C. Isolated plantlets with healthy shoot and root systems recovered through synseeds, 6 week-old culture
Figure 64
A B
Explanation of Figure 64 Successfully hardened plantlets obtained from synseeds
A. Group of acclimatized plantlets obtained through synseeds in soilrite B. A twig of regenerated plantlet of S. mauritiana showing flower heads
Chl a (mg g-1) Chl b (mg g-1) Total Chl (mg g-1) 1.8 a
1.6 ) -1 b 1.4 a
1.2 c cd d b 1.0
c c 0.8 c Chlorophyll content (mg l (mg content Chlorophyll a
0.6 b c d 0.4 d
0.2 0 7 14 21 28 Acclimatization period (days)
Figure 65. Change in chlorophyll contents (a, b & Total) (mg g-1) during acclimatization. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Carotenoids (mg l-1) 0.40 a
) 0.35 ab -1
0.30
bc
0.25 c
c
Carotenoids content (mg g (mg content Carotenoids 0.20
0.15 0 7 14 21 28 Acclimatization period (days)
Figure 66. Change in carotenoids content (mg g-1) during acclimatization. The line represents mean ± S. E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
-2 -1 Net Photosynthetic Rate (PN) (µmol CO2 m s ) 8 ) -1
s a 7 -2 m 2 b 6 ) (µmol CO
N 5
c 4
3
d d 2 NetPhotosynthetic Rate (P
1 0 7 14 21 28 Acclimatization period (days)
-2 -1 Figure 67. Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization. The bars represents mean ± S. E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
Figure 68
LP AD
SB
A B
C
Explanation of Figure 68 Histological study of regenerating tissues
A. Section showing subsequent development of the bulging into well developed shoot bud with apical dome (AD) and leaf primordia (LP) B. Shoot tip explant of S. mauritiana showing induction of shoot bud (SB) in the form of bulging from axillary region after 7 days of incubation on BA (1.0 µM) + IAA (0.5 µM) C. Leaf tissue of S. mauritiana showing adventitious shoot bud development without any intermittent callus on BA (2.5 µM) + IAA (0.5 µM) after 1 week of incubation.
4.3 Decalepis hamiltonii
4.3.1 Seed germination and collection of explants
The effect of different concentrations of GA on in vivo and in vitro seed germination of D. hamiltonii was examined. The sterilized seeds were failed to germinate in soil without pre-soaking of seeds in GA. The pre-soaking of seeds in GA induced germination from 3.60 ± 0.50 to 9.60 ± 0.67% (Table 52). Since the germination percentage was not much effective to initiate the experiment, therefore in vitro seed germination was also performed. Addition of GA to the culture medium significantly enhanced the seed germination frequency. The full-strength MS basal medium revealed better response as compared to half-strength. Highest germination (83.20 ± 1.82%) was observed on full-strength MS basal medium amended with 2.5 µM. The seeds inoculated on to 2.5 µM GA showed emergence of radical 7.60 ± 0.40 days of incubation (Fig. 69 A & B). Further increasing (5.0 µM) or decreasing (1.0 µM) the GA concentration, germination was delayed to 14.40 ± 0.67 and 14.60 ± 0.67 days respectively and only 74.20 ± 2.28 and 61.60 ± 2.11% germination were recorded on 5.0 and 1.0 µM GA supplemented full-strength MS media respectively. A similar trend was also noticed on half-strength MS medium supplemented with varying concentrations of GA however the germination was lesser as compared to the full-strength MS medium with similar concentrations of GA. The least germination (29.00 ± 2.44%) was observed on 1.0 µM GA supplemented half-strength MS basal medium wherein germination initiated within 17.20 ± 0.86 days of incubation (Table 53). The nodal segments (1-1.5 cm), shoot tips (0.8-1.5 cm), leaves and cotyledon were excised from 3 week-old aseptic seedlings and used as explants for multiple shoots regeneration.
4.3.2 Nodal segment culture
The nodal segments remained green and fresh but no shoot regeneration was noticed on MS basal medium and necessitated the PGR supplementation for axillary bud sprouting. Thus, different concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 7.5 µM) of various cytokinins (BA, Kn, 2-iP and TDZ) were tested with or without varying concentrations (0.1, 0.5 and 1.0 µM) of auxin (NAA, IAA and IBA).
4.3.2.1 Effect of adenine-based cytokinins on shoot regeneration
In the presence of adenine-based cytokinins bud breakage and induction of multiple shoots was observed after 10-12 days of incubation. Among three cytokinins tested, BA was found to be the most effective (Fig. 70 A, B & C). All the parameters i.e., percentage response, mean number of shoots per explant and mean shoot length were increased gradually up to 5.0 µM BA. 5.0 µM BA was proved as the most critical concentration for inducing maximum bud break (84.20 ± 1.31%), shoot number per explant (4.20 ± 0.37) and shoot length (5.30 ± 0.09 cm) after 4 weeks of culture (Fig. 70 D). Higher concentration of BA (7.5 µM) significantly reduced the regeneration efficiency and only 2.20 ± 0.20 shoots per explant were induced after an incubation period of 4 weeks. Other two adenine-based cytokinins viz., Kn and 2-iP also induced bud break, but with reduced frequency (Table 54). Only 3.40 ± 0.24 and 3.60 ± 0.40 shoots per explant were noticed on 5.0 µM Kn and 2-iP supplemented MS media.
4.3.2.2 Effect of cytokinin-auxin combinations on shoot regeneration
The combination of BA (5.0 µM) and auxins (NAA, IAA and IBA) significantly enhanced the morphogenic response where shoot induction was possible within 1 week of incubation. Among three auxins tested, IAA at 0.5 µM exhibited maximum (94.00 ± 1.87%) response, highest number (5.80) of shoots per explant and longest shoots (5.80 ± 0.37 cm) after 4 weeks of culture. At higher concentration of IAA, the regeneration response and number of shoots per explant slightly reduced and only 4.60 ± 0.24 shoots per explant were noticed after 4 weeks of culture. The MS medium supplemented with BA and NAA showed similar response as shown by BA and IAA combinations however regeneration efficiency was slightly reduced. After 4 weeks of incubation, a maximum of
5.00 ± 0.31 shoots per explant were induced on 5.0 µM BA and 0.5 µM NAA combination. While, the combinations of BA and IBA did not improve bud break and shoot sprouting satisfactorily as compared to IAA and NAA due to high frequency of callogenesis and only 4.40 ± 0.24 shoots per explant were recorded on 5.0 µM BA and 0.5 µM IBA (Fig. 71 A-C, Table 55). The shoots regenerated on cytokinins alone as well as with auxin showed rudimentary leaf development and premature abscission; therefore to prevent the premature leaf fall and to increase leaf area with enhanced shoot proliferation, different additives such as adenine sulphate (ADS), glutamine (Glu) and phloroglucinol (PG) were supplemented to the optimized combination of BA (5.0 µM) and IAA (0.5 µM).
4.3.2.2.1 Effect of growth additives on shoot proliferation and growth
The inclusion of Glu to the shoot multiplication medium exhibited no considerable effect on shoot multiplication and their growth, however a significant influence on premature leaf fall was observed. 30.0 µM Glu resulted in slightly improved growth with no leaf fall whereas intermediate leaf fall occurred at 20.0 µM Glu. In contrast, the lowermost treatment of Glu (10.0 µM) exhibited poor shoot growth with heavy leaf fall. The addition of ADS reinforced the overall shoot growth. The shoot multiplication was increased significantly with the increase in the ADS concentration from 10.0 to 30.0 µM. Among all the concentrations tested, 30.0 µM ADS induced a maximum of 8.80 ± 0.37 shoots per explant (1.5 time increase) with the highest shoot length of 6.46 ± 0.11 cm after 4 weeks of incubation. Moreover, ADS at this level prevented an early leaf fall within 2 weeks of incubation and also increased the stem girth, thus making the shoots healthiest among all the additives (Fig. 72 A). The inclusion of PG instead of ADS checked leaf fall and slightly enhanced the shoot multiplication as compared to additive-free shoot regeneration medium. The most suitable concentration of PG was 30.0 µM that induced nearly 7.20 shoots per explant (1.24 times increase) with 6.30 cm shoot length (Fig. 72 B). Thus, PG exhibited lesser effective role in shoot multiplication as compared to ADS containing medium (Table 56).
4.3.2.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Different TDZ concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) were also assessed for shoot regeneration from the nodal segments, but explants did not exhibit any shoot development on TDZ supplemented MS medium. They remained green up to 3 week, thereafter tissue turned brown and died.
4.3.2.4 Effect of different pH on shoot regeneration
The effect of different pH (5.0, 5.4, 5.8, 6.2 and 6.6) on regeneration was tested with MS medium supplied with an optimal combination of BA (5.0 µM), IAA (0.5 µM) and ADS (30.0 µM). Among a range of pH tested, 5.8 was proved to be optimal for the shoot multiplication and elongation where a maximum of 8.80 ± 0.37 shoots per explant with highest shoot length (6.46 ± 0.11 cm) was obtained and thereafter a gradual decreasing trend was observed. On lowering or increasing the pH range, the shoot multiplication rate was severally affected. Only 3.00 ± 0.54 and 3.80 ± 0.37 shoots per explant having 3.40 ± 0.13 and 4.40 ± 0.13 cm shoot length were induced at 5.4 and 6.6 pH respectively, after 4 weeks of incubation whereas at 5.0 pH all the nodal segments turned brown and failed to exhibit morphogenic response might be due to the acidic nature of medium (Fig. 73).
4.3.2.5 Effect of different carbon sources on shoot regeneration
Three carbon sources i.e., sucrose, fructose and glucose were also tested with MS basal medium having BA (5.0 µM), IAA (0.5 µM) and ADS (30.0 µM) at optimum 5.8 pH. Among three carbon sources, sucrose found to be the best for shoot regeneration as induced a maximum of 8.80 ± 0.37 shoots per explant and 6.46 ± 0.11 cm shoot length after 4 weeks of culture. MS medium supplemented with fructose showed somewhat similar response and induced 5.60 ± 0.67 shoots per explant and 5.32 ± 0.14 cm shoot length. Whereas, glucose was found the least effective for shoot regeneration as regenerated only 2.40 ± 0.24 shoots per explant with 3.40 ± 0.13 cm shoot length (Fig. 74).
4.3.2.6 Effect of different culture media on shoot regeneration
The effect of different basal media (MS, B5 and WPM) amended with an optimal concentration of BA (5.0 µM), IAA (0.5 µM) and ADS (30 µM) on culture establishment and shoot regeneration was evaluated. The results revealed a significant effect of media formulation on percentage bud break, shoot number and shoot length. Among the different basal media tested, MS medium was found best for the growth of cultures (Fig.). The number of shoots (8.80 ± 0.37) and their length (6.46 ± 0.11) were highest on MS medium followed by WPM (6.20 ± 0.58 shoots) and B5 (5.00 ± 0.31 shoots) media in deceasing order after 4 weeks of culture. Consequently, in all subsequent experiments only MS medium was used (Fig. 75).
4.3.3 Shoot tip culture
The morphogenetic response of shoot tip explant to various cytokinins (BA, Kn, 2-iP and TDZ) and auxins (NAA, IAA and IBA) was evaluated.
4.3.3.1 Effect of adenine-based cytokinins on shoot regeneration
Shoot tips remained green, but ceased to grow on PGR-free MS Basal medium (control). However, supplementation of BA (1.0, 2.5, 5.0 and 7.5 µM) triggered stimulatory response and supplementation of 5.0 µM BA proved to be critical concentration for the regeneration of 3.20 ± 0.20 shoots per explant with 4.84 ± 0.10 cm shoot length in 81.40 ± 1.86% culture (Fig. 76 A). At this treatment new leaves emerged out after 7th day of inoculation and after 12 days shoot differentiation were noticed. On increasing or decreasing the concentration of BA beyond optimal level, differentiation of shoots was reduced. Kn and 2-iP were lesser effective at 5.0 µM in comparison with BA as induced only 2.20 ± 0.20 and 2.40 ± 0.24 shoots per explant respectively (Table 57). Simultaneously yellowish-brown, nodular organogenic tissue was also initiated from the basal cut end of the explants after 2 weeks of incubation on optimum concentration of BA (5.0 µM). On further culturing, few green shoot buds were regenerated after 4 weeks. Basal clumps of shoot buds were cut from the mother explants
and then transferred to different shoot regeneration media for their multiplication and elongation. In contrast, Kn and 2-iP induced light-brown, fleshy but non-organogenic tissues.
4.3.3.2 Effect of cytokinin-auxin combinations on shoot regeneration
To evaluate the synergistic effect of cytokinins-auxin combination on multiple shoot induction through shoot tip explants, different concentrations (0.1, 0.5 and 1.0 μM) of auxin (NAA, IAA and IBA) were added to the optimized concentration of BA (5.0 μM). Among the auxins tested, IAA was found to be most effective followed by NAA and IBA which were comparatively lesser effective (Table 58). Shoot tip explants cultured on MS medium augmented with 5.0 µM BA and 0.5 µM IAA exhibited maximum percent (92.00 ± 1.22%) response and induced 3.80 ± 0.37 shoots per explant haiving 5.34 ± 0.10 cm shoot length after 4 weeks of culture. A gradual decrease in the number of shoots per explant was observed at lower (0.1 µM) and higher concentration (1.0 µM) of IAA, NAA and IBA due to intense callusing (Fig. 76 B & C). Again it was noticed that on cytokinin-auxin combinations, all the regenerated shoots exhibited poor leaf development and premature leaf fall (Fig. 76 D); therefore different growth additives were tested with the optimized combination of BA and IAA in order to improve shoot growth. Similar to 5.0 µM BA alone, combination of 5.0 µM BA and 0.5 IAA also induced basal nodular tissues after 2 weeks of culture. For shoot bud initiation and their further proliferation, these nodular tissues (induced on cytokinin alone and in combination with auxin) were cut and then transferred to the optimized combination of PGRs and growth additive.
4.3.3.2.1 Effect of growth additives on shoot regeneration
To study the effect of different growth additives on shoot growth, shoots regenerated on MS medium supplemented with 5.0 µM BA and 0.5 µM IAA were transferred on to the MS medium supplemented with Glu, ADS, PG (10, 20, 30 and 40 µM) in addition to similar combination of BA and IAA. All the additives significantly
improved shoot multiplication and their growth. Addition of 30.0 µM Glu to the optimized combination of BA and IAA exhibited slightly improved growth and induced an average of 4.80 ± 0.20 shoots per explant and 6.22 ± 0.08 cm shoot length with no leaf fall (Fig. 76 E). On lowering the Glu concentration to 20.0 µM leaf fall occurred at low frequency, whereas 10.0 µM failed to check early leaf fall. Contrary to this, ADS exerted the best response in terms of all the evaluated parameters i.e., arresting the early leaf fall, shoot number per explants and shoot length. Among different concentrations of ADS, 30.0 µM proved to be the critical as induced a maximum 8.20 ± 0.37 shoots per explants and 6.54 ± 0.08 cm shoot length after 4 weeks of transfer. At this concentration, leaves were shiny and well expanded, exhibited vigorous production of shoots (Fig. 76 F). PG had similar effect on shoot multiplication and leaf fall. Among tested concentrations, 30.0 µM PG induced an average of 6.40 ± 0.50 shoots per explant and attained 6.22 ± 0.08 cm shoot length after 4 week of incubation (Table 59). As ADS was found the best growth additive for axillary bud multiplication as compared to Glu and PG, therefore for adventitious shoot regeneration only ADS was selected. Basal nodular tissues were cut from two mother sources i.e., 5.0 µM BA with and without 0.5 µM IAA (Fig. 77 A-C) and then transferred to different concentrations of GA (0.5, 1.0, 2.5 and 5.0 µM) and ADS (10.0, 20.0, 30.0, 40.0 and 50.0 µM) were added to the MS medium with 5.0 µM BA and 0.5 µM IAA. Tissue regenerated on BA and IAA combination showed better response as compared to the tissue regenerated on BA alone. Among different ADS regimes, 30.0 µM induced an average of 7.60 ± 0.50 and 4.40 ±
0.24 shoots per culture when transferred from 5.0 µM BA with IAA 0.5 µM (N2 tissue)
and 5.0 µM BA (N1 tissue) containing MS medium respectively (Fig. 78 A & B).
Whereas 1.0 µM GA induced 3.00 ± 0.31 and 4.80 ± 0.37 shoots per culture from N1 and
N2 tissues respectively. Further increasing or decreasing the GA and ADS concentrations beyond the optimum level reduced organogenesis was noticed from both the tissues. However, on combination of optimized concentrations of GA (1.0 µM) and ADS (30.0 µM) considerably reinforced shoot regeneration and shoot elongation. A maximum of 15.40 ± 0.67 shoots per culture and 4.56 ± 0.06 cm shoot length was recorded on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 1.0 µM GA and 30.0 µM ADS
from N2 tissue while an average of 8.00 ± 0.63 shoots per culture with 4.20 ± 0.09 cm shoot length was induced on similar combination of PGR and growth additive (Fig. 78 C & D, Table 60).
4.3.3.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Shoot tip explants failed to regenerate shoots on TDZ supplemented MS medium, only grew in to a single shoot of 2-3 cm after 3 weeks of culture.
4.3.4 Leaf culture
4.3.4.1 Effect of adenine-based cytokinins on shoot regeneration
Leaf explants were inoculated on different concentrations of (1.0-7.5 µM) three adenine-based cytokinins i.e., BA, Kn and 2-iP for shoot regeneration. On all the treatemnets, callusing was started from petiolar cut end of the leaf explants after 2 weeks of incubation. Among the treatments, only 5.0 µM BA supplemented MS medium induced single leaf along with huge callusing from the petiolar cut of the explant after 4 weeks of incubation (Fig. 79 A). However, rest of the treatments induced only callogenesis (greenish-white and compact) even after 4 weeks of culture (Table 61).
4.3.4.2 Effect of cytokinin-auxin combinations on shoot regeneration
Leaf explants were also inoculated on various cytokinin-auxin combinations. Different concentrations (0.1, 0.5 and 1.0 µM) of three auxins (NAA, IAA and IBA) were added to 5.0 µM BA containing MS media. Similar to BA, Kn and 2-iP, cytokinin-auxin combinations induced huge callusing from the basal petiolar cut end of the leaf explants. However, callusing was started 1 week earlier on cytokinin-auxin combination than cytokinin alone. Only 0.5 and 1.0 µM IAA and 1.0 µM NAA exhibited shoot regeneration however the percent regeneration and mean number of shoots per explant were very less. Among the treatments, on 5.0 µM BA and 1.0 µM IAA supplemented MS medium a maximum of 1.80 ± 0.37 shoots per explant were noticed in 17.00 ± 3.00%
cultures after 4 weeks of culture. Shoots attained rudimentary growth therefore data was not recorded in terms of shoot length (Fig. 79 B & C, Table 62).
4.3.4.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Leaf explants failed to induce any organogenic behavior on TDZ supplemented MS medium (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM). Only slight enlargement in leaf size was noticed after 2 weeks of culture thereafter they remained as such even after 4 weeks of incubation (Fig. 79 D).
4.3.5 Cotyledon culture
4.3.5.1 Effect of adenine-based cytokinins on shoot regeneration
Cotyledons were found to be non-responsive on BA, Kn and 2-iP supplemented nutrient media (1.0-7.5 µM). The cultured cotyledons remained green on all the treatments (1.0-7.5 µM) of cytokinin. Neither caulogenesis nor callogenesis was possible through cotyledon explants even after 4 weeks of culture.
4.3.5.2 Effect of cytokinin-auxin combinations on shoot regeneration
Different concentrations (0.1, 0.5 and 1.0 µM) of three auxins (NAA, IAA and IBA) were added to 5.0 µM BA containing MS media to see the morphogenic response of cotyledons. Similar to cytokinins, no regeneration was noticed on cytokinin-auxin combinations through cotyledons. Only a little enlargement in explant size was noticed after 4 weeks of incubation.
4.3.5.3 Effect of urea-based cytokinin (TDZ) on shoot regeneration
Similar to the leaf explants, all the treatments of TDZ (0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µM) failed to induce caulogenesis or callogenesis through cultured cotyledons. All the cotyledons remained green on TDZ supplemented MS medium but did not give any sign for shoot differentiation up to 4 weeks of incubation.
4.3.6 Effect of sub-culturing on shoot proliferation
The regenerating tissues derived from axillary/apical bud multiplication (through nodal and shoot tip explants) were cut into two pieces and then sub-cultured to the fresh culture medium containing 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS after every 4 week for further shoot proliferation. Sub-culturing was continued up to the six culture passages (Fig. 80). Multiplication rate was increased up to the third culture passage (14.60 ± 0.50 and 14.00 ± 0.54 shoots per nodal segment and shoot tip explant respectively) thereafter, the proliferation rate stabilized at fourth passage and subsequently it declined due to callus formation. While, shoot length increased gradually after every sub-culture passage for both the explant type. As the result implicit that during sub-culturing, optimized combination of PGRs and ADS was not found beneficial after third sub-culture passage, therefore tissues were again sub-cultured on to lower concentration of BA (2.5 µM) along with 0.5 µM IAA and 30.0 µM ADS. This combination proved effective to maintain the cultures up to two year. On the other hand, shoots derived from basal nodular tissues of shoot tip explants
(N1 and N2) were sub-cultured on to the fresh MS medium supplemented with 5.0 µM BA, 0.5 µM IAA, 1.0 µM GA and 30.0 µM ADS. Each regenerating tissue (derived from single shoot tip) was cut into four pieces and sub-cultured as a separate unit on the fresh regeneration medium, but the data was noted as aggregate of all the four pieces. Sub- culturing gradually increased the organogenic efficiency of N1 and N2 tissues up to fifth and fourth passage respectively. Maximum of 20.20 ± 0.80 and 25.40 ± 0.50 shoots per
explant were induced from N1 and N2 tissues respectively after fifth and fourth sub- culture passage, thereafter proliferation stabilized. Although shoot length was consistently enhanced after each sub-culture passage and resulted in 5.40 ± 0.05 and 6.00
± 0.07 cm long shoots from N1 and N2 tissues respectively, after sixth sub-culture passage (Fig. 81). Like the regenerating cultures obtained from axillary buds multiplication (shoot tip and nodal segments), shoots induced from basal nodular tissues were maintained on GA-free MS medium supplemented with 2.5 µM BA, 0.5 µM IAA and 30.0 µM ADS up to one year.
4.3.7 In vitro rooting of microshoots
To obtain complete plantlets, regenerated microshoots (3.0-4.0 cm) were transferred to full and half-strength MS basal medium with or without different concentrations (1.0, 2.5 and 5.0 µM) of auxins (NAA, IAA and IBA). Shoots failed to induce root on both the strength of MS medium without any auxin, only basal callusing was noticed from the cut ends of the microshoots. Exogenous addition of auxins induced healthy root formation differentially depending upon type and the concentration of auxin used. Among the treatments tested, microshoots were best rooted on half-strength MS medium supplemented with 2.5 µM NAA which induced a maximum of 7.80 ± 0.37 roots per shoot with 6.40 ± 0.13 cm root length after 4 week of transfer followed by 4.00 ± 0.31 roots per shoot having 5.22 ± 0.10 cm root length on 2.5 µM IAA supplemented to the half-strength MS medium. On increased and decreased concentrations of NAA beyond the optimal level mean number of roots per shoot was significantly reduced, but the longest roots (17.56 ± 0.42 cm long) were noticed on 1.0 µM NAA (Fig. 82 A-D). On the other hand, IBA was found to be the least effective. Only 2.5 µM IBA induced single root with 3.98 ± 0.08 cm length after 4 week of incubation, while rest of two treatments of IBA induced only high frequency of callogenesis (Table 63).
4.3.8 Acclimatization of plantlets
Tissue culture raised plantlets with 5-7 fully expanded leaves and well developed root system were removed from the culture tubes, washed carefully with tap water, transferred to various planting substrates and hardened off (Fig. 83 A & B), adopting the procedure given in materials and method. Among three planting substrates tested, percent survival of in vitro raised plants was 88.40% in vermi-compost; however 95.10% plantlets survived in soilrite after 2 month of their field transfer. On the other hand, garden soil and farmyard mixture (2:1) did not found suitable for acclimatization (Fig. 84).
4.3.9 Synseed production
The results related to successful synseed formation and their germination in different culture regimes are summarized under the following subheads.
4.3.9.1 Effect of Na2-alginate concentration on synseed formation
In order to optimize the concentration of Na2-alginate for an ideal synseed development, five concentrations of Na2-alginate (1, 2, 3, 4 and 5%) were tested with 100
mM CaCl2·2H2O. Among different concentrations tested 4% Na2-alginate proved to be the critical for synseed production and an average of 68.60 ± 1.86% germination was noticed when placed to MS basal medium (Fig. 85 A & B). On increasing the concentration to 5% relatively hard synseeds were formed which delayed their successful germination; whereas 3% Na2-alginate resulted in deformed and very delicate synseed production those were difficult to handle. On further lowering the concentration to 1.0 and 2.0% only fragile synseeds were obtained (Table 64).
4.3.9.2 Effect of CaCl2·2H2O concentration on synseed formation
After optimizing the Na2-alginate concentration, effect of different concentrations
of CaCl2·2H2O (25, 50, 75, 100 and 200 mM) on synseed formation was also assessed.
Amongst, 100 mM CaCl2·2H2O with 4% Na2-alginate resulted in successful synseed development (68.60 ± 1.86%). On decreasing and increasing the concentration, similar
features in synseed morphology were noticed as observed with Na2-alginate (Table 65).
4.3.9.3 In vitro plantlet regeneration from synseeds on culture medium
Synseeds were placed aseptically on different treatments of PGRs and ADS. PGR-free MS basal medium (control) revealed the least germination (68.60 ± 1.86%). Among the PGR combinations tested, maximum seed germination (77.00 ± 2.09%) was achieved on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS (Fig. 85 C & D) followed by 74.20 ± 1.90% germination on 5.0 µM BA and 0.5 µM IAA containing MS medium. Latter treatment induced weaker shoot as compared to the BA,
IAA and ADS combination. Addition of NAA in place of IAA slightly reduced the germination frequency in all the tested combinations (Table 66). It was also noticed that on IAA combinations shoots emerged out without any intervening callus, while all the treatments with NAA exhibited callus associated shoot formation. However, microshoots regenerated from the alginate synseeds of D. hamiltonii failed to induce rooting on the germination medium, therefore microshoots were separated from the gel matrix of synseed and then transferred to root induction media. For in vitro root induction different concentrations of NAA (1.0, 2.5 and 5.0 µM) were tested rather than IAA and IBA as NAA revealed its promotive effect on root induction in D. hamiltonii microshoots regenerated from nodal and shoot tip explants. The shoots were best rooted on 2.5 µM NAA added to half-strength MS medium and an average of 4.60 ± 0.50 roots per microshoot with 5.26 ± 0.08 cm root length induced after 4 week of transfer (Fig. 85 E, Table 67).
4.3.9.4 In vitro germination of synseeds and naked nodal segments after low temperature storage
Encapsulated (having MS and DDW gel matrix) and non-encapsulated nodal segments were stored at 4 ˚C for 1, 2, 4, 6 and 8 weeks to analyze the effect of storage on tissue survival. For synseeds prepared with MS nutrient gel matrix, germination frequency was linearly reduced up to 4 week of storage (47.00 ± 1.54); thereafter a drastic loss in tissue survival noticed and only 14.00 ± 1.37% germination was achieved after 8 week of storage. In contrast, synseeds with DDW gel matrix did not find suitable for storage purpose. Whereas, non-encapsulated nodal segments could be stored only for 2 week, thereafter tissue did not survive (Table 68).
4.3.9.5 Acclimatization of plantlets
Complete plantlets with well developed shoot and root systems were transferred to thermocol cups containing sterilized soilrite, moistened with normal tap water as required and followed similar pattern of acclimatization as described in materials and method. New leaves emerged out after 2 week of acclimatization, thereafter shoots
attained active growth in the ambient conditions. Successfully acclimatized plantlets were then transferred to the field conditions where more than 80% survival rate was recorded.
4.3.9.6 Ex vitro sowing of synseeds on various planting substrates for the recovery of plantlets
Encapsulated nodal segments of D. hamiltonii were failed to germinate when they were directly sown to different planting substrate.
4.3.10 Physiological study
4.3.10.1 Chlorophyll a, b and total chlorophyll content during acclimatization
Chlorophyll a, b and total chlorophyll content showed similar trend during various phases of acclimatization. Plants when taken out of culture tubes and acclimatized showed decrease in chl a (0.93 ± 0.01 to 0.84 ± 0.02 mg g-1), b (0.43 ± 0.01 to 0.33 ±0.02 mg g-1) and total chlorophyll (1.23 ±0.03 to 1.03 ±0.05 mg g-1) content during first week of transfer. Thereafter a linear increase was noticed with each passing week. After 14 days of transfer considerable increase in chlorophyll a (0.94 ± 0.02 mg g-1), b (0.50 ±0.01 mg g-1) and total chlorophyll (1.30 ± 0.03 mg g-1) content was seen, this further increased slightly during fourth week stabilizing ultimately to chl a 1.38 ±0.02 mg g-1. chl b 0.64 ±0.01 mg g-1 and total chl 1.46 ± 0.02 mg g-1 (Fig. 86).
4.3.10.2 Carotenoids content during acclimatization
Carotenoids content showed similar increasing trend during successive acclimatization like that of chlorophyll. After a slight decrease, carotenoids increased gradually up to 28 days of acclimatization. Marked increase in carotenoids amount (0.23 ± 0.01 to 0.33 ± 0.01 mg g-1) was noticed just after 14 days of transfer. Similarly a steep rise in carotenoids content (0.39 ±0.02 to 0.49 ± 0.01 mg g-1) was also witnessed after 21 and 28 days of acclimatization (Fig. 87).
4.3.10.3 Net photosynthetic rate (PN) during acclimatization
During acclimatization, net CO2 gas exchange rate was measured after 7, 14, 21 and 28 days of transplant. During the first week after transfer the net photosynthetic rate
(PN) in D. hamiltonii plantlets decreased (3.34 ± 0.13 to 2.39 ± 0.03). Net photosynthetic rate increased again to 4.48 ± 0.14 after 14 days of acclimatization. After next two week of acclimation it increased to 4.95 ± 0.12 and 5.32 ± 0.09 (Fig. 88).
4.3.11 Histological study
Histology of shoot tip explants of D. hamiltonii was conducted to ascertain the mode of regeneration and origin of de novo shoot formation. Sections of yellowish brown nodular tissue induced at basal cut end of the shoot tip explants on BA (5.0 µM) and IAA (0.5 µM) after 2 weeks of incubation was studied. During the study the unorganized nature of parenchymatous cells revealed callogenic nature of the tissue (Fig. 89 A). During later stages of development the callus cells showed distinct meristematic regions with cluster of actively dividing cells of the nodule characterized by densely stained regions consisting of small cells (Fig. 89 B). These regions were referred as meristemoids. Meristemoids in the calli later started initiating shoot buds in the form of bulging (Fig. 89 C). These bulging later developed into well differentiated shoot buds consisting of meristematic apical dome with two leaf primordia flanking both the sides of the dome (Fig. 89 D).
Table 52. Effect of pre-soaking of seeds in GA on in vivo seed germination in D. hamiltonii after 3 weeks of sowing GA (µM) Mean no. of days to Germination frequency (%) germination Control 00.00 ± 0.00f 0.00 ± 0.00h GA (0.25) 15.80 ± 1.42ab 5.00 ± 0.31fg GA (0.5) 11.40 ± 0.97cd 9.60 ± 0.67f GA (1.0) 10.60 ± 0.60de 3.60 ± 0.50fg Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 53. Effect of different GA concentrations on in vitro seed germination of D. hamiltonii after 3 weeks of culture Treatment (µM) Mean no. of days to Germination frequency germination (%) MS 19.40 ± 0.60a 43.00 ± 1.54d MS + GA (1.0) 14.60 ± 0.67bc 61.60 ± 2.11c MS + GA (2.5) 7.60 ± 0.40e 83.20 ± 1.82a MS + GA (5.0) 14.40 ± 0.67bc 74.20 ± 2.28b ½ MS 0.00 ± 0.00f 00.00 ± 0.00h ½ MS + GA (1.0) 17.20 ± 0.86ab 29.00 ± 2.44e ½ MS + GA (2.5) 11.60 ± 1.02cd 58.00 ± 2.54c ½ MS + GA (5.0) 14.60 ± 0.67bc 63.00 ± 1.84c Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 54. Effect of different cytokinins on shoot induction through nodal segments of D. hamiltonii after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 00.00 ± 0.00h 0.00 ± 0.00g 0.00 ± 0.00e - BA (1.0) 53.00 ± 1.54fg 2.00 ± 0.37def 4.06 ± 0.15d - BA (2.5) 64.00 ± 1.37de 3.00 ± 0.31abcd 4.68 ± 0.09bc - BA (5.0) 84.20 ± 1.31a 4.20 ± 0.37a 5.30 ± 0.09a + BA (7.5) 67.80 ± 1.49bcd 2.20 ± 0.20cdef 4.34 ± 0.12cd + + Kn (1.0) 51.00 ± 1.18g 1.20 ± 0.20fg 4.64 ± 0.06bc - Kn (2.5) 58.80 ± 2.03ef 2.00 ± 0.31def 4.90 ± 0.10ab - Kn (5.0) 72.40 ± 1.43b 3.40 ± 0.24abc 5.30 ± 0.07a + + Kn (7.5) 64.40 ± 1.12de 1.60 ± 0.24abc 4.32 ± 0.09cd + + 2-iP (1.0) 48.20 ± 0.91g 1.40 ± 0.24f 4.20 ± 0.09cd - 2-iP (2.5) 63.40 ± 1.02de 2.80 ± 0.20bcde 5.06 ± 0.08ab + 2-iP (5.0) 71.40 ± 1.16bc 3.60 ± 0.40ab 5.20 ± 0.09a + 2-iP (7.5) 65.60 ± 1.69cd 2.40 ± 0.24bcdef 4.34 ± 0.14cd + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 55. Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through nodal segments of D. hamiltonii after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 80.40 ± 1.63bc 3.80 ± 0.22bcd 5.38 ± 0.08ab - NAA (0.5) 85.60 ± 1.69b 5.00 ± 0.31ab 5.00 ± 0.07bc + NAA (1.0) 77.60 ± 1.12c 3.80 ± 0.37bcd 4.30 ± 0.09d + IAA (0.1) 86.00 ± 1.88ab 4.40 ± 0.24bc 5.12 ± 0.05abc - IAA (0.5) 94.00 ± 1.87a 5.80 ± 0.37a 5.48 ± 0.13a + + IAA (1.0) 81.20 ± 1.59bc 4.60 ± 0.24ab 4.92 ± 0.04c + + IBA (0.1) 55.60 ± 1.69e 3.20 ± 0.20cd 4.80 ± 0.11c + + IBA (0.5) 66.00 ± 1.37d 4.40 ± 0.24bc 4.18 ± 0.09d + + IBA (1.0) 52.60 ± 1.66e 2.60 ± 0.24d 3.88 ± 0.10d + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 56. Effect of glutamine (Glu), adenine sulphate (ADS) and phlouroglucinol (PG) supplemented with 5.0 µM BA and 0.5 µM IAA on shoot multiplication through nodal segments of D. hamiltonii on MS medium after 4 weeks of transfer Glu ADS PG Mean no. of Mean shoot length Remarks (µM) shoots/explant (cm) 10 5.80 ± 0.37b 5.64 ± 0.06d - 20 6.00 ± 0.31b 6.02 ± 0.06bcd + 30 6.00 ± 0.31b 6.34 ± 0.06ab + + + 40 6.00 ± 0.00b 5.68 ± 0.08d + + 10 7.00 ± 0.31b 6.16 ± 0.12abc + + + 20 7.40 ± 0.24ab 6.18 ± 0.06abc + + + 30 8.80 ± 0.37a 6.46 ± 0.11a + + + + 40 7.20 ± 0.20ab 6.02 ± 0.08bcd + + + 10 6.00 ± 0.44b 5.78 ± 0.08cd + 20 6.80 ± 0.37b 6.10 ± 0.08abc + + 30 7.20 ± 0.48ab 6.30 ± 0.09ab + + + 40 6.40 ± 0.50b 6.02 ± 0.06bcd + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. - poor growth with heavy leaf fall. + moderate growth with intermediate leaf fall. + + moderate growth with no leaf fall. + + + slightly improved growth with no leaf fall. + + + + vigorous growth with no leaf fall.
Table 57. Effect of different cytokinins on shoot induction through shoot tip explants of D. hamiltonii after 4 weeks of culture Cytokinin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis Control 00.00 ± 0.00g 0.00 ± 0.00f 0.00 ± 0.00f - BA (1.0) 59.60 ± 1.63de 1.20 ± 0.20de 4.08 ± 0.08e - BA (2.5) 63.80 ± 1.62cd 2.00 ± 0.31bcd 4.44 ± 0.10de - BA (5.0) 81.40 ± 1.86a 3.20 ± 0.20a 4.84 ± 0.10abcd + BA (7.5) 72.20 ± 1.01b 1.20 ± 0.20de 4.66 ± 0.08cd + + Kn (1.0) 54.40 ± 1.28ef 1.00 ± 0.00e 4.42 ± 0.13de - Kn (2.5) 61.20 ± 1.15cd 1.20 ± 0.20de 4.74 ± 0.08bcd - Kn (5.0) 72.60 ± 1.66b 2.20 ± 0.20bc 5.22 ± 0.08a + + Kn (7.5) 62.00 ± 0.94cd 2.00 ± 0.00bcd 4.62 ± 0.09cd + + 2-iP (1.0) 49.60 ± 1.63f 1.00 ± 0.00e 4.46 ± 0.09de - 2-iP (2.5) 58.20 ± 0.91de 1.20 ± 0.20de 4.96 ± 0.07abc + 2-iP (5.0) 66.60 ± 1.02bc 2.40 ± 0.24ab 5.20 ± 0.09ab + 2-iP (7.5) 61.40 ± 1.07cd 1.40 ± 0.24cde 4.60 ± 0.13cd + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 58. Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through shoot tip explants of D. hamiltonii after 4 weeks of culture Auxin % Response Mean no. of Mean shoot Frequency of (µM) shoots/explant length (cm) callogenesis NAA (0.1) 77.60 ± 1.12b 2.20 ± 0.00cd 4.42 ± 0.13cd - NAA (0.5) 85.60 ± 1.69a 3.20 ± 0.20ab 4.92 ± 0.12abc + + NAA (1.0) 71.40 ± 1.32bc 2.00 ± 0.31cd 4.20 ± 0.09de + + + IAA (0.1) 85.60 ± 1.69a 2.20 ± 0.20bcd 5.00 ± 0.09ab - IAA (0.5) 92.00 ± 1.22a 3.80 ± 0.37a 5.34 ± 0.10a + IAA (1.0) 75.00 ± 1.48b 2.80 ± 0.20abc 4.72 ± 0.09bcd + IBA (0.1) 57.00 ± 2.42d 1.60 ± 0.24d 3.86 ± 0.09ef + IBA (0.5) 65.60 ± 1.69c 2.20 ± 0.20bcd 4.54 ± 0.12bcd + + IBA (1.0) 49.00 ± 1.18e 1.20 ± 0.20d 3.60 ± 0.13f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. -, +, + +, + + +, indicate no, slight, moderate, intense callusing respectively.
Table 59. Effect of glutamine (Glu), adenine sulphate (ADS) and phlouroglucinol (PG) supplemented with 5.0 µM BA and 0.5 µM IAA on shoot multiplication through shoot tip explants of D. hamiltonii on MS medium after 4 weeks of transfer Glu ADS PG Mean no. of Mean shoot Remarks (µM) shoots/explant length (cm) 10 4.20 ± 0.37c 5.64 ±0.06de - 20 4.60 ± 0.24c 5.80 ± 0.07bcde + 30 4.80 ± 0.20bc 6.22 ± 0.08ab + + + 40 4.00 ± 0.44c 5.68 ± 0.09cde + + 10 4.60 ± 0.24c 5.94 ± 0.16bcde + + 20 6.40 ± 0.50b 6.20 ± 0.13abc + + + 30 8.20 ± 0.37a 6.54 ± 0.08a + + + + 40 6.40 ± 0.50b 6.04 ± 0.18abcd + + 10 4.20 ± 0.20c 5.48 ± 0.09e + 20 5.60 ± 0.24bc 5.90 ± 0.08bcde + + 30 6.40 ± 0.50b 6.22 ± 0.08ab + + + 40 4.40 ± 0.24c 5.62 ± 0.08de + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. - poor growth with heavy leaf fall. + moderate growth with intermediate leaf fall. + + moderate growth with no leaf fall. + + + slightly improved growth with no leaf fall. + + + + vigorous growth with no leaf fall.
Table 60. Effect of PGR, GA and ADS on shoot regeneration through shoot tips derived nodular tissues of D. hamiltonii after 4 weeks of transfer * * BA IAA GA ADS N1 tissue N2 tissue (µM) Mean no. of Mean shoot Mean no. of Mean shoot shoots/culture length (cm) shoots/culture length (cm) 5.0 - 0.00 ± 0.00d 0.00 ± 0.00f 0.0 ± 0.00e 0.00 ± 0.00e 5.0 0.5 0.00 ± 0.00d 0.00 ± 0.00f 0.0 ± 0.00e 0.00 ± 0.00e 5.0 0.5 0.5 - 1.60 ± 0.24cd 2.24 ± 0.09de 3.00 ± 0.31d 3.48 ± 0.09cd 5.0 0.5 1.0 - 3.00 ± 0.31bc 3.02 ± 0.06b 4.80 ± 0.37cd 3.72 ± 0.09bc 5.0 0.5 2.5 - 2.80 ± 0.37bc 3.16 ± 0.12b 4.20 ± 0.48cd 4.06 ± 0.08ab 5.0 0.5 5.0 2.00 ± 0.31c 2.48 ± 0.13cd 3.60 ± 0.24d 3.04 ± 0.17d 5.0 0.5 10 2.40 ± 0.40c 1.96 ± 0.11e 4.40 ± 0.24cd 3.12 ± 0.11d 5.0 0.5 20 3.20 ± 0.37bc 2.30 ± 0.07de 4.80 ± 0.37cd 3.24 ± 0.09cd 5.0 0.5 30 4.40 ± 0.24b 2.80 ± 0.07bc 7.60 ± 0.50b 3.34 ± 0.15cd 5.0 0.5 40 3.00 ± 0.31bc 2.08 ± 0.08de 5.80 ± 0.73bc 3.24 ± 0.11cd 5.0 0.5 1.0 30 8.00 ± 0.63a 4.20 ± 0.09a 15.40 ± 0.67a 4.56 ± 0.06a Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. * N1- tissue regenerated on 5.0 µM BA and N2 – tissue regenerated on 5.0 µM and 0.5 µM IAA.
Table 61. Effect of different cytokinin concentrations on shoot induction through leaf explants of D. hamiltonii after 4 weeks of culture Cytokinin ((µM) % callogenesis Type of callus Control 00.00 ± 0.00f No callogenesis BA (1.0) 00.00 ± 0.00f No callogenesis BA (2.5) 24.00 ± 1.87cd Greenish-white, compact BA (5.0) 34.60 ± 2.03a Greenish-white, compact BA (7.5) 30.20 ± 2.69abc Greenish-white, compact Kn (1.0) 00.00 ± 0.00f No callogenesis Kn (2.5) 07.60 ± 1.43ef Greenish-white, compact Kn (5.0) 20.60 ± 1.16d Greenish-white, compact Kn (7.5) 24.60 ± 2.03bcd Greenish-white, compact 2-iP (1.0) 00.00 ± 0.00f No callogenesis 2-iP (2.5) 11.40 ± 0.97e Yellowish-white, compact 2-iP (5.0) 32.40 ± 1.46ab Yellowish-white, compact 2-iP (7.5) 27.00 ± 3.00abcd Yellowish-white, compact Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 62. Effect of different combinations of auxin (NAA, IBA and IAA) with 5.0 µM BA on shoot induction through leaf explants of D. hamiltonii after 4 weeks of culture Auxin (µM) % regeneration Mean no. of Type of callus shoots/explant NAA (0.1) 0.0 ± 0.00c 0.0 ± 0.00c No callogenesis NAA (0.5) 0.0 ± 0.00c 0.0 ± 0.00c Greenish-white, compact NAA (1.0) 7.60 ± 1.02b 1.40 ± 0.24ab Greenish-white, compact IAA (0.1) 0.0 ± 0.00c 0.0 ± 0.00c Greenish-white, compact IAA (0.5) 10.00 ± 0.63b 1.00 ± 0.00b Greenish-white, compact IAA (1.0) 17.00 ± 3.00a 1.80 ± 0.37a Greenish-white, compact IBA (0.1) 0.0 ± 0.00c 0.0 ± 0.00c No callogenesis IBA (0.5) 0.0 ± 0.00c 0.0 ± 0.00c No callogenesis IBA (1.0) 0.0 ± 0.00c 0.0 ± 0.00c No callogenesis Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
Table 63. Effect of different auxins concentrations on in vitro root induction in D. hamiltonii after 4 weeks of transfer Auxin % Response Mean no. of Mean root Frequency (µM) roots/shoot length (cm) of callogenesis MS 0.00 ± 0.00f 0.00 ± 0.00e 0.00 ± 0.00f + + ½ MS 0.00 ± 0.00f 0.00 ± 0.00f 0.00 ± 0.00f + ½ MS + NAA (1.0) 80.40 ± 1.63b 2.40 ± 0.24c 17.56 ± 0.42a + ½ MS + NAA (2.5) 96.40 ± 1.56a 7.80 ± 0.37a 6.40 ± 0.13b + ½ MS + NAA (5.0) 72.20 ± 1.01c 1.40 ± 0.24d 4.30 ± 0.09d + + + ½ MS + IAA (1.0) 82.00 ± 1.37b 1.00 ± 0.00d 4.20 ± 0.09de + ½ MS + IAA (2.5) 79.40 ± 1.16b 4.00 ± 0.31b 5.22 ± 0.10c + + ½ MS + IAA (5.0) 53.60 ± 1.56d 1.20 ± 0.20d 3.52 ± 0.13e + + + ½ MS + IBA (1.0) 0.00 ± 0.00f 0.00 ± 0.00e 0.00 ± 0.00f + + ½ MS + IBA (2.5) 46.20 ± 1.24e 1.00 ± 0.00d 3.98 ± 0.08de + + + ½ MS + IBA (5.0) 0.00 ± 0.00f 0.00 ± 0.00e 0.00 ± 0.00f + + + Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability. +, + +, + + +, indicate slight, moderate, intense callusing respectively.
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Table 64. Effect of sodium alginate concentration on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture on MS medium Sodium alginate (% w/v) Conversion response (%) into plantlets 1.0 Fragile beads 2.0 Fragile beads 3.0 64.40 ± 1.91a (but soft to handle) 4.0 68.60 ± 1.86a 5.0 33.60 ± 1.56b Different concentrations of sodium alginate and 100 mM CaCl2·2H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
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Table 65. Effect of calcium chloride concentration on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture on MS medium Calcium chloride (mM) Conversion response (%) into plantlets 25 Fragile beads 50 Fragile beads 75 42.00 ± 0.94b(but soft to handle) 100 68.60 ± 1.86a 200 24.40 ± 1.28c Different concentrations of sodium alginate and 100 mM CaCl2·2H2O were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
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Table 66. Effect of different treatments on conversion of encapsulated nodal segments of D. hamiltonii after 6 weeks of culture Treatment (µM) Conversion response (%) into plantlets MS 68.60 ± 1.86b MS + BA (5.0) 70.60 ± 1.80ab MS + BA (5.0) + NAA (0.5) 71.80 ± 1.85ab MS + BA (5.0) + IAA (0.5) 74.20 ± 1.90ab MS + BA (5.0) + NAA (0.5) + ADS (30.0) 74.80 ± 1.77ab MS + BA (5.0) + IAA (0.5) + ADS (30.0) 77.00 ± 2.09a Different concentrations of CaCl2·2H2O and 4% (w/v) sodium alginate were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
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Table 67. Effect of different NAA concentrations supplemented to half-strength MS basal medium on in vitro root induction in microshoots recovered through synseeds of D. hamiltonii after 4 weeks of incubation NAA (µM) % Response Mean no. of roots/shoot Mean root length (cm) 1.0 82.40 ± 1.86b 2.20 ± 0.20b 4.90 ± 0.11a 2.5 90.20 ± 1.80a 4.60 ± 0.50a 5.26 ± 0.08a 5.0 79.20 ± 1.01b 1.60 ± 0.24c 4.00 ± 0.17b
Different concentrations of CaCl2·2H2O and 4% (w/v) sodium alginate were added to MS medium. Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
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Table 68. Effect of different storage durations on conversion of encapsulated and non-encapsulated nodal segments of D. hamiltonii into plantlets Storage Conversion of Conversion of Conversion of duration encapsulated nodal encapsulated nodal non-encapsulated (weeks) segments into plantlets segments into plantlets nodal segments (%) (encapsulation (%) (encapsulation into plantlets (%) matrix prepared in MS matrix prepared in basal medium) distilled water)
0 77.00 ± 2.09a 16.00 ± 1.37a 99.60 ± 0.40a
1 73.20 ± 2.51a 00.00 ± 0.00b 34.60 ± 2.03b
2 63.80 ± 1.35b 00.00 ± 0.00b 14.40 ± 1.69c
4 47.00 ± 1.54c 00.00 ± 0.00b 00.00 ± 0.00d
6 25.00 ± 1.84d 00.00 ± 0.00b 00.00 ± 0.00d
8 14.00 ± 1.37e 00.00 ± 0.00b 00.00 ± 0.00d
Data represents Mean ± SE of 20 replicates per treatment in three repeated experiments. Mean value followed by the same alphabets are not significantly different according to Tukey’s Test at 5% probability.
68
Figure 69
A B
Explanation of Figure 69 Seed germination in D. hamiltonii
A. Seeds of D. hamiltonii B. Aseptic seedlings grown on full-strength MS medium supplemented with 2.5 µM GA, 2 week-old culture
69
Figure 70
A B C D
Explanation of Figure 70 Shoot regeneration through nodal segments
A, B. Nodal segment cultured on MS medium supplemented with 1.0 µM BA, 2 & 4 week-old culture A. Nodal segment cultured on MS medium supplemented with 2.5 µM BA, 4 week-old culture B. Nodal segment cultured on MS medium supplemented with 5.0 µM BA, 4 week-old culture
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Figure 71
A B C
Explanation of Figure 71 Shoot regeneration through nodal segments
A. Shoot regeneration on 5.0 µM BA and 0.5 µM IAA supplemented MS medium, 3 week-old culture B. Shoot regeneration on 5.0 µM BA and 0.5 µM IBA supplemented MS medium, 3 week-old culture C. Poor shoot regeneration on 5.0 µM BA and 1.0 µM IAA supplemented MS medium, 4 week-old culture
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Figure 72
Explanation of Figure 72 Shoot regeneration through nodal segments
A. Culture showing healthy shoot regeneration (with broad leaves) through nodal segments on 5.0 µM BA + 0.5 µM IAA + 5.0 µM BA + 30.0 µM ADS, 3 week-old culture
B. Shoot regeneration through nodal segments on 5.0 µM BA + 0.5 µM IAA + 5.0 µM BA + 30.0 µM PG, 3 week- old culture
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14 8 Mean shoot no./explant Mean shoot length (cm) 12 a
6 10 b a
c 8 4 d b 6
bc Mean shoot no./explant shoot Mean 4 c lengthshoot Mean (cm) 2
2
e d 0 0 5.0 5.4 5.8 6.2 6.6
pH of the culture medium
Figure 73. Effect of different pH of the culture medium supplemented with BA (5.0 µM) + IAA (0.5 µM) + ADS (30 µM) on shoot regeneration through nodal segments of D. hamiltonii after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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8 14 Mean shoot no./explant Mean shoot length (cm) a 12
6 10 b a
8 bc c 4 6 Mean shoot no./explant shoot Mean 4 length shoot Mean (cm) c
2 2
0 Sucrose Fructose Glucose
Carbon source
Figure 74. Effect of different carbon sources on shoot regeneration through nodal segments of D. hamiltonii after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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8 14 Mean shoot no./explant a Mean shoot length (cm) 12 6 b 10 a
c 8 b 4
6 b Mean shoot no./explant shoot Mean Mean shoot lengthshoot Mean (cm) 4 2
2
0 0 MS WPM B5 Culture medium
Figure 75. Effect of different culture media on shoot regeneration through nodal segments of D. hamiltonii supplemented with BA (5.0 µM) + IAA (0.5 µM) + ADS (30 µM) after 4 weeks of culture. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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Figure 76
A B C
D E F
Explanation of Figure 76 Shoot regeneration through shoot tip explants
A. Shoot regeneration on 5.0 µM BA containing MS medium, 3 week-old culture B. Shoot regeneration on 5.0 µM BA and 1.0 µM IAA containing MS medium, 4 week-old culture C. Culture showing poor shoot regeneration due to intense callusing on 5.0 µM BA and 1.0 IBA containing MS medium, 3 week-old culture D. Poor growth of regenerated shoot on 5.0 µM BA + 0.1 µM IBA containing MS medium, 3 week-old culture E. Shoot tip cultured on 5.0 µM BA + 0.5 µM IAA + 30.0 µM GA containing MS medium, 1 week-old culture F. Culture showing healthy shoot regeneration 5.0 µM BA + 1.0 µM IAA + 30.0 µM ADS containing MS medium, 4 week-old culture
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Figure 77
A B C
Explanation of Figure 77 Regeneration of basal nodular tissue through shoot tip explants
A. Nodular tissue regenerated from basal cut end of the shoot tip on 5.0 µM BA containing MS medium, 3 week- old culture B. Enlarged view of the nodular tissue of the above culture C. Nodular tissue regenerated from basal cut end of the shoot tip on 5.0 µM BA and 0.5 µM IAA containing MS medium, 3 week-old culture
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Figure 78
A
B C D
Explanation of Figure 78 Shoot regeneration through nodular tissue
A. Shoot regeneration form nodular tissue induced on BA (5.0 µM) + IAA (0.5 µM) when cultured on 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS, 2 week-old culture B. Shoot regeneration form nodular tissue induced on BA (5.0 µM) when cultured on 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS, 2 week-old culture C. High frequency shoot regeneration form nodular tissue induced on BA (5.0 µM) + IAA (0.5 µM) when cultured on 5.0 µM BA, 0.5 µM IAA, 1.0 µM GA and 30.0 µM ADS, 3 week-old culture D. Shoot regeneration form nodular tissue induced on BA (5.0 µM) when cultured on 5.0 µM BA, 0.5 µM IAA, 1.0 µM GA and 30.0 µM ADS, 3 week-old culture
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Figure 79
A B
C D
Explanation of Figure 79 Leaf culture
A. Leaf emerged from petiolar cut end of the explant along with intense callusing on 5.0 µM BA, 4 week-old culture B. Shoot regeneration from leaf explant along with intense callusing on 5.0 µM BA and 0.5 µM IAA, 4 week-old culture C. Leaf-derived callus on 5.0 µM BA and 1.0 µM IBA, 4 week-old culture D. Leaf cultured on TDZ (0.5 µM) showing enlargement in size, 4 week-old culture
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Nodal segments Shoot tip explants Nodal segments Shoot tip explants
20 8 a a a a a ab bc b a a a 7 15 a a a a a a ab a b ab bc b 6 10 c
5 Mean shoot length (cm) length shoot Mean Mean no. of shoots/explant of no. Mean 5
4
0 1 2 3 4 5 6 Sub-culture passage
Figure 80. Effect of sub-culturing on shoot proliferation through nodal and shoot tip explants of D. hamiltonii. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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N1 tissue
N2 tissue
N1 tissue
N2 tissue 40 a a ab b 6 c d a 30 ab 5 c bc cd d a a a a 4 a b a a 20 b ab 3 b c 2 Mean shoot length (cm) length shoot Mean
Mean no. of shoots/explant of no. Mean 10
1
0 0 1 2 3 4 5 6 Sub-culture passage
Figure 81. Effect of sub-culturing on shoot proliferation through shoot tip derived N1 and N2 tissues of D. hamiltonii. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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Figure 82
A C
B D
Explanation of Figure 82 In vitro root induction
A. In vitro root induction on half-strength MS medium supplemented with 2.5 µM NAA, 3 week-old culture B. Enlarged view of above culture C. In vitro root induction on half-strength MS medium supplemented with 5.0 µM NAA, 3 week-old culture D. Enlarged view of above culture
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Figure 83
A B
Explanation of Figure 83 Acclimatization of micropropagated plantlet
A. Plantlets with shoot and root systems B. An acclimatized plantlet in soilrite, 4 week-old
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100 Soilrite Vermi-compost Garden soil + farmyard 80
60 % Survival % 40
20
0 Soilrite Vermi-compost Garden soil + farmyard (2:1)
Types of planting substrate
Figure 84. Effect of different planting substrates on survival percentage of regenerated plantlets after 2 months of field transfer. The bars represent the results of 300 plantlets for each substrate.
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Figure 85
A B
C
D E
Explanation of Figure 85 Synseed production and their germination
A. Encapsulated nodal segments placed on MS basal medium, 2 day-old culture B. Shoot bud sprouting on MS basal medium, 1 week-old culture C & D. Synseed germination on MS medium supplemented with 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS, 2 and 4 week-old culture E. Root induction in synseed derived shoot on half-strength MS medium supplemented MS medium, 4 week-old culture
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Chl a (mg g-1) Chl b (mg g-1) Total Chl (mg g-1) 1.6 a
ab a ) 1.4 b -1 b b 1.2 c
1.0 c c c 0.8 a
Chlorophyll content (mg g (mg content Chlorophyll 0.6 b bc c 0.4 d
0.2 0 7 14 21 28 Acclimatization period (days)
Figure 86. Change in chlorophyll content (a, b & total) (mg g-1) during acclimatization. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test
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Carotenoids (mg g-1) 0.55
a 0.50 ) -1 0.45
b 0.40
0.35 bc
d 0.30 Carotenoids content (mg g (mg content Carotenoids
0.25 cd
0.20 0 7 14 21 28 Acclimatization period (days)
Figure 87. Change in carotenoids content (mg g-1) during acclimatization. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
87
-2 -1 Net Photosynthetic rate (PN) (µmol CO2 m s ) 6.0
) a
-1 5.5 s
-2 ab 5.0 b 4.5 ) (µmol CO2 m N 4.0
d 3.5
3.0
c 2.5 Net Photosynthetic rate (P rate Photosynthetic Net
2.0 0 7 14 21 28 Acclimatization period (days) -2 -1 Figure 88. Change in Net Photosynthetic rate (µmol CO2 m s ) during acclimatization. The bars represent mean ± S.E. Bars denoted by the same letter within response variables are not significantly different (P=5%) using Tukey’s Test.
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Figure 89
MS
A B
LP
SB
LP
C D
Explanation of Figure 89 Histological study of regenerating tissues
A. Section of shoot tip derived basal nodular tissue of D. hamiltonii showing undifferentiated parenchymatous cells revealing the callogenic nature of nodular tissue. B. Callus cells showing distinct meristematic regions (arrows) referred as meristemoids (MS) with cluster of actively dividing cells C. Initiation of shoot bud from meristemoid in the form of bulging (arrow) D. Development of well differentiated shoot bud (SB) from the meristemoid with a pair of leaf primordial (LP)
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Chapter 5 Discussion
Chapter 5
DISCUSSION
Organogenesis is a developmental process that is in some ways unique to plants. Animal cells follow developmental paths that normally involve irreversible differentiation towards a specific cell or tissue type. Plant cells, however, may retain the ability to dedifferentiate from their current structural and functional state and to begin a new developmental path towards a number of other morphogenic end points. At first sight, it is not clear whether there are stem cells in plants. During vegetative propagation, a new plant can arise from a definite cell that terminated its growth or several cells, from one or several tissues. Hence, all the plant cells are totipotent and an entire plant can be formed from a single cell. This process is regulated by a system of cell interactions through the synthesis and transport of phytohormons and other compounds. The uppermost cells of the root and shoot apical meristem are considered as stem cells. They are similar in many features to the stem cells of animals. But unlike animals, the stem cells can repeatedly arise in plants during morphogenesis and regeneration or in tissue culture from actively diving or differentiated cells (Ivanov 2003). Plant tissues in vitro may produce many types of primordial those will eventually differentiate into embryos, flowers, leaves, shoots and roots. These primordials originate de novo from a cellular dedifferentiation process followed by initiation of a series of events that results in their formation. The cell or cells thought to be the direct progenitors are somehow stimulated to undergo a number of rapid cell division leading to the formation of a meristemoid (meristem-like cells). Early in their development, meristemoids are thought to be morphogenically plastic and capable of developing into a number of different primordials. This unique developmental flexibility has been widely used by plant propagators. Two organogenic events comprise a common approach to in vitro plant propagation. The first event is to regenerate multiple shoot meristems followed by their growth and development into microshoots of a suitable size for the second event, which is the induction of de novo root meristem production. It is believed that under in vitro culture conditions, these organogenic events can be the result of two differing ontogenic pathways.
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According to Hicks (1980), two developmental sequences lead to organogenesis. They differ in the presence or absence of callus in the organogenic events. A developmental sequence involving an intervening callus stage is termed as “indirect” organogenesis. While, “direct” organogenesis is accomplished without an intervening proliferating callus. Direct shoot regeneration is preferred since it reduces the possibility of somaclonal variations common in plants regenerated from cultured cells or tissues (Misra and Datta 2001, Liu and Bao 2003, Dayal et al. 2003, Mujib 2005, Sharma et al. 2009).
PRIMARY ORGAN Direct MERISTEMOID EXPLANTS FORMATION organogenesis
CALLUS Indirect organogenesis
The process of organogenesis includes three main stages. Firstly, the cells of the explants have to acquire the ability to respond to hormonal signals. Secondly, competent cells are determined for specific organ formation under the influence of exogenous hormonal signals. And lastly, morphogenesis is completed independently of the exogenously supplied growth regulators. Thus, the process of organogenesis has been broken into following phases (adapted from Christianson and Warnick 1985);
COMPETANCE DETERMINATION
+ + EXPLANT ORGAN
1 2 3
DEDIFFERENTIATION INDUCTION DIFFERENTIATION
Initial two phases are the keys those precede the differentiation phase. These phases encompass events which begin with dedifferentiation and result in the attainment of “competence” followed by induction which culminates in the fully “determined” state. The morphological differentiation and development of nascent shoot or root then proceed, eventually resulting in a functional organ. 117
In recent years, advances in plant genetic engineering have opened new avenues for crop improvement and various plants with novel agronomic traits have been produced. The lack of efficient regeneration protocols in some important threatened, endangered or rare valuable plants is a major impediment for their improvement via genetic engineering; therefore, it is important to continue research towards understanding the factors involving in in vitro plant regeneration. In the present investigation, three medicinally important threatened/endangered plants viz. Spilanthes acmella, Spilanthes mauritiana and Decalepis hamiltonii have been assessed in order to achieve rapid and reproducible in vitro regeneration system. The results obtained during the present study have been discussed in the light of existing literature. To make this chapter easy to understand, discussion has been categorized under the following sub-heads:
5.1 Seedling establishment
Considering the advantages of seedling explants for transformation and micropropagation studies due to easy planning of experiments, reduction in contamination during tissue culture, reduction in labour and maintenance costs, in the present study aseptic seedlings used as the explant source (nodal segments, shoot tips, leaves and cotyledons). During the present investigation, seed germination was performed under in vivo and in vitro culture conditions but results indicate that in vitro culture conditions germination was more effective than in vivo germination even after the pre-soaking of seeds in GA. In S. acmella and S. mauritiana, the highest germination (94.00 ± 2.44 and 86.00 ± 1.87% respectively) was observed with 1.0 µM GA supplemented half- strength MS medium. On 1.0 µM GA, seeds showed emergence of radicals within 4.20 ± 0.37 and 5.80 ± 0.58 days of incubation in S. acmella and S. mauritiana respectively. However, significant reduction in seed germination was noticed when nutrient strength was increased to full (full-strength MS medium). The results were in consonance with that of Kishore et al. (2006) & Pandey and Agarwal (2009) who also succeeded in the seed germination of Ascocenda species and S. acmella on half- strength MS basal medium. In contrast to Spilanthes species, full-strength MS medium supplemented with 2.5 µM GA yielded optimum seed germination (83.20 ± 1.82%) in D. hamiltonii.
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There is a circumstantial evidence that ABA is involved in regulating the onset of seed dormancy and maintaining dormant state. ABA accumulation in developed seeds is low during early stages and is greatest during mid-development, when reserves are being synthesized and decline as the seed undergoes maturation/drying (Berry and Bewley 1992). In general, GA is known to obviate the requirement of seed for various environmental clues as it promotes germination and counteracts the inhibitory effect of ABA (Bewley and Black 1994). The promotive role of GA on breaking of seed dormancy and germination has been studied time to time (Saba et al. 1997, Bose and Sharma 2000, Husain et al. 2007b, Joseph et al. 2011).
5.2 Direct organogenesis through shoot tip explants and nodal segments
For successful micropropagation nodal segments and shoot tips are preferred as pre-existing meristem easily develops into shoots. Nodal segments containing axillary buds have quiescent or active meristems depending upon the physiological stage of the plant. In nature these buds remain dormant for a specific period depending on the growth pattern of the plant. However, using tissue culture, the rate of shoot multiplication can be greatly enhanced by performing axillary bud culture in a nutrient medium containing suitable PGR. In the present study, nodal segments of all the plant species i.e., S. acmella, S. mauritiana and D. hamiltonii failed to induce multiple shoot buds on PGR-free basal MS medium (control) therefore, it was mandatory to augment the culture medium with cytokinin alone or in combination with auxin to induce multiple shoot buds. Cytokinins were reported to play a key role in DNA synthesis and cell division, which might be the reason for induction of multiple shoots (Khan et al. 2011). In S. acmella and S. mauritiana, only BA and Kn induce shoot formation from nodal segments. Maximum of 2 shoots per nodal segment were noticed in 89.00 ± 0.70 and 99.60 ± 0.40% cultures of S. acmella and S. mauritiana respectively when cultured on 1.0 µM BA supplemented MS medium. While, 2-iP at all the tried concentrations proved to be ineffective. As far as the nodal segments of D. hamiltonii were concerned, all adenine-based cytokinins (BA, Kn and 2-iP) were capable of shoot induction. However, BA was found to be significantly most effective than other cytokinins. Comparatively higher concentration of BA i.e., 5.0 µM exhibited critical response as
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induced a maximum of 4.20 ± 0.37 shoots per explant in 84.20 ± 1.31% cultures after 4 weeks of inoculation. Further increasing or decreasing BA concentration beyond the critical level, percent response and mean number of shoots per explant decreased. Similar observations with different cytokinins have been reported in other plant species of Asclepiadaceae such as Celastrus paniculatus (Nair and Seeni (2001), Holostemma ada-kodien (Martin 2002), Tylophora indica (Faisal et al. 2007) and Ceropegia spiralis (Murthy et al. 2010). Generally, a cytokinin is required for in vitro axillary shoot induction and proliferation. However, optimal cytokinin concentration varies with the plant species (Park et al. 2008). After optimizing the cytokinin concentration, various auxins (IAA, IBA and NAA) were examined with the optimized cytokinin to maximize the shoot regeneration though nodal segments in all the plant species. Although in Spilanthes species none of the cytokinin-auxin combinations effectively enhanced the regeneration efficiency of nodal segment. Only, in D. hamiltonii both the regeneration frequency and mean number of shoots per explant enhanced significantly as compared to the cytokinin alone. The combination of 5.0 µM BA and 0.5 µM IAA was found to be the best combination as it maximized the production to 5.80 ± 0.37 shoots per explant with 5.48 ± 0.13 cm shoot length in 94.00 ± 1.87% cultures. The synergistic effect of auxin in combination with an optimal cytokinin has been demonstrated in medicinal plants of Asclepiadaceae, namely Hemidesmus indicus (Sreekumar et al. 2000), Holostemma ada-kodien (Martin 2002), Tylophora indica (Faisal et al. 2007) and Sida cordifolia (Sivanesan and Jeong 2007). These results indicate that cytokinins could ensure in vitro regeneration and synergism of cytokinin-auxin combination was extremely favourable for their multiplication. The morphogenetic potential of shoot tip explants was compared with nodal segments on similar treatments of PGRs. The shoot tip explants of Spilanthes species were found to be more responsive as compared to the nodal segments. This is in agreement with the report of Sujatha and Ranjitha Kumari (2007) who documented high frequency of shoot regeneration from shoot tip explants than nodal segments in Artemisia vulgaris. Shoot tip exert strong apical dominance in woody plant species which inhibited bud sprouting and shoot multiplication even in the presence of BA (George and Sherrington 1984, Marks and Simpsons 1994, Lakshmanan et al. 1997). It is widely recognised that apical dominance is caused by the action of basipetally transported auxin from the apex that consequently inhibiting axillary bud growth 120
(Cline 1994). Contrary to this, the shoot tip explants of D. hamiltonii were lesser effective for multiple shoot regeneration when compared to its nodal segments. This differential response of nodal and shoot tip explants has been attributed to the differences between the physiological status of shoot buds on different regions of a stem (Vieitez et al. 1985). Results indicate that among various cytokinins tested, 1.0 µM BA was optimal with 66.0 ± 2.44 and 59.80 ± 1.56% response in from shoot tip explants of S. acmella and S. mauritiana respectively. In S. acmella, a maximum of 8.0 ± 0.31 shoots per shoot tip and 3.9 ± 0.30 cm shoot length was induced on 1.0 µM BA after 4 weeks of culture. On similar treatment of BA (1.0 µM), a maximum of 6.00 ± 0.44 shoots per shoot tip and 2.36 ± 0.14 cm shoot length was achieved after similar 4 week of incubation in S. mauritiana. Shoot tip explants of D. hamiltonii required comparatively higher concentration of BA (5.0 µM) for inducing a maximum of 3.80 ± 0.37 shoots per explant and 5.34 ± 0.10 cm shoot length in 92.00 ± 1.22% cultures. The superiority of BA over other cytokinins with respect to multiple shoot bud induction through shoot tip explants has also been documented for several medicinal and aromatic plant species such as Ocimum basilicum (Begum et al. 2002), Picrorhiza kurroa (Chandra et al. 2004), Coleus blumei (Rani et al. 2006) and Stevia rebaudiana (Debnath 2008, Sharma and Shahzad 2011). The promotory effect of BA over other cytokinins could be due to its easy permeability, increased affinity for active cell uptake, less resistance to the cytokinin oxidase or receptor abundance in its perception apparatus which interacts with the coupling elements in the signal transduction chain. Other reasons for superiority of BA may be attributed to the ability of plant tissues to metabolize BA more readily than other synthetic PGRs or to the ability of BA to induce production of natural hormones such as zeatin within the tissue (Malik et al. 2005). However, the exact mode of action and the reason for the variability in its performance still remain elusive (Dal Cin et al. 2007). Similar to the nodal segments, to examine the synergistic effect of cytokinin- auxin combination on shoot regeneration, three auxins (NAA, IBA and IAA) in addition to the optimized concentration of cytokinin (1.0 and 5.0 µM BA) were tested with the shoot tip explants. Among the combinations tested, 1.0 µM BA and 0.1 µM NAA induced the highest number of 33.0 ± 1.09 shoots per explant and 5.2 ± 0.09 cm shoot length in 96.0 ± 2.44% shoot tip cultures of S. acmella. The results are in accordance with earlier findings of several workers, where the addition of low level of 121
NAA with BA promoted shoot proliferation from shoot tip explants as reported in Santolina canescens (Casado et al. 2002) and Gynura procumbens (Keng et al. 2009). In contrast to this, Haw and Keng (2003) reported multiple shoot formation (2.6 shoots per explant) from axillary bud in S. acmella on BA (6.0 mg l-1) supplemented medium while addition of auxin did not show any improvement in shoot proliferation. This intricacy might be because of explants origin as Haw and Keng (2003) used the explants from in vivo grown plants of S. acmella. During the investigation, it was found that BA and IAA combination was significantly the most effective in S. mauritiana and D. hamiltonii. The MS medium amended with 1.0 µM BA and 0.5 µM IAA enhanced all the evaluated parameters i.e., regeneration frequency (98.0 ± 2.00%), mean number of shoots per explant (18.80 ± 0.48 cm) and mean shoot length (4.26 ± 0.10) in S. mauritiana. However, in D. hamiltonii optimum response (3.80 ± 0.37 shoots in 92.00 ± 1.22% cultures) was found at 5.0 µM BA with 0.5 µM IAA. Contrary to the present findings, Giridhar et al. (2003) reported a maximum number of shoots (6.4 ± 0.8) from single node containing shoot tip explant on 31.08 µM BA and 14.68 µM phenyl acetic acid (PAA). Thereafter, they (2005) achieved almost similar number of shoots (6.5 ± 0.4)) from shoot tip explants on 4.92 µM 2-iP supplemented MS medium after 8 weeks of culture. They also reported that BA alone or in combination with IAA (0.6 µM) resulted in profuse callusing from the basal cut end of explants. However, Reddy (2002) documented 20 µg l-1 triacontanol (TRIA) for best axillary shoot proliferation in similar plant species. Noteworthy response in the present study was that the cytokinin-auxin combination improved shoot proliferation from both the nodal segments and shoot tip explants in D. hamiltonii, but shoots exhibited rudimentary leaf development and premature abscission; therefore to prevent premature leaf fall and to increase leaf area without degradation in rate of shoot proliferation, the addition of different adjuvants like ADS, Glu and PG to an optimal combination of BA (5.0 µM) and IAA (0.5 µM) proved to be of significant. The data reveals that 30.0 µM ADS with 5.0 µM BA and 0.5 µM IAA showed best response over Glu and PG. Addition of ADS not only prevented an early leaf fall within 1 week of incubation but also improved the overall shoot growth with continuous increase in shoot number and maximum 8.80 ± 0.37 and 8.20 ± 0.37 shoots per explant with the highest shoot length of 6.46 ± 0.11 and
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6.54 ± 0.08 cm were achieved from nodal segment and shoot tip explant respectively, after 4 weeks of culture. Moreover, ADS at this level increased the stem girth, thus making the shoots healthiest among all the additives. Adenine in the form of ADS can stimulate cell growth and greatly enhance shoot formation (Murashige 1974). It provides an available source of nitrogen to the cells, which can generally be taken up more rapidly than the inorganic nitrogen as suggested by Husain et al. (2008) for Pterocarpus marsupium and achieved highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) from nodal explants on MS medium amended with 4.0 µM BA, 0.5 µM IAA and 20 µM ADS. In Melia azedarach, Husain and Anis (2008) reported best regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) from nodal explants on MS medium amended with 5.0 µM BA, 0.5 µM -1 -1 IAA and 30 µM ADS while addition of 250 mg l (NH4)2SO4, and 100 mg l
K2SO4, prevented defoliation and tip burning without affecting the number of shoots. Reinforce effect of ADS on shoot bud proliferation and quality of shoots has been reported time to time in different woody species like, Tectona grandis (Devi et al. 1994), Bauhinia vahlii (Dhar and Upreti 1999), Acacia catechu (Kaur and Kanta 2000) and Jatropha curcas (Datta et al. 2007). Besides N6-substituted adenine derivatives (BA, Kn and 2-iP), various diphenylurea type cytokinins have also been employed for in vitro morphogenetic response in various plant species. Among different diphenylurea type cytokinins, TDZ has been proved to be the most potent bioregulant for micropropagation studies. There is growing evidence that TDZ may be involved in increasing the biosynthesis or accumulation of endogenous cytokinin (Murthy et al. 1995). Therefore, the goal of this segment of the current study was to determine the role of TDZ as cytokinin for morphogenesis through shoot tips and nodal segments in all the three plant species. Nodal segments of all the three plant species and shoot tip explants of only D. hamiltonii failed to exert their morphogenic response on TDZ supplemented MS medium. They remained green up to 3 weeks, thereafter tissue turned brown and died. Inhibitory role of TDZ on multiple shoot formation through shoot tip explants of D. hamiltonii has already been reported by Giridhar et al. (2005). On the other hand, shoot tip explants of S. acmella and S. mauritiana were more responsive on TDZ supplemented medium than adenine-based cytokinins (BA, Kn and 2-iP) containing medium. Observations clearly reveal the difference in mode of regeneration and 123
indicate that TDZ induced de novo multiple shoot formation from basal cut end of the shoot tip explants rather than through multiplication of apical shoot buds as observed on adenine-based cytokinins. TDZ was effective only at low concentration (0.25 µM) than adenine-based cytokinins (1.0 µM BA). Higher concentrations (0.5-5.0 µM) greatly reduced the regeneration efficiency. On 0.25 µM TDZ maximum of 30.00 ± 0.30 and 26.20 ± 1.35 shoots per shoot tip explant were recorded in S. acmella and S. mauritiana respectively. Similar response with the low concentration of TDZ has been reported by Huetteman and Preece (1993), Lu (1993), Banerjee et al. (2004), Khurana et al. (2005), Ahmad et al. (2006a & b), Ahmad and Anis (2007), Sunagawa et al. (2007), He et al. (2007) and Shirani et al. (2009). The possible reason for the higher activity of individual TDZ treatment might be its high stability due to its resistance to cytokinin oxidase and suggesting its substitutive activity for both auxin and cytokinin required for shoot formation as already reported by Visser et al. (1992) and Huetteman and Preece (1993). Although TDZ promoted higher frequency shoot regeneration in both Spilanthes species but shoots were rosette like and failed to elongate. The reason for poor internodal elongation with TDZ might be due to an apical dominance release that accelerates axillary bud formation and adventitious shoot production. TDZ has been reported to inhibit shoot elongation on prolonged exposure (Huetteman and Preece 1993, Faisal et al. 2005a & b, Khurana et al. 2005, Ahmad and Anis 2007). Thus, TDZ derived shoots were transferred to adenine based cytokinin (BA) and auxin (NAA or IAA) combinations to improve shoot growth. Ahmed and Anis (2007) also suggested the use of BA and NAA combination to improve the shoot growth of TDZ derived cultures of Vitex negundo. However, in the present study, on cytokinin-auxin combinations shoot regeneration efficiency was enhanced but shoot elongation did not improve effectively. Cultures showing glassy and brittle shoot due to hyperhydricity. This deleterious effect might be due to the enhanced endogenous level of cytokinins by suppressing the activity of cytokinin oxidase by TDZ. This was also confirmed by Murthy et al. (1995) and Hutchinson et al. (1996). TDZ has a stronger effect on hyperhydricity in Mesembryanthemum crystallinum (Sunagawa et al. 2007). Therefore, PGR-free nutrient medium was tried for healthy shoot proliferation and elongation from TDZ derived tissues. Hyperhydricity was completely eliminated on full and half-strength basal MS media. On full-strength MS basal medium, highest number of 63.40 ± 1.10 and 64.00 ± 1.37 shoots per explants with 5.30 ± 0.10 and 124
5.36 ± 0.13 cm shoot length were noticed in S. acmella and S. mauritiana respectively. In Ocimum basilicum, Siddique and Anis (2008) also noticed fasciated and distorted shoots on TDZ containing MS medium and suggested repeated sub- culturing of TDZ regenerating tissues on PGR-free MS medium for further multiplication and elongation of shoots Plant cells and tissues require an optimum hydrogen ion concentration (pH) for an effective growth and development in cultures. The pH affects nutrient uptake as well as enzymatic and hormonal activities in plants (Bhatia and Ashwath 2005). The optimal pH level regulates the cytoplasmic activity that affects cell division and the growth of shoots and it does not interrupt the function of cell membrane and the buffered pH of cytoplasm (Brown et al. 1979). The pH also influences the solidification of the culture medium; a pH higher than 6 produces a very hard medium and a pH lower than 5 does not solidified the medium satisfactorily (Bhatia and Ashwath 2005). Therefore, it is necessary to optimize the pH level for maximum shoot regeneration because pH level directly influence shoot regeneration. In the plant species, among various pH levels, maximum response was achieved at 5.8 pH. Results are in agreement with many reports where optimum regeneration was achieved at 5.8 pH (Faisal et al. 2007, Shahzad et al. 2011, Sahai et al. 2010). In contrast to this, some plant species require acidic pH for maximum shoot regeneration as reported by Bhatia and Ashwath (2005) and Naik et al. (2010). Plant Cells and tissues in a culture medium lack autotrophic ability and therefore, need external carbon for energy (Razdan 1993). The addition of an external carbon source to the medium enhances the proliferation of cells and regeneration of green shoots (Nowak et al. 2004, Gürel and Gülşen 1998). Many authors have examined the effect of different carbon sources on the morphogenic reaction and in vitro tissue growth of different plant species (Pati et al. 2006, Sivanesan and Jeong 2007, Dobránszki and da Silva 2010, Mohamed and Alsadon 2010, Abou-Rayya et al. 2010 and Siridhar and Naidu 2011). During the present investigation, sucrose showed best response in all the plant species as compared to fructose and glucose. Similarly, in most of the plant species sucrose containing medium exhibited maximum shoot regeneration (Fuentes et al. 2005, Sivanesan and Jeong 2007 and Sujatha and Ranjitha Kumari 2008). Optimum regeneration on sucrose than other carbon sources might be due to efficient uptake across the plasma membrane (Borkowska and Szezebra 1991).
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Moreover, the success in cell, tissue and organ culture depend up on the selection or development of the culture medium according to the metabolic needs of the cultured cells and tissue. A sub-optimal culture medium may cause physiological disorder or death of tissue (Nas and Read 2000). Chemical composition of the culture medium has been reported to affect all types of morphogenic responses, including caulogenesis (Morard and Henry 1998), axillary bud proliferation (Schnapp and Preece 1986), plant regeneration (Shahrawat et al. 1999) and embryogenesis (Gyulai et al. 1992). According to a recent report of Al-Khayri (2011), basal salt requirements differ according to culture stage and cultivar type. Therefore, it was mandatory to optimize the best culture medium composition for in vitro response. In the present study, MS medium revealed maximum regeneration in all the three plant species which was in accordance to the reports of Komalavalli and Rao (2000), Faisal et al. (2007), Husain et al. (2008) and Husain and Anis (2009).
5.3 Direct organogenesis through leaves and cotyledons
The process of organogenesis provides the basis for asexual plant propagation largely from non-meristematic somatic tissues. Moreover, direct de novo or adventitious shoot regeneration is most reliable approach for transgenic experiments. Therefore, in the present investigation young leaves and cotyledons were selected to induce de novo organogenesis. It has been found that the leaf explants cultured on control treatment only enlarged in size, but did not give any sign for organogenesis and indicated the requirement of PGR supply to the explants for adventitious or de novo organogenesis. Even after the augmentation of PGRs to the culture medium, leaf explants of D. hamiltonii was found almost non-responsive in terms of shoot formation. However, leaf explants of S. acmella and S. mauritiana induced multiple shoot buds only at their proximal cut ends adjacent to petiole, but distal ends including leaf margin and leaf surface did not exhibit any sign of differentiation. High frequency of shoot regeneration from proximal end as compared to distal end of the leaf explants has also been emphasized in Ocimum basilicum (Phipeen and Simon 2000), Beta vulgaris (Zhang et al. 2001), Tagetes erecta (Misra and Datta 2001), Anthurium andraeanum (Martin et al. 2003), Euphorbia nivulia (Martin et al. 2005), Ophiorrhiza prostrata (Beegum et al. 2007), Spilanthes mauritiana (Sharma et al. 2009b) and Lysimachia
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species (Zheng et al. 2009). The regenerative capacity at the base of petiole, however, could be ascribed to basipetal transport of endogenous auxins and/or carbohydrates (Dubois and de Vries 1995) or position of the regenerative target cells (Margara 1982). To clarify the fact for high regeneration potential of proximal end in comparison to distal end, Welander (1998) reported that there is a physiological gradient in the leaf explant from proximal to distal end for de novo regeneration due to difference in the maturity. Leaves attain maturity first at tip (distal end) and subsequently in a basipetal progression. The results reveal that among three adenine-based cytokinins (BA, Kn and 2- iP), 2.5 µM BA was the best cytokinin for direct organogenesis from leaf explants followed by Kn and 2-iP. In S. acmella, 2.5 µM BA induced a maximum of 6.20 ± 0.48 shoots per explant and 2.82 ± 0.10 cm shoot length in 85.00 ± 1.84% cultures after 4 weeks of incubation. However, a maximum of 5.60 ± 0.40 shoot per explant and 3.44 ± 0.16 cm shoot length with 87.00 ± 2.00% response frequency was noticed in S. mauritiana leaf cultures on similar concentration of BA i.e., 2.5 µM. On exceeding the concentration of BA beyond the optimal level, callogenesis was increased that negatively affected caulogenic response. The stimulating effect of BA on multiple shoot formation from leaf explants has been reported in various medicinal plants including Sansevieria cylindrica (Anis and Shahzad 2005), Ophiorrhiza prostrata (Beegum et al. 2007), Chicorium intybus (Yucesan et al. 2007), Spilanthes acmella (Saritha and Naidu 2008) and Coleus forskohlii (Sahai and Shahzad 2010). Cytokinin-auxin combinations further enhanced the caulogenic efficiency of leaf explants as compared to cytokinin alone. Among the combinations tested, 2.5 µM BA along with 0.5 µM NAA exhibited a synergetic effect on multiple shoot formation (27.80 ± 1.01) in S. acmella. The present investigation also corroborate with those of Kumar et al. (1998) in Pauwolnia fortunei, Echeverrigaray et al. (2000) in Chaememlum species, Azad et al. (2005) in Phellodendron species, Dhar and Joshi (2005) in Saussuraea species, Agrawal and Sardar (2006 and 2007) in Cassia angustifolia and Mohapatra et al. (2008) in Centella asiatica where the combination of NAA and BA proved optimum for shoot differentiation from leaf explants. This existence of synergistic and additive interactions of auxin and cytokinin involves a complex web of signal interactions such as increased sensitization, receptivity, feedback inhibition and modulation of gene expression resulting in variable translation of mRNA population (Cline 1991, Eklof et al. 1997, Schmulling et al. 127
1997, Armstrong et al. 2004, Rashotte et al. 2005, Woodword and Bartel 2005, Hirose et al. 2007). Now it is a well established fact that the two hormone definitely “cross-talk” at molecular level for evoking morphogenesis in callus tissue in vitro, however, no central regulator of this molecular cross-communication has been identified to date (Skoog and Miller 1957, Koroch et al. 2002, Coenen et al. 2003). Supporting the role of NAA over other auxins, Nordstrom et al. (2004) advocated that NAA has more affinity for easy penetration through plasma membrane even without an active uptake. On the other hand, 2.5 µM BA with 0.5 µM IAA was found to be critical in S. mauritiana that induced maximum of 15.00 ± 0.31 shoots per leaf explant. In S. acmella, direct regeneration from leaf explants has also been reported by Saritha and Naidu (2008). However, they recommended the composition of 3.0 mg dm-3 BA and 1.0 mg dm-3 IAA for maximum shoot regeneration (20 shoots per explant). This differential requirement of auxin in comparison to the present findings might be due to variation in genotype of similar plant species. When different concentrations of TDZ were evaluated for organogenesis, only the leaf explants of S. mauritiana induced direct shoot buds while in S. acmella leaf explants induced indirect organogenesis. In S. mauritiana, on 0.5 µM TDZ leaf explants induced a little callusing on their surface that ceased to grow further. After scarping the callus tissue, a maximum of 8.40 ± 0.50 shoots per explant directly induced from leaf explants on 0.5 µM TDZ. TDZ has also been successfully exploited for direct shoot regeneration from leaf explant in a number of plant species (Feyissa et al. 2005, Ma et al. 2011). However, shoots did not attain satisfactory shoot length on TDZ supplemented medium; therefore they were transferred to other BA containing medium with or without auxin. Of the various combinations tested, 1.0 µM BA in conjunction with 0.5 µM IAA proved to be the best shoot proliferation medium, giving a maximum of 12.40 ± 0.74 shoots per explant, but still the shoot length did not improve successfully (2.20 ± 0.07 cm). Moreover, further culturing on cyotkinin- auxin combination, shoot became hyperhydric might be due to continuous exposure of PRGs. The shoots were sub-cultured on PGR-free MS medium on which a maximum of 15.00 ± 0.31 shoots per explant with 2.62 ± 0.04 cm of shoot length were induced. Thus, results of the present findings indicate that an optimum exposure time of the explants in TDZ supplemented MS medium followed by the withdrawal of PGR effectively triggered shoot multiplication through leaf in S. mauritiana.
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The cotyledons cultured on control treatment enlarged in size, but did not give any sign for organogenesis. Similar to the leaf explants, cotyledons of Spilanthes species were found to be responsive but all the cultured cotyledons of D. hamiltonii did not induce any shoot fromation even after 4 weeks of culture. Among the cytokinins tested, only BA and Kn exhibited organogenesis from the cotyledons at their petiolar cut ends while 2-iP supplemented medium proved to be totally ineffective. The best response was obtained on 2.5 µM BA in both the species of Spilanthes as induced a maximum of 7.2 ± 0.37 and 4.80 ± 0.37 shoots per cotyledon in S. acmella and S. mauritiana respectively. This was in accordance with other studies in which shoots were obtained on BA containing nutrient medium from cultured cotyledons (Sul and Korban 2004, Joshi and Kothari 2007). Addition of 0.5 µM NAA (S. acmella) or 0.5 µM IAA (S. mauritiana) to the optimum cytokinin concentration (2.5 µM BA) revealed a significant synergism on regeneration efficiency of the cultured cotyledons. Maximum of 17.40 ± 0.87 and 17.80 ± 1.01 shoots were induced from cotyledons when cultured on 2.5 µM BA with 0.5 µM NAA and 2.5 µM BA with 0.5 µM IAA in S. acmella and S. mauritiana respectively after 4 weeks of culture. A series of experiments with cotyledons indicate that the requirements of cytokinin-auxin combination for high frequency shoot regeneration (Singh et al. 2002, Otroshy et al. 2011). Similar to the other explants, different concentrations of TDZ were also assessed with cotyledons in all the selected plant species, but TDZ failed to induce direct and indirect organogenesis through this explant.
5.4 Indirect organogenesis
The shoot tip explants of D. hamiltonii induced nodular organogenic nodular calli from basal cut end of the explant on 5.0 µM BA alone or in combination with 0.5 µM IAA simultaneously with the multiplication in apical bud. Between these two calli, callus obtained on 5.0 µM BA and 0.5 µM IAA was found to be highly competent as induced a maximum of 15.40 ± 0.67 shoots per culture with 4.56 ± 0.06 cm shoot length when transferred on 5.0 µM BA, 0.5 µM IAA, 1.0 µM GA and 30.0 µM ADS containing MS medium. However, in the absence of GA only 7.60 ± 0.50 shoots per culture were noticed on 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS. Thus, suggesting a significant role of GA for the induction and elongation of shoot buds
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from callus tissue. Kumar et al. (2007) and Misra and Chakrabarty (2009) also reported the significant role of GA on shoot elongation in Capsicum frutescens and Rosa clinophylla respectively. As far as the effect of urea derived cytokinin on indirect organogenesis is concerned, TDZ induced non-organogenic calli from leaf explants in D. hamiltonii. Only the leaf explants of S. acmella exhibited indirect organogenesis on TDZ supplemented MS medium. On 2.5 µM TDZ leaf explants produced greenish-white lucid calli from their surface from which few bud-like proturbances were noticed after 4 weeks of culture. While, higher concentration of TDZ led to non-organogenic callogenesis. Further growth of shoot buds (induced from callus tissue) was possible on TDZ and BA combinations. On 1.0 µM TDZ and 1.0 µM BA a maximum of 14.00 ± 0.54 shoots per culture with 1.20 ± 0.11 cm shoot length was induced. On further sub-culturing of these stunted shoots on similar combination of TDZ and BA, hyperhydricity was noticed in regenerated shoots. It was observed that all the TDZ derived shoots were greenish-white in colour and distorted. This deleterious effect of TDZ could be overcome by transferring the shoots on PGR-free MS basal medium on which a maximum of 20.40 ± 0.74 healthy shoots per explant with 3.54 ± 0.08 cm of shoot length were induced. Leaves were well expanded and greener than on TDZ and BA combination. TDZ mediated indirect organogenesis through leaf explants has also been reported in Ocimum basilicum (Phippen and Smion 2000) and Cimicifuga racemosa (Lata et al. 2002). While, Abbasi et al. (2010) investigated the morphogenic potential of leaf derived callus in Silybum marianum on 5.0 mg l-1 BA supplemented MS medium.
5.5 In vitro rooting of microshoots
The success of in vitro regeneration relies on an efficient rooting in regenerated shoot and their subsequent acclimatization which is often problematic in some plant species as reported for D. hamiltonii by Bais et al. (2000). During the present investigation, in vitro rooting was easier in both the Spilanthes species than in D. hamiltonii. In fact, in S. acmella and S. mauritiana rooting was possible on PGR- free half-strength MS medium (although roots were very thin and delicate) while in D. hamiltonii microshoots necessitated the requirement of auxin for adventitious rooting. For healthy root induction half-strength MS medium was selected in combination
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with different concentrations of three auxins viz., IAA, IBA and NAA. The requirement of half-strength culture medium for root induction has also been reported in many plant species including Melia azedarach (Shahzad et al. 2001), Pterocarpus marsupium (Husain et al. 2007a) and Acacia sinuata (Shahzad et al. 2006). However, the strength of nutrient medium did not affect the rooting frequency in some plant species (Sharma and Chandel 1992, Anis and Faisal 2005, Pandey et al. 2006). Among different concentrations of auxins tested, rooting was best achieved on 2.5 µM NAA in all the tested plant species. On this optimal treatment maximum of 30.40 ± 0.50 roots with 18.60 ± 0.18 cm root length and 26.40 ± 1.12 roots with 8.02 ± 0.17 cm root length were induced in S. acmella and S. mauritiana respectively. Similarly, 2.5 µM NAA supplemented half-strength MS medium induced highest of 7.80 ± 0.37 roots per shoot and 6.40 ± 0.13 cm root length in D. hamiltonii. The stimulatory effect of NAA on root formation has also been previously reported in many medicinal plants like Tagetes erecta (Misra and Datta 2001), Carthamus tinctorius (Radhika et al. 2006), Trichosanthes dioica (Malek et al. 2007) and Lysimachia species (Zheng et al. 2009). However, the requirement of IAA for best rooting has been reported by Sujatha and Ranjitha Kumari (2007), Ahmed et al. (2007), Anbazhagan et al. (2010) and Gantait et al. (2010). In comparison to our findings, Saritha et al. (2002), Deka and Kalita (2005), Pandey and Agrawal (2009) reported best rooting response on IBA containing medium in S. acmella while Haw and Keng (2003) surprisingly achieved rooting on BA supplemented medium in this plant species. Recently, Singh and Chaturvedi (2010) documented optimum rooting on half-strength MS medium (only major salts reduced to half-strength) with 50 g l-1 sucrose in S. acmella. Similar to their study, Serres et al. (1990) also observed that the percentage of rooting and number of roots per shoot is positively correlated with sucrose concentrations in chestnut microcuttings. High sucrose concentration increases the osmotic pressure of the medium, which in turn stimulates the mitochondria to generate more energy to facilitate rooting (Bonga and Von Aderkas 1992). In contrary to the present findings, Bais et al. (2000) and Reddy et al. (2001) used AgNO3 for in vitro root formation and elongation in D. hamiltonii. Later, Giridhar et al. (2005) suggested the addition of phenolic compounds to auxin (IBA) containing medium for rooting in this woody climber. But the present protocol
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provides an easy rooting and thus alleviates the need of complex chemicals (like
AgNO3 and phenolic compounds) for root induction in D. hamiltonii. In the present study, in vitro flowering was also noticed in S. acmella simultaneous with the root induction on NAA containing nutrient medium. In vitro flowering is an important feature of micropropagation studies. Singh (1991) used this approach for Carthamus oxyacantha to overcome the problem of asynchrony in ex vitro flowering. In vitro flowering has also been reported by various researchers (Tejovathi and Anwar 1984, Nikam and Shitole 1999). Sivanesan and Jeong (2007) reported continued flowering (for more than two years) in Pentanema indicum. They observed that both BA and IAA strongly affected the flower bud induction. Flowering ability was further increased with IBA when applied to the plantlets previously treated with BA, IAA and ADS. However, high IBA concentrations were ineffective to from reproductive buds.
5.6 Acclimatization of plantlets
The period of transition during the process of hardening after transfer from in vitro to ex vitro environment is considered to be the most important step in tissue culture. Tissue culture regenerated plantlets are usually very fragile and mostly die if they are directly transferred to the field because of transplantation shock. Moreover, due to heterotrophic mode of nutrition, lack of adaptation or exposure to the outside environment, during lab to land transfer micropropagated plants are first placed in the hardening chamber. In general, during the period of hardening care is taken over the physical (temperature, light intensities, relative humidity, air current, atmosphere
CO2) and other factors (mineral nutrition and pH). One important factor during acclimatization is the type of potting material used. The use of sufficiently porous substratum that allows adequate drainage and aeration has been recommended for fast acclimatization of in vitro regenerated plants (Dunstan and Turner 1984). During the present study, among three planting substrates (soilrite, vermi-compost and garden soil with farmyard), the highest survival of micropropagated plantlets was achieved in soilrite. After successful acclimatization, there was no detectable variation among the plantlets with respect to morphology and growth. All of them were free from external defects. In most of the micropropagation protocols, soilrite exhibited a good percentile of survived plantlets (Siddique et al. 2008, Sharma et al. 2009 a & b,
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Parveen and Shahzad 2011, Khan et al. 2011). In contrast, other planting substrates like vermiculite (Reddy et al. 2001, Faisal et al. 2007), leaf mould: garden soil (3:1) (Misra and Datta 2001), cocopeat (Sivaram and Mukundan 2003) and vermi-compost (Mohapatara et al. 2008) have also been suggested for acclimatization.
5.7 Synseeds development and their germination under in vitro and ex vitro conditions
Recent advances in plant biotechnology have made the synthesis of synseed a reality. Initially, the synseed development is restricted to somatic embryos. Due to low success and high cost of somatic embryo production, axillary buds, shoot tips, bulbs, protocorm like bodies (PLBs) or other meristematic have been suggested for encapsulation (Pond and Cameron 2003). This has opened up new vistas for germplasm conservation, storage or means to reduce the need for transplanting and sub-culture during off-season periods. In the present study, only juvenile nodal segments, excised from aseptic seedlings were used for encapsulation as the availability of nodal segments was more than shoot tips per seedling. Similarly, the encapsulation of nodal segments for synseeds production (axillary buds) has been documented for many plant species like Pimpinella pruatjan (Roostika et al. 2006), Punica granatum (Naik and Chand 2006), Hibiscus moscheutos (Preece and West 2006, West et al. 2006), Morus species (Kavyashree 2006), Rauvolfia tetraphylla (Faisal et al. 2006a), Tylophora indica (Faisal and Anis 2007), Psidium guajava (Rai et al. 2008), Cannabis sativa (Lata et al. 2009) and Vitex negundo (Ahmad and Anis 2010). Comparatively, fewer reports are available on encapsulation of shoot tips such as Chonemorpha grandiflora (Nishitha et al. 2006), Spilanthes acmella (Singh et al. 2009b), Solanum tuberosum (Nyende et al. 2003), Phyllanthus amarus (Singh et al. 2006a), Withania somnifera (Singh et al. 2006b) and Psidium guajava (Rai et al. 2008). Nodal segments and shoot tips have been found suitable for encapsulation studies as they are the excellent plant materials for preparation of sysneeds as possessing meristematic tissues. However, lack of root apex and the inability of the propagules to form roots is a major bottleneck to recover the complete plantlet from synseed, especially in woody plant species.
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To produce synseeds, excised nodal segments mixed with Na2-alginate (an
encapsulation matrix) and dropped into CaCl2·2H2O solution (a complexing agent). An ion-exchange process takes place during this period resulting in the replacement of Na+ ions by Ca++ ions forming the Ca-alginate beads which are refereed as “synseeds”. The morphology of synseeds in respect to shape, texture, transparency and rigidity may vary with different concentrations of Na2-alginate and CaCl2·2H2O and also depends on propagule type and plant species (Rai et al. 2009). Therefore, as an initial experiment of synseed, the concentrations of these two encapsulating chemicals were optimized for all three plant species.
Irrespective of the plant species, 4% Na2-alginate with 100 mM CaCl2·2H2O was found best combination for gel complexion which produced firm, clear and
isodiametric synseeds. Lower concentrations of Na2-alginate and CaCl2·2H2O were not suitable because synseeds were fragile and difficult to handle during transfer to regeneration medium. The reduction in the gelling ability of lower concentrations of
Na2-alginate after exposure to high temperature during autoclaving has already been reported by Larkin et al. (1998). On the contrary, high concentrations of both the encapsulating chemicals resulted in too hard beads and showed considerable delay in germination. This corroborates with the findings of Kavyashree et al. (2006), Swamy
et al. (2009) and Sudararaj et al. (2010) who reported 4% Na2-alginate as critical concentration for synseed development in Morus alba, Pogostemon cablin and Zingiber officinale respectively. In comparison to the present findings, Singh et al. (2009b) reported optimum synseed formation by encapsulating shoot tips of S.
acmella in 3% Na2-alginate and 100 mM CaCl2·2H2O. In most of the reports 3%
Na2-alginate and 100 mM CaCl2·2H2O has been proved as the best combination for an ideal synseed fromation (Ahmad and Anis 2010, Tabassum et al. 2010, Ozudogru
et al. 2011). However, Pinker et al. (2005) reported 3% Na2-alginate and 75 mM
CaCl2·2H2O as best combination for encapsulating the nodal segments of Dendranthema × grandiflora. But Lata et al. (2009) achieved optimum synseed
fromation in Cannabis sativa with 5% Na2-alginate and 50 mM CaCl2·2H2O. This
variation in Na2-alginate concentration for bead formation in different plant species might be due to the variation in commercial source from which the chemicals were purchased as reported earlier by Ghosh and Sen (1993) and Mandal et al. (2000).
Synseeds obtained with 4% Na2-alginate and 100 mM CaCl2·2H2O showed the highest of 74.80 ± 1.46, 74.40 ± 1.93 and 68.60 ± 1.86% germination for S. 134
acmella, S. mauritiana and D. hamiltonii respectively, when placed on PGR-free MS basal medium (control) for 6 weeks. To further enhance the germination frequency of synseeds, PGRs (cytokinin and auxin) were added to the MS basal medium. In S. acmella, combination of 1.0 µM BA and 0.5 µM NAA exhibited maximum germination (87.80 ± 1.15%) after 6 weeks of culture. While, 1.0 µM BA and 0.5 µM IAA yielded optimum germination (83.00 ± 2.09%) in S. mauritiana. In both the species of Spilanthes, complete plantlets with healthy shoot and root systems were recovered from the synseeds on same germination media. Thus, a separate experiment for root induction did not require prior to acclimatization. Similar responses have been documented in Pimpinella pruatjan (Roostika et al. 2006), Tylophora indica (Faisal and Anis 2007) and Vitex negundo (Ahmad and Anis 2010). In D. hamiltonii, maximum germination (77.00 ± 2.09%) was noticed on 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS supplemented MS medium. Although synseeds failed to induce rooting on germination medium therefore, an additional experiment was required to induce rooting in microshoots obtained from synseeds. The best rooting was achieved on half-strength MS medium comprising 2.5 µM NAA and a maximum of 4.60 ± 0.50 roots per microshoot with 5.26 ± 0.08 cm root length was induced in 90.20 ± 1.80% of cultures after 4 weeks of trasnfer. Similarly, Gangopadhyay et al. (2005) devised a two step method to achieve maximum synseed conversion into complete plantlets in Ananus comosus; firstly, shoots were retrieved from synseed and in the second step, these microshoots were rooted in liquid medium (supplemented with IBA and Kn) supported with Luffa-sponge. Bekheet (2006) and Lata et al. (2009) achieved rooting in Allium sativum and Cannabis sativa on MS medium containing IAA and IBA respectively. In contrast, Swamy et al. (2009) reported rooting on PGR-free half-strength MS basal medium in microshoots retrieved from synseeds of Pogostemon cablin. Synseed technology also acts as a tool of germplasm exchange between countries. For this purpose synseed storage is a critical factor which determines their successful germination after transportation abroad. During cold storage, synseeds require no transfer to fresh medium, thus reduces the cost of maintaining germplasm cultures (West et al. 2006). Therefore, appropriate storage conditions and definite storage period are prerequisites to maintain synseed viability during transportation that leads to successful commercialization of synseed technology. In the present study, two types of syneed (one with MS gel matrix and other with DDW matrix) and 135 naked nodal segments were stored at low temperature (4 ºC) to see the effect of storage on tissue viability.
The Na2-alginate combined with MS nutrients demonstrated significant superiority over DDW with respect to shoot growth. In all the plant species, with an increase in storage time to more than 4 weeks, the germination frequency decreased gradually, thereafter a drastic loss in germination was noticed for synseeds having encapsulation matrix of MS medium. Decline in conversion response could be attributed to inhibition of tissue respiration by the alginate matrix or a loss of moisture due to partial desiccation during storage as reported earlier (Danso and Ford-Llyod 2003, Faisal et al. 2006a, Faisal and Anis 2007). After 4 weeks of storage 73.60 ± 1.56, 75.40 ± 1.43 and 47.00 ± 1.54% conversions of synseeds into complete plantlets were noticed for S. acmella, S. mauritiana and D. hamiltonii respectively. On the other hand, in S. acmella and S. mauritiana synseeds prepared with DDW did not survive after just after 2 weeks of storage while in D. hamiltonii such synseeds failed to store. These findings suggest that the MS nutrients are essential ingredients of Na2- alginate matrix for plantlet conversion. Synseeds prepared with MS nutrients were viable (46.00 ± 1.87% in S. acmella, 41.00 ± 1.18% in S. mauritiana and 14.00 ± 1.37% in D. hamiltonii) even after 8 weeks of cold-dark storage while only 9.20 ± 0.37 and 4.20 ± 0.58% conversion frequencies were observed for non-encapsulated nodal segments of S. acmella and S. mauritiana after similar 8 weeks of storage. Only 14.40 ± 1.69% germination was noticed just after 2 weeks of storage for non-encapsulated nodal segments of D. hamiltonii, thereafter none of the nodal segments survived. The observation with cold stored synseeds of these threatened and endangered species are in accordance with previous reports on other species (Kinoshita and Saito 1992, Adriani et al. 2000, Tsvetkov et al. 2006, Faisal et al. 2006a, Faisal and Anis 2007). However, the temperature requirement for optimum viability varies from plant to plant. Generally, 4 ºC temperature is found to be most suitable for synseeds storage (Saiprasad and Polisetty 2003, Kavyashree et al. 2006, Faisal et al. 2007, Singh et al. 2007, Pintos et al. 2008, Sharma et al. 2009a & b, Ikhlaq et al. 2010, Tabassum et al. 2010). Few investigations revealed the requirement of higher temperature (25 ºC) rather than low temperature for amenable storage of synseeds in certain tropical and sub-tropical crops. Sundararaj et al. (2010) observed 100% re-growth ability for
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Zingiber officinale synseeds incubated at 25 ºC while no re-growth was observed for synseeds stored at 4 ºC, in dark. Ex vitro sowing of synseeds provides a commercially important and cost effective technique for direct recovery of plantlets. High-frequency conversion of synseeds into complete plantlets on simple planting substrates such as sand/soil/soirirte is necessary for their use in commercial-scale propagation. However, to date only a few reports are available on direct conversion of encapsulated somatic embryos or shoot buds on such substrates (Redenbaugh et al. 1986, Mathur et al. 1989, Pattnaik et al. 1995). In the present study, among various planting substrates, soilrite moistened with quarter-strength MS nutrient medium was found to be the most suitable planting substrate for ex vitro sowing of synseeds in S. acmella (63.00 ± 2.00%) and S. mauritiana (63.40 ± 1.02) while ex vitro germination was not successfully achieved for the synseeds of D. hamiltonii.
5.8 Physiological study of acclimatized plantlets
Upon transfer to natural conditions, micropropagated plants might fail to cope with the stressful situation because of sudden changes in environmental conditions, the latter resulting in physiological impairment leading to high mortality. Hence, a slow and gradual ex vitro transfer process is necessitated during the period in which a sustained acclimatization is effected to correct the physiological anomalies and deficiencies. During the period of stress alleviation, substantial changes in plants are expected to occur which would affect different physiological and biological parameters of in vitro raised plants. The stress adaptation in plants is more accurately monitored by studying the behavior of photosystem II (PSII) because of its sensitivity to different stresses (Strasser and Tsimilli- Michael 2001). PSII fluorescence changes in relation to the adaptation of in vitro-grown plants to ex vitro conditions are yet to be properly reported. The results of these investigations demonstrated that significant changes in the photosynthetic and fluorescence parameters of the plants occurred as a response to the transplantation shock during adaptation under indoor environmental ambience as well as at the initial stage of transfer to the outdoor condition. A precocious ex vitro adaptation of plants inside the culture room could facilitate primary hardening of plants.
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It is known that, shortly after transplantation, plantlets show low photosynthetic capabilities and have to be kept under high humidity conditions to prevent water loss through stomata until they recover photosynthetic competence (Shacke et al. 1990, Preece and Sutter 1991, Kirdmanee et al 1996). Considering the importance of this step of acclimation, various physiological parameters, like chlorophyll and carotenoids contents and net photosynthetic rate was studied during the acclimatization period in the present study. The information generated herein could be useful for optimizing the key physiological parameters so that the in vitro- derived plants could face negligible mortality during due course of ex vitro acclimatization thereby rendering micropropagation more meaningful and profitable for a range of economically important plant groups. The chlorophyll (chl a, b & total) and carotenoids contents in Spilanthes species and D. hamiltonii decreased initially during first week of transfer from in vitro to ex vitro conditions. After that steady increase in their contents was noticed. Similar trend was observed for chlorophyll content s in Tobacco by Kadleček et al. (1998) where after an abrupt initial decrease of the chlorophyll a and b content during first week of transplantation, slow increase was witnessed. Decrease in chlorophyll and carotenoids contents during initial days of transplantation was accompanied by poorly developed chloroplast and disorganized grana (Siddique and Anis 2008). After 14 days of transplantation a steep rise in chlorophyll content was observed which was associated with the formation of new leaves. Lu and Zhang 1998 and Sopher et al. 1999 suggested loss of chlorophyll during acclimatization means that the leaves were damaged due to photo-inhibition. However, an increase in carotenoids level after 14 days of acclimatization specifies that plants sustained the light stress. Such an increase in carotenoids further reflects the functional response of photosynthetic apparatus to the different light environment, since the plant protective role of carotenoids against photooxidative damage is well documented (Donnelly and Vidaver 1984, Young 1991, Huylebroeck et al. 2000).
Net photosynthetic rate (PN) in all the species decreased in the first week after transplantation and increased thereafter. The decline in photosynthetic rate during the first week after transfer from in vitro to ex vitro condition indicates that climatic conditions create stress in micropropagated plants. Increase in PN after ex vitro transfer is usually associated with the formation of new leaves. Similar results were
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observed earlier in Solanum tuberosum and Spathiphyllum floribundum ( Baroja 1993, Baroja et al. 1995, Van Huylenbroeck and Debergh 1996).
5.9 Histological study of morphogenic tissues
Histological investigations were undertaken to study the mode and origin of regeneration under the effect of exogenous phytohormones during morphogenesis through various explants. The direct origin of shoot buds was traced to the compressed axillary region of the shoot tip explants in both S. acmella and S. mauritiana. Studies revealed the bud initiation in the form of bulge which later transformed into well differentiated shoot bud. In case of leaf explants of S. acmella petiolar cut end proved responsive by marking initial swelling which gave rise to shoot buds in the later stages. Similar origin of shoot buds was traced directly from petiolar cut end of leaf segments during histogenesis in Tagetes erecta (Misra and Datta 2001). Leaf derived callus of S. acmella induced on TDZ (2.5 µM) showed early vascularization of cells resulting in early response in the form of multiple shoot bud induction from marginal cells of the calli. In case of D. hamiltonii histological investigation proved useful in ascertaining the callogenic nature of the regenerating nodular tissue formed at the basal cut end of shoot tip explant. Sequential regeneration process was revealed through the histological sections of nodular tissue at various stages of development. Formation of meristemoids in the developing callus ensured high frequency adventitious shoot regeneration. Similar kind of meristematic regions were observed in leaf derived tissue of Ruta graveolens (Ahmad et al. 2010). Meristemoid mediated shoot regeneration was also recorded through basal callus of shoot tip and nodal explants in Bixa orellana (Parimalan et al. 2009), nodal explants of Lotus corniculatus (Nikolić et al. 2010) and in cotyledonary node explant of Faba bean (Aly and Hattori 2007).
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Chapter 6 Summary & Conclusions
Chapter 6
SUMMARY AND CONCLUSIONS
Human beings have been utilizing plants for basic preventive and curative health care since time immemorial. In India, thousands of medicinal plant species are known to have medicinal value to cure specific ailments. However, it is estimated that about 15,000 species of medicinal plants are globally threatened; the causes include loss of habitat, over harvesting and pollution. Conservation of medicinal plants is therefore important to ensure sustainable human development. Conservation of plant diversity requires in situ and ex situ strategies that have been widely used by scientists to establish efficient conservation programmes. In vitro culture is an efficient method for ex situ conservation of plant diversity (Krogstrup et al. 1992, Fay 1994). Especially, in vitro propagation of endangered plants can offer considerable benefits for the rapid cultivation of at risk species which have a limited reproductive capacity and exist in threatened habitats (Fay 1992). In the present study, one threatened i.e., Spilanthes acmella and two endangered i.e., Spilanthes mauritiana and Decalepis hamiltonii medicinal plants were selected for their propagation and conservation by adopting the plant tissue technique. Fig. 90 represents a sketch of all the experiments of micropropagation, conducted for the selected plant species. In all the selected plant species, seedlings were treated as explant source. The results obtained during the present study are summarized below;
6.1 Spilanthes acmella
In vitro seed germination was significantly more effective than in vivo germination. Among the treatments, seeds were best germinated (94.00 ± 2.44%) on half-strength MS medium with 1.0 µM GA. Four explants viz., nodal segments, shoot tips, leaves and cotyledons were excised from these 4 week-old aseptic seedlings to determine their ability to induce multiple shoots. Nodal segments were least effective for shoot regeneration even in the presence of different cytokinins (BA, Kn, 2-iP and TDZ) and auxins (NAA, IAA and IBA). Whereas, shoot tip explants exhibited high frequency shoot regeneration as a
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Axenic Seedlings
(Explant Source) Micropropagation Synseed development
NS ST L CL NS
Culture Ideal bead formation Direct Indirect organogenesis organogenesis
Shoot induction Beads having Beads having MS nutrient gel DDW gel ti ti
Shoot proliferation
Sub-culturing and culture Ex vitro In vitro Low temperature maintenance sowing germination storage In vitro rooting
Hardening & acclimatization
Field140 transfer Figure 90 Figure 90. Flow chart showing overall design of conducted experiments in the present study
result of apical bud multiplication and from their basal cut ends depending on the type of cytokinin used. On adenine-based cytokinins (BA, Kn and 2-iP) shoot regeneration occurred due to multiplication in apical bud while urea based cytokinin (TDZ) favored direct adventitious shoot induction from the basal cut ends of the explant. Among BA, Kn and 2-iP, 1.0 µM BA supplemented MS medium induced a maximum of 8.0 ± 0.31 shoots per shoot tip explant with a mean shoot length of 3.9 ± 0.30 cm. Addition of auxin at low concentration to this optimized concentration of BA further enhanced the regeneration efficiency. With the combination of 1.0 µM BA and 0.1 µM NAA, a maximum of 33.0 ± 1.09 shoots per shoot tip explant with a mean shoot length of 5.2 ± 0.09 cm were formed in 96.0 ± 2.44% cultures after 4 weeks of incubation. Comparatively lower concentration of TDZ i.e., 0.25 µM induced a maximum of 30.00 ± 0.30 shoots per shoot tip explant in 98.00 ± 2.00% cultures, however shoots were stunted (1.00 ± 0.10 cm). Thus, to enhance the shoot length regenerating tissues were firstly transferred to BA (1.0 µM) and NAA (0.5 µM) containing medium then cultured on PGR-free full-strength MS medium to avoid any deleterious effect of TDZ. Shoot regeneration was significantly affected by pH of the medium, media formulation and type of energy source. During the present study, best shoot regeneration was achieved on MS medium supplemented with 3% sucrose at 5.8 pH. Direct adventitious shoot regeneration was also possible from cultured leaves and cotyledons. Both these explants showed regeneration from their basal petiolar cut ends while entire leaf margin and leaf surface remained as such. The leaves explants were more responsive in terms of direct organogenesis than cotyledons. Among adenine-based cytokinins, BA at 2.5 µM induced maximum of 6.20 ± 0.48 and 7.2 ± 0.37 shoots per leaf and cotyledon explants respectively. Addition of reduced concentration of auxin considerably enhanced organogenesis. Highest of 27.80 ± 1.01 and 17.40 ± 0.87 shoots per explant were formed from leaf and cotyledon explants respectively when cultured on 2.5 µM BA and 0.5 µM NAA supplemented MS medium.
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On TDZ supplemented nutrient medium, leaf explants showed indirect organogenesis while cotyledons induced non-organogenic callus. 2.5 µM TDZ supplemented MS medium induced greenish-white lucid callus that were highly competent on TDZ (1.0 µM) and BA (1.0 µM) combination and induced a maximum of 14.00 ± 0.54 shoots in rosette fashion due to poor internodal growth. Moreover, all the shoots were greenish-white in color and crumpled due to their vigorous production. MS medium amended with 1.0 µM BA improved the shoot growth of these TDZ derived shoots satisfactorily. Sub-culturing had a significant effect on regenerative capacity of responsive explants. From shoot tip explants, the frequency of shoot multiplication increased drastically from first sub-culture (42.20 ± 1.50 shoots per explant) to sixth sub-culture passage (77.20 ± 0.86 shoots per explant) and remained almost consistent at seventh sub-culture, but declined thereafter. However, through leaf and cotyledon explants the regeneration efficiency consistently increased up to fifth sub-culture passage (73.00 ± 0.80 and 41.00 ± 1.18 shoots per leaf and cotyledon respectively). Nevertheless, a gradual increase in shoot length was observed after every passage of sub-culturing for all the explant types. Of the different auxins, 2.5 µM NAA supplemented to half-strength MS medium showed optimum rooting with a maximum of 30.40 ± 0.50 roots per shoot and root length of 18.60 ± 0.18 cm. Plantlets rooted on this treatment resulted in maximum survival rate after transplantation, as the roots induced directly from the basal cut end in absence of intervening callus. Complete plantlets with healthy shoot and root systems were successfully hardened off. Amongst three planting substrates, maximum survival percentage was recorded in soilrite (96.60%) followed by vermi- compost (81.60%) and mixture of garden soil with farmyard (66.60%). After successful acclimatization, they were transferred to field conditions where they grew well, attained maturity and flowered normally.
Nodal segments encapsulated with a combination 4% Na2-alginate and 100 mM CaCl2·2H2O were treated as ideal synseeds exhibited optimum rigidity. On MS basal medium only 74.80 ± 1.46% germination was observed from sysneeds. Germination frequency was further enhanced with PGR supplementation to the basal nutrient medium. Maximum conversion frequency of synsseds (87.80 ± 1.15%) was achieved on 1.0 µM BA and 0.5 µM NAA supplemented MS medium. Healthy rooting was also induced on similar germination medium, thus escaping the 141
requirement of an additional experiment for root induction of microshoots recovered from synseed. Low temperature storage (4 °C) of synseeds was also carried out to examine the tissues’ viability to revive physiological activity leading to plantlet development after 1, 2, 4, 6 and 8 weeks. Synseeds prepared with MS nutrients and DDW as gel matrices were compared with non-coated nodal segments for this purpose. Encapsulation with MS nutrients showed significant superiority over DDW for successful synseed germination as MS nutrients provided an ‘artificial endosperm’ around the propagule. Up to 4 week of low temperature storage, viability of nodal segments decreased gradually for synseed prepared in MS nutrient gel matrix whereas a significant loss in the morphogenetic potential was observed for naked nodal segments. On the other hand, synseeds having DDW gel matrix did not survive after 2 weeks of storage. Ex vitro sowing of synseeds was also performed as it provides a commercially important and cost effective technique for direct recovery of plantlets. Among various planting substrates, soilrite moistened with quarter-strength MS nutrient medium was most suitable for ex vitro sowing. Various photosynthetic parameters like chlorophyll (a, b & Total), carotenoids content and net photosynthetic rate were evaluated during subsequent weeks of acclimatization. Histological studies undertaken, clearly established the mode of shoot regeneration through responsive explants.
6.2 Spilanthes mauritiana
Similar to S. acmella, highest seed germination (86.00 ± 1.87%) was achieved on half-strength MS medium supplemented with 1.0 µM GA under in vitro condition. Nodal segments, shoot tips, leaves and cotyledons were excised from 4 week-old aseptic seedlings. Nodal segments did not show effective shoot regeneration on all the tested treatments of PGRs. However, rest of the explants induced multiple shoots effectively on different combinations of cytokinin and auxins. Adenine-based cytokinins showed multiplication in pre-existing apical bud of shoot tip explants while urea-based cytokinin (TDZ) induced de novo shoot formation from the basal cut end of shoot tip explants. Among BA, Kn and 2-iP, BA (1.0 µM) was proved to be optimal with a maximum of 6.00 ± 0.44 shoots per shoot tip explant
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measuring 2.36 ± 0.14 cm. However, additions of auxin at lower concentration enhanced shoot number and shoot length. Highest number of 18.80 ± 0.48 shoots per shoot tip explant and 4.26 ± 0.10 cm was achieved on MS medium containing a combination of BA (1.0 µM) and IAA (0.5 µM). Reduced concentration of TDZ (0.25 µM) proved optimal for high frequency shoot regeneration (26.20 ± 1.35 shoots per shoot tip explant). Although the mean number of shoots per explant was quite higher on TDZ containing MS medium than BA containing MS medium but growth in shoots was insignificant even after 4 weeks of culture. The shoot stunting effect of TDZ could be overcome by transferring the shoots on 1.0 µM BA and 0.5 µM IAA supplemented MS medium and followed by PGR-free medium. A maximum of 64.00 ± 1.37 shoots per shoot tip explant with mean shoot length of 3.72 ± 0.08 cm was recorded on PGR free full-strength MS basal medium. Among different pH levels, carbon sources and culture media tested for shoot tip explant, optimum shoot proliferation and elongation was achieved on MS medium comprising an optimal combination of BA (1.0 µM) and IAA (0.5 µM) with sucrose at 5.8 pH wherein a maximum of 18.80 ± 0.48 shoots per explant with 4.26 ± 0.10 cm of shoot length were induced after 4 weeks of culture. Leaf and cotyledon explants induced direct adventitious shoot organogenesis from their petiolar cut ends within one week of incubation. 2.5 µM BA revealed optimum regeneration over Kn and 2-iP supplemented MS media with the highest of 5.60 ± 0.40 and 4.80 ± 0.37 shoot per leaf and cotyledon explant respectively. Addition of 0.5 µM IAA to 2.5 µM BA significantly enhanced the shoot regeneration from both the explants. The highest number of 15.00 ± 0.31 and 14.0 ± 1.09 shoots per explant were noticed from leaf and cotyledons respectively when placed on optimized combination of BA (2.5 µM) and IAA (0.5 µM). The leaf explants implanted on MS basal medium having 0.25 and 0.5 µM TDZ induced calli first then started differentiation directly. Callus did not differentiate in any shoot however, after scarping the callus tissue leafy shoots emerged directly from leaf surface on similar treatments of TDZ. Among different concentrations, 0.5 µM TDZ proved optimal concentration as induced a maximum of 8.40 ± 0.50 mean number of shoots per explant. These regenerated shoots did not grow further on the same culture medium. Of the various combinations tested, 1.0 µM BA in conjunction with 0.5 µM IAA proved to be best shoot proliferation medium, giving a maximum of 143
12.40 ± 0.74 shoots per explant with 2.20 ± 0.07 cm shoot length. Further, PGR-free MS basal medium enhanced shoot proliferation (15.00 ± 0.31shoots per explant) and overall shoot growth (2.62 ± 0.04 cm). Contrary to the leaf explants, none of the tried concentration of TDZ showed caulogenic response (either direct or indirect) through cotyledons. Sub-culturing gradually increased the regeneration efficiencies’ of all the explant types. For shoot tip explants, frequency of direct shoot multiplication increased considerably from first (34.00 ± 1.37 shoots per explant) to sixth sub- culture (73.20 ± 1.82 shoots per explant) and remained almost consistent at seventh sub-culture passage (72.20 ± 0.83 shoots per explant), but declined thereafter (64.00 ± 1.37 shoots per explant). For the leaf explants, the regeneration efficiency continuously increased up to fifth sub-culture passage (70.20 ± 0.80 shoots per explants), remained same at sixth passage. Thereafter, a gradual loss in shoot number was observed during seventh (67.40 ± 0.87) and eight sub-culture passage (65.60 ± 0.67). On the other hand, for cotyledonary leaf explant maximum number of shoots (39.80 ± 0.80) was obtained after fifth sub-culture passage. Irrespective to the explant types, a continuous increase in shoot length was noticed for all the explant types after every sub-culture passage. The half-strength MS basal medium augmented with NAA was best for healthy root induction as compared to IAA and IBA. A maximum of 26.40 ± 1.12 roots per microshoot with 8.02 ± 0.17 cm root length was achieved without an intervening callogenesis on half-strength MS medium augmented with 2.5 µM NAA. Amongst three planting substrates tested, maximum survival percentage of regenerated plantlets was observed for soilrite (92.60%) followed by vermi-compost (83.30%) and garden soil mixed with farmyard (34.00%). After acclimatization plantlets were transferred to field condition where they grew well and flowered successfully.
Among, various concentrations of Na2-alginate and CaCl2·2H2O, 4% Na2-
alginate with 100 mM CaCl2·2H2O produced clear and uniform beads within an ion
exchange duration of 20 min. Nodal segments encapsulated in Na2-alginate (4%) and
CaCl2·2H2O (100 mM) exhibited re-growth within one week of inoculation. Maximum conversion response (83.0 ± 2.09%) was observed on MS medium supplemented with BA (1.0 µM) and IAA (0.5 µM). Complete plantlets (with shoot and root systems) were recovered from synseeds on same germination medium. 144
Different storage time (0, 1, 2, 4, 6 and 8 week) was also found to influence the regeneration potential of encapsulated propagules. Synseeds having MS nutrients could survive after 8 weeks while DDW containing sysneeds died after 2 weeks of storage. A gradual decrease in regeneration frequency up to 4 weeks of storage (75.40 ± 1.43% germination) was noticed for synseed with MS nutrients, thereafter the percent germination decreased considerably. Non-encapsulated nodal segments showed satisfactory germination (42.60 ± 1.66%) only after 2 weeks of storage, after that germination greatly reduced. Various planting substrates were also assessed for direct conversion of synseeds into complete plantlets under ex vitro conditions. Among different planting substrates, soilrite moistened with quarter-strength MS nutrient medium was found to be the most suitable for ex vitro sowing of synseeds. Physiological investigations were undertaken in terms of photosynthetic pigments (Chl a, b & Total and carotenoids) estimation and net photosynthetic rate evaluation during transfer of in vitro raised plants to ex vitro conditions and their subsequent acclimatization. Histological studies were conducted in S. mauritiana to confirm the mode of shoot regeneration from responsive explants.
6.3 Decalepis hamiltonii
In vitro seed germination was found more effective than in vivo germination even after the pre-soaking of seeds in GA. In contrast to Spilanthes species, full- strength MS medium supplemented with 2.5 µM GA yielded optimum seed germination (83.20 ± 1.82%) in D. hamiltonii. The possibility of mass propagation through nodal and shoot tip explants as was compared in D. hamiltonii. No caulogenic response was observed on control treatment through both the explant. Among three adenine-based cytokinins, BA was more potent for axillary bud break. Supplementation of 5.0 µM BA to the MS medium resulted in maximum multiplication. The nodal segments produced higher number of shoots 4.20 ± 0.37 per explant with mean shoot length of 5.30 ± 0.09 cm. On similar treatment, shoot tips produced 3.20 ± 0.20 shoots per explant with 4.84 ± 0.10 cm shoot length after 4 weeks of culture. Among different cytokinin-auxin combinations attempted, enhanced proliferation was noticed on 5.0 µM BA plus 0.5 µM IAA for both the explant types,
145 thus exhibited similar hormonal requirement for morphogenesis. On this treatment a maximum number of 5.80 ± 0.37 and 3.80 ± 0.37 shoots per explant were obtained in 94.00 ± 1.87 and 92.00 ± 1.22% cultures through nodal segments and shoot tip explants respectively. Although the cytokinin-auxin combination improved shoot proliferation from both the explants in D. hamiltonii, but shoots exhibited rudimentary leaves showing premature abscission; therefore different adjuvants like ADS, Glu and PG were added to the regeneration medium. Among different adjuvant, 30.0 µM ADS with 5.0 µM BA and 0.5 µM IAA proved to be of significance. Addition of ADS not only prevented an early leaf fall within 2 weeks of incubation but also improved overall total shoot growth with continuous increase in shoot number and maximum of 8.80 ± 0.37 and 8.20 ± 0.37 shoots per explant with the highest shoot length of 6.46 ± 0.11 and 6.54 ± 0.08 cm were achieved from nodal segment and shoot tip explants respectively. In addition, ADS at this level increased the stem girth, thus making the shoots healthiest among all the additives. The shoot tip explants induced highly potent nodular organogenic calli from their basal cut ends on 5.0 µM BA and 0.5 µM IAA supplemented MS medium, simultaneously with multiplication in apical bud. A maximum of 15.40 ± 0.67 shoots per explant with 4.56 ± 0.06 cm shoot length obtained when such callus was cultured on a combination of BA (5.0 µM), IAA (0.5 µM)), GA (1.0 µM) and ADS (30.0 µM). Among a range of pH, energy source and culture media tested for nodal segments, the optimum shoot multiplication and elongation was observed at 5.8 pH with sucrose containing MS medium where maximum (8.80 ± 0.37) shoots per explant with highest shoot length (6.46 ± 0.11 cm). The nodal segments and shoot tip explants failed to exert their morphogenetic response on TDZ supplemented MS medium. While, leaf explants and cotyledons did not exhibit any shoot organogenesis at all the tried combinations of cytokinins (either adenine or urea based) and auxins. During sub-culturing of mother tissue on optimized combination of PGRs and adjuvant, multiplication rate was increased up to third sub-culture passage; thereafter, the proliferation rate stabilized at fourth passage and subsequently it declined due to callus formation. While, shoot length increased gradually after every sub-culture
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passage for both the explant type. After third sub-culture tissues were again sub- cultured on to lower concentration of BA (2.5 µM) along with IAA (0.5 µM) and ADS (30.0 µM). Sub-culturing gradually increased the organogenic efficiency of shoot tip derived callus up to fourth sub-culture passage. Maximum of 25.40 ± 0.50 shoots per explant were induced after fourth sub-culture passage, thereafter proliferation stabilized. However, shoot length enhanced consistently after each sub-culture passage. Like the regenerating cultures obtained from apical or axillary buds multiplication, shoots induced from callus tissues were maintained on GA-free MS medium supplemented with BA (2.5 µM), IAA (0.5 µM) and ADS (30.0 µM). Among the auxins tested, microshoots were best rooted on half-strength MS medium supplemented with 2.5 µM NAA which induced a maximum of 7.80 ± 0.37 roots per shoot with 6.40 ± 0.13 cm root length after 4 weeks of transfer. Among three planting substrates tested, highest survival was noticed for soilrite from which 95.10% plantlets were survived when transferred to the normal field conditions. The established plantlets did not show any variation in morphology and growth characteristic when compared to the mother plant.
Among the combinations, 4% sodium alginate and 100 mM CaCl2·2H2O were found best for gel complexion which produced firm, clear and isodiametric beads by encapsulating nodal segments. Synseeds showed highest of 68.60 ± 1.86% germination when placed on PGR-free MS basal medium. Augmentation of PGRs considerably enhanced the germination frequency of synseeds. A combination of 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS yielded maximum germination (77.00 ± 2.09%), although microshoots failed to induce roots on similar germination medium. Therefore, an additional rooting experiment was required for complete plantlets’ recovery. The best rooting was achieved on half-strength MS medium comprising 2.5 µM NAA. Two types of syneed (one with MS gel matrix and other with DDW matrix) and naked nodal segments were stored at low temperature (4 ºC) to see the effect of storage on tissue viability. The sodium alginate combined with MS nutrients demonstrated significant superiority over DDW with respect to shoot growth. Synseeds prepared with DDW failed to store. With an increase in storage time to more than 4 weeks, germination frequency decreased gradually thereafter a drastic
147 loss in germination was noticed for synseeds having encapsulation matrix of MS medium. These synseeds were viable (14.00 ± 1.37%) even after 8 weeks of storage. In comparison to the encapsulated nodal segments, only 14.40 ± 1.69% germination was noticed for non-encapsulated nodal segments after 2 weeks of storage, thereafter they died. On the other hand, direct sowing of synseeds under ex vitro regimes found unsuitable in D. hamiltonii. Physiological investigations were undertaken in terms of photosynthetic pigments’ (Chl a, b & Total and carotenoids) estimation and net photosynthetic rate evaluation during acclimatization. Histological study was also conducted in D. hamiltonii to confirm the mode of shoot regeneration from basal nodular tissues of shoot tip explants.
Conclusions
Thus, in the present investigation following conclusions have been drawn;
• In vitro seed germination was best achieved on half-strength MS medium with 1.0 µM GA in S. acmella and S. mauritiana while full-strength MS medium supplemented with 2.5 µM GA yielded optimum seed germination in D. hamiltonii. • In Spilanthes species shoot tips were more responsive than nodal segments. Contrary to this, nodal segments exhibited maximum shoot regeneration in D. hamiltonii. • Leaves were more responsive than cotyledons for direct de novo organogenesis in Spilanthes species while these explants were not suitable for organogenesis in D. hamiltonii. • For culture initiation, MS medium with BA (1.0 µM) in Spilanthes species and BA (5.0 µM) in D. hamiltonii was found optimum for shoot tip and nodal segments respectively. However, leaves required 2.5 µM BA for shoot regeneration in Spilanthes species. • Addition of low concentration of auxin to the optimized concentration of cytokinin significantly enhanced the regeneration efficiency of all the responsive explants.
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• Best shoot multiplication was achieved on MS medium containing BA (1.0 µM) and NAA (0.1 µM) through shoot tip explants of S. acmella and BA (1.0 µM) with IAA (0.5 µM) through shoot tip explants of S. mauritiana. In D. hamiltonii, maximum shoot regeneration was noticed on BA (5.0 µM), IAA (0.5 µM) and ADS (5.0 µM). • The combination of BA (2.5 µM) with NAA (0.5 µM) and BA (2.5 µM) with IAA (0.5 µM) induced maximum regeneration from leaf explants of S. acmella and S. mauritiana respectively. • MS medium containing 0.25 µM TDZ induced high frequency direct shoot regeneration (but stunted shoots) from basal cut end of the shoot tip explants in Spilanthes species. • TDZ containing MS medium induced indirect (2.5 µM) and direct organogenesis (0.5 µM) from leaf explants of S. acmella and S. mauritiana respectively. • Rooting was best achieved on half-strength MS medium supplemented with 2.5 µM NAA in all the plant species tested.
• A combination 4% Na2-alginate and 100 mM CaCl2·2H2O was found best for gel complexion which produced firm, clear and isodiametric synseeds. • Under in vitro conditions, synseeds were best germinated on 1.0 µM BA plus 0.5 µM NAA and 1.0 µM BA plus 0.5 µM IAA in S. acmella and S. mauritiana respectively. While, in D. hamiltonii synseeds showed maximum germination on 5.0 µM BA, 0.5 µM IAA and 30.0 µM ADS. • Under ex vitro condition, recovery of complete plantlets from synseeds was highest on soilrite moistened with quarter-strength MS nutrient medium in both the Spilanthes species while in D. hamiltonii ex vitro germination was not possible on any of the planting substrates tested. • Soilrite was the most suitable planting substrate for acclimatization of regenerated plantlets for all the plant species. • During acclimatization chlorophyll, carotenoids contents and net photosynthetic rate increased after an initial decline during first week of acclimatization. • Histological sections clearly revealed shoot regeneration pathway.
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List of publications form the thesis
Sharma S, Shahzad A, Shahid M and Jahan N (2011). An efficient in vitro
production of shoots from shoot tips and antifungal activity of Spilanthes
acmella (L.) Murr. International Journal of Plant Developmental Biology
(accepted).
Sharma S, Shahzad A, Jan N and Sahai A (2009a). In vitro studies on shoot regeneration through various explants and alginate-encapsulated nodal segments of Spilanthes mauritiana DC., an endangered medicinal herb. International Journal of Plant Developmental Biology 3: 56-61.
Sharma S, Shahzad A and Sahai A (2009b). Artificial seeds for propagation and preservation of Spilanthes acmella (L.) Murr., a threatened pesticidal plant species. International Journal of Plant Developmental Biology 3: 62-65.
Other publications
Sharma S and Shahzad A (2011). High frequency clonal multiplication of Stevia rebaudiana Bertoni, sweetener of the future. Journal of Functional and Environmental Botany 1: 70-76.
Sharma S, Shahzad A and Anis M (2010). In vitro shoot organogenesis and regeneration of plantlets from nodal explants of Murraya koenigii (L.) Spreng. (Rutaceae), a multipurpose aromatic medicinal plant. Medicinal and Aromatic Plant Sciences and Biotechnology 4: 33-36.
Sahai A, Shahzad A and Sharma S (2010). Histology of organogenesis and somatic embryogenesis in excised root cultures of an endangered species Tylophora indica (Asclepiadaceae). Australian Journal of Botany 58: 198- 205.