In Vitro Studies on Growth and Morphogenesis in Selected Medicinally Important Cassia Spp

In vitro studies on growth and morphogenesis in selected medicinally important Cassia spp.

THESIS

SUBMITTED FOR THE AWARD OF THE DEGREE OF

DOCTOR of Philosophy IN BOTANY

SHAHINA PARVEEN

Department of Botany Aligarh Muslim University Aligarh, UP (INDIA) 2012

Dedicated To My Parents

Plant Biotechnology Dr. Anwar Shahzad Ph.D. (AMU) Laboratory, Department of Assistant Professor Botany, Aligarh Muslim DST Young Scientist University , Aligarh-202002 Member Academic Council, UP (India) AMU [email protected] [email protected]

Dated: ……………….

Certificate

This is to certify that the thesis entitled “In vitro studies on growth and morphogenesis in selected medicinally important Cassia spp.” 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. Shahina Parveen under my guidance and supervision and has not been submitted in part or full for the award of any other degree of this or any other university.

(DR. ANWAR SHAHZAD) (Supervisor)

Contact no. +91- 9837061683 ACKNOWLEDGEMENTS

Begins with the name of Almighty Allah the most beneficent and merciful. My all praises are for Almighty Allah, for giving me this glorious opportunity to share my efforts with you in the form of thesis and blessing me with the strength to accomplish this goal.

At this moment of accomplishment, I am deeply appreciative and indeed indebted to my supervisor, Dr. Anwar Shahzad, Assistant Professor, Department of Botany, A.M.U. Aligarh, for his valuable suggestions, constant vigilance, persistent patience and timely help which has made this work possible. It is no exaggeration to say without his generous sharing of insight, experiences and encouragements this thesis could not have been in its present form.

I sincerely express my deepest sense of gratitude to Prof. Mohammad Anis, Chairman, Department of Botany, A.M.U. Aligarh, for his indispensable guidance, encouragement and providing all the necessary facilities to complete the present work efficiently.

It’s my immense pleasure to acknowledge Prof. Sayeed A. Siddiqui, former Chairman, Department of Botany, A.M.U. Aligarh, for their motivation and concern throughout the experimental work.

I am delighted to express my thanks to my seniors Dr. Mohammad Faisal (Assistant Professor, KSU Riyadh), Dr. Naseem Ahmad (Young Scientist, DST-Fast Track) and Dr. Iram Siddique (Assistant Professor, KSU Riyadh) for their critical suggestions, guidance and co-operation throughout the work.

I am highly thankful to my colleagues Dr. Aastha Sahai, Dr. Shiwali Sharma, Ms. Arjumend Shaheen (Project Fellow - UGC) and Ms. Taiba Saeed (Research Assistant - UPCST) for their untiring help, co-operation, support and providing conducive environment to sort out the intricacies and complications of my work.

My special thanks to my friends Ms. Ankita Varshney, Dr. Anushi Arjumend Jahan, Dr. Zeba Khan, Mrs. Honey Gupta, Mrs. Razzaquia Khan, Mrs. Seemab Mukhtar, Dr. Hamid Iqbal, Mr. Abid Rafiq, Dr. Faheem Ahmad and Mr. Hilal Ahmad for all their support, encouragement, understanding and for being with me through thick and thin.

My heart-felt thanks to all my labmates Ms. Nigar Fatima, Ms. Ruphi Naaz, Ms. Shahnaz Perveen, Ms. Afshan Naaz, Mr. Imran Khan, Mr. Rafique Ahmad and Mr. Saad Bin Javed.

Finally with deep adoration, I acknowledge my Parents, elder brothers (Mr. Mohammad Saleem, Mr. Mohammad Sadiq, Mr. Abdul Majid, Mr. Mohammad Sajid) and younger sister Ms. Shazia Hasan for their love, affection, inspiration, blessings and moral support without which this work would have been impossible.

I take this opportunity to sincerely acknowledge the University Grants Commission (UGC) for providing financial assistance under the scheme of NON-NET Fellowship and Maulana Azad National Fellowship (MANF) in the form of Senior Research Fellowship (SRF).

Dated: (Shahina Parveen)

ABBREVIATIONS

ABA : Abscisic acid AdS : Adenine sulphate

AgNO3 : Silver nitrate

B5 : Gamborg et al. medium BA : 6-Benzyladenine CA : Chlorengenic acid CW : Coconut water ++ : Ca2 Calcium ion

CaCl2∙2H2O : Calcium chloride (dihydrated) CH : Casein hydrolysate Chl : Chlorophyll cm : Centimeter °C : Degree centigrade DDW : Double distilled water 2,4-D : 2, 4-Dichlorophenoxyacetic acid DPU : Diphenylurea EDTA : Ethylene diamine tetra acetic acid FAA : Formalin acetic acid g : Gram

GA 3 : Gibberellic acid h : Hour HCl : Hydrochloric acid

HgCl2 : Mercuric chloride 2iP : 2-Isopentenyl adenine IAA : Indole-3-acetic acid IBA : Indole-3-butyric acid IRGA : Infra-Red Gas Analyzer Kn : Kinetin

L2 : Philips and Collins medium µg : Microgram µl : Microlitre

iii

µm : Micrometer µM : Micromolar M : Molarity mg : Milligram mg/g : Milligram per gram mg/l : Milligram per litre min : Minute ml : Millilitre mm : Millimeter mM : Millimolar MS : Murashige and Skoog medium N : Normality % : Percentage NAA : α-Naphthalene acetic acid NOA : Naphthoxy acetic acid NaCl : Sodium chloride + NH4 : Ammonium ion nM : Nanomolar - NO3 : Nitrate ion OH- : Hydroxyl ion PG : Phloroglucinol PGR : Plant growth regulator

PN : Net photosynthetic rate RH : Relative humidity s : Second TDZ : Thidiazuron 2,4,5-T : 2,4,5-Trichlorophenoxyacetic acid UV : Ultraviolet V : Volt v/v : Volume by volume W : Watt w/v : Weight by volume WPM : Woody plant medium YE : Yeast extract iv

CONTENTS

Page no.

ACKNOWLEDGEMENTS i-ii ABBREVIATIONS iii-iv

CHAPTER 1 INTRODUCTION 1-14

1.1 India: A mega biodiversity centre 1.2 Plants in traditional medicine 1.3 Threat to medicinal plants 1.4 Conservation of plants through in vitro techniques 1.5 Plant description 1.5.1 Cassia angustifolia Vahl. 1.5.1.1 Distribution 1.5.1.2 Morphological characters 1.5.1.3 Medicinal uses 1.5.1.4 Active compounds 1.5.1.5 Commercial products 1.5.1.6 Other uses 1.5.1.7 Propagation 1.5.2 Cassia sophera Linn. 1.5.2.1 Distribution 1.5.2.2 Morphological characters 1.5.2.3 Medicinal uses 1.5.2.4 Propagation 1.6 Plant tissue culture studies on C. angustifolia and C. sophera till date 1.7 Objectives

CHAPTER 2 REVIEW OF LITERATURE 15-65

2.1 A brief history of plant tissue culture 2.2 Micropropagation or in vitro propagation 2.3 Tissue culture studies in some medicinally important Cassia spp. 2.3.1 Shoot induction and multiplication 2.3.1.1 C. angustifolia Vahl. 2.3.1.2 C. auriculata L. 2.3.1.3 C. obtusifolia L. 2.3.1.4 C. alata L. 2.3.1.5 C. siamea Lam. 2.3.1.6 C. sophera Linn. 2.3.1.7 C. tora L. 2.3.2 Rooting and acclimatization 2.4 Different strategies of micropropagation in other plant species 2.4.1 Direct regeneration 2.4.1.1 Apical meristem/axillary bud proliferation 2.4.1.1.1 Effect of cytokinins and auxins on shoot multiplication 2.4.1.1.2 Effect of TDZ on shoot multiplication 2.4.1.1.3 Effect of subculture passages 2.4.1.2 Adventitious shoot regeneration 2.4.2 Indirect organogenesis 2.4.3 Somatic embryogenesis 2.5 Rooting in microshoots 2.5.1 In vitro rooting 2.5.2 Ex vitro rooting 2.6 Synthetic seeds 2.7 Acclimatization 2.8 Physiological studies 2.9 Different factors affecting in vitro regeneration 2.9.1 Nature of explant: source, type and age of the explant 2.9.2 Media composition 2.9.3 Sources of carbohydrate and their concentration 2.9.4 pH of the medium

CHAPTER 3 MATERIALS AND METHODS 66-79

3.1 Plant material and explant source 3.2 Culture media 3.2.1 Inorganic nutrients 3.2.2 Organic supplements 3.2.3 Preparation of stock solutions of different media 3.3 Plant Growth Regulators (PGRs) or plant hormones 3.3.1 Preparation of stocks of different PGRs 3.4 Carbon and energy source 3.5 Adjustment of pH and gelling of the medium 3.6 Filling of the medium 3.7 Sterilization 3.7.1 Sterilization of the medium 3.7.2 Sterilization of glasswares and instruments 3.7.3 Sterilization of plant material (seeds) 3.7.4 Sterilization of laminar air flow hood 3.8 Inoculation of seeds and establishment of aseptic seedlings 3.9 Collection of explants and establishment of cultures 3.10 Rooting in microshoots 3.11 Hardening and acclimatization 3.12 Synthetic seeds 3.12.1 Explant source 3.12.2 Encapsulation matrix 3.12.2.1 Encapsulation of explants 3.12.3 Planting media and culture conditions 3.12.4 Low temperature storage 3.12.5 Ex vitro conversion of synthetic seeds into plantlets 3.13 Physiological studies 3.13.1 Chlorophyll and carotenoids estimation 3.13.2 Leaf gas exchange measurements 3.14 Histological studies 3.14.1 Fixation and storage 3.14.2 Embedding, sectioning and staining 3.15 Chemicals and glasswares used 3.16 Statistical analysis

CHAPTER 4 OBSERVATIONS AND RESULTS 80-142

4.1 C. angustifolia Vahl. 4.1.1 In vitro seed germination 4.1.2 Direct shoot regeneration

4.1.2.1 Cotyledonary node (CN) explant 4.1.2.1.1 Effect of explant age on multiple shoot regeneration 4.1.2.1.2 Effect of cytokinins on multiple shoot regeneration 4.1.2.1.3 Effect of cytokinin-auxin combinations 4.1.2.1.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.1.2.1.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.1.2.1.5 Effect of different media 4.1.2.1.6 Effect of pH 4.1.2.1.7 Effect of sucrose concentrations

4.1.2.2 Nodal segment (NS) explant 4.1.2.2.1 Effect of explant age on multiple shoot regeneration 4.1.2.2.2 Effect of cytokinins on multiple shoot regeneration 4.1.2.2.3 Effect of cytokinin-auxin combinations 4.1.2.2.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.1.2.2.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.1.2.2.5 Effect of different media 4.1.2.2.6 Effect of pH 4.1.2.2.7 Effect of sucrose concentrations

4.1.2.3 Shoot tip (ST) explant 4.1.2.3.1 Effect of explant age on multiple shoot regeneration 4.1.2.3.2 Effect of cytokinins on multiple shoot regeneration 4.1.2.3.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration 4.1.2.3.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.1.2.3.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.1.2.3.5 Effect of different media 4.1.2.3.6 Effect of pH 4.1.2.3.7 Effect of sucrose concentrations 4.1.2.4 Effect of subculture passages on shoot multiplication and proliferation

4.1.3 Indirect Organogenesis 4.1.3.1 Cotyledonary leaf (CL) explant 4.1.3.1.1 Effect of explant age on callus production 4.1.3.2 Root (R) explant 4.1.3.2.1 Effect of explant age on callus production 4.1.3.3 Effect of different auxins on callus induction from cotyledonary leaf (CL) and root (R) explants 4.1.3.4 Effect of different cytokinins on callus induction from cotyledonary leaf (CL) and root (R) explants 4.1.3.5 Shoot differentiation from cotyledonary leaf derived callus 4.1.3.5.1 Effect of cytokinins on multiple shoot differentiation 4.1.3.5.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation 4.1.3.6 Shoot differentiation from root derived callus 4.1.3.6.1 Effect of cytokinins on multiple shoot differentiation 4.1.3.6.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation 4.1.3.7 Effect of subculturing and maintenance of cultures

4.1.4 Somatic embryogenesis 4.1.5 Rooting in microshoots 4.1.5.1 In vitro rooting 4.1.5.2 Ex vitro rooting 4.1.6 Hardening and acclimatization 4.1.7 Synthetic seeds 4.1.7.1 Effect of different concentrations of sodium alginate on beads formation 4.1.7.2 Effect of different concentrations of calcium chloride on beads formation 4.1.7.3 Effect of PGRs on conversion of synthetic seeds into plantlets 4.1.7.4 Low temperature storage 4.1.7.5 Ex vitro germination of synthetic seeds 4.1.8 Physiological studies 4.1.8.1 Chlorophyll a, b and total chlorophyll content 4.1.8.2 Carotenoids content 4.1.8.3 Net photosynthetic rate (PN ratio) 4.1.9 Histological studies

4.2 C. sophera Linn. 4.2.1 In vitro seed germination 4.2.2 Direct shoot regeneration

4.2.2.1 Cotyledonary node (CN) explant 4.2.2.1.1 Effect of explant age on multiple shoot regeneration 4.2.2.1.2 Effect of cytokinins on multiple shoot regeneration 4.2.2.1.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration 4.2.2.1.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.2.2.1.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.2.2.1.5 Effect of different media 4.2.2.1.6 Effect of pH 4.2.2.1.7 Effect of sucrose concentrations

4.2.2.2 Nodal segment (NS) explant 4.2.2.2.1 Effect of explant age on multiple shoot regeneration 4.2.2.2.2 Effect of cytokinins on multiple shoot regeneration 4.2.2.2.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration 4.2.2.2.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.2.2.2.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.2.2.2.5 Effect of different media 4.2.2.2.6 Effect of pH 4.2.2.2.7 Effect of sucrose concentrations

4.2.2.3 Shoot tip (ST) explant 4.2.2.3.1 Effect of explant age on multiple shoot regeneration 4.2.2.3.2 Effect of cytokinins on multiple shoot regeneration 4.2.2.3.3 Effect of cytokinin-auxin combination on multiple shoot regeneration 4.2.2.3.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration 4.2.2.3.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation 4.2.2.3.5 Effect of different media 4.2.2.3.6 Effect of pH 4.2.2.3.7 Effect of sucrose concentrations 4.2.2.4 Effect of subculture passages

4.2.3 Indirect organogenesis 4.2.3.1 Cotyledonary leaf (CL) explant 4.2.3.1.1 Effect of explant age on callus production 4.2.3.2 Leaf (L) explant 4.2.3.2.1 Effect of explant age on callus production 4.2.3.3 Effect of different auxins on callus induction from cotyledonary leaf (CL) and leaf (L) explants 4.2.3.4 Effect of different cytokinins on callus induction from cotyledonary leaf (CL) and leaf (L) explants 4.2.3.5 Shoot differentiation from cotyledonary leaf derived callus 4.2.3.5.1 Effect of cytokinins on multiple shoot differentiation 4.2.3.5.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation 4.2.3.6 Effect of subculture passages and maintenance of cultures

4.2.4 Rooting in microshoots 4.2.4.1 In vitro rooting 4.2.4.2 Ex vitro rooting 4.2.5 Hardening and acclimatization 4.2.6 Synthetic seeds 4.2.6.1 Effect of different concentrations of sodium alginate on beads formation 4.2.6.2 Effect of different concentrations of calcium chloride on beads formation 4.2.6.3 Effect of PGRs on conversion of synthetic seeds into plantlets 4.2.6.4 Low temperature storage 4.2.6.5 Ex vitro germination of synthetic seeds 4.2.7 Physiological studies 4.2.7.1 Chlorophyll a, b and total chlorophyll content 4.2.7.2 Carotenoids content 4.2.7.3 Net photosynthetic rate (PN ratio) 4.2.8 Histological studies

CHAPTER 5 DISCUSSION 143-178

5.1 Source of explants 5.2 In vitro seed germination 5.3 Direct regeneration 5.3.1 Type of explant 5.3.2 Age of explant 5.3.3 Effect of cytokinins and auxins on multiple shoot regeneration 5.3.4 Effect of TDZ on multiple shoot regeneration 5.3.5 Effect of subculture passages on proliferation of shoots 5.4 Effect of different media, pH and sucrose concentrations 5.5 Indirect organogenesis 5.5.1 Type of explant 5.5.2 Age of explant 5.5.3 Effect of cytokinins and auxins on callus induction 5.5.4 Effect of cytokinins and auxins on differentiation of shoots from callus 5.5.5 Effect of subculture passages and maintenance of cultures 5.6 Somatic embryogenesis 5.7 Rooting in microshoots 5.7.1 In vitro rooting 5.7.2 Ex vitro rooting 5.8 Acclimatization of regenerated plantlets 5.9 Synthetic seeds 5.10 Physiological studies 5.11 Histological studies

CHAPTER 6 SUMMARY AND CONCLUSIONS 179-192

6.1 Cassia angustifolia Vahl. 6.2 Cassia sophera Linn.

REFERENCES 193-280

Chapter 1 Introduction

Chapter 1 INTRODUCTION

Plants have been used in traditional healthcare system from ancient time, particularly among tribal communities. The World Health Organization (WHO) has registered 20,000 medicinal plants globally and India’s contribution is 15- 20%. According to the WHO estimate, about 80% of the population in the developing countries depends directly on plants for its medicines. In India about 2,000 drugs used are of plant origin (Laloo et al. 2006). Almost all cultures from ancient times till today have used plants as medicine. In recent years there has been renewed interest in natural medicines that are derived from plant parts or plant extracts. The global demand for herbal medicine is not only large, but growing (Srivastava 2000). The international market for traditional products is amount US $ 62 billion and is expected to reach US $ 5 trillion by 2050. The market for Ayurvedic medicines is estimated to be expanding at 20% annually in India (Subrat 2002), while the quantity of medicinal plants obtained from just one province of China (Yunnan) has grown by 10 times in the last 10 years (Pei Shengji 2002). Today medicinal plants are vital to the global economy, as approximately 85% of traditional medicine preparations include the use of plants or plant extracts (Nalawade and Tsay 2004).

1.1 India: A mega biodiversity centre India, on the tropic of cancer, is one of the twelve mega biodiversity centre with 8% of the global biodiversity in 2.4% land. The country is 10th amongst plant rich countries of the world and 4th amongst the countries of Asia. Two of the world’s 25 hot spots – are in India. The Western Ghats as a whole is an abode of rich diverse flora; the natural forests of the region are the birthplace of nearly 500 medicinal plants used for traditional and folk medicinal practices (Krishnan et al. 2011).

India is sitting on a gold mine of well recorded and traditionally well practiced knowledge of herbal medicines. The country is perhaps the largest producer of medicinal herbs and is rightly called the “Botanical garden of the world”. There are very few medicinal herbs of commercial importance which are not found

1 here. Medicinal plants as a group include approximately 8,000 species and account for about 50% of all the higher flowering plant species of India. This proportion of medicinal plants is the maximum known in any other country against the existing flora of that country (Kala et al. 2006). Very small amounts of the medicinal plants are lichens, ferns, algae etc; the majority of the medicinal plants are higher plants (Sharma et al. 2010). India is having 45,000 plant species; its diversity is supreme due to the 16 different agroclimatic zones, 10 vegetative zones and 15 biotic provinces (Samy and Gopalakrishnakone 2007). The country has rich floral diversity (Table 1).

Table 1. Floral diversity in India. Species Numbers

Higher plants 15,000-18,000 Fungi 23,000 Algae 25,000 Lichens 1,600 Bryophytes 1,800 Microorganisms 30 million

1.2 Plants in traditional medicine Plants have been used in traditional medicine for several thousand years. The knowledge of medicinal plants has been accumulated in the course of many centuries based on different medicinal systems such as Ayurveda, Unani, Sidha and Homeopathy. There are considerable economic benefits in the production of indigenous medicines and in the use of medicinal plants for the treatment of various diseases. Due to less communication means, poverty, ignorance and unavailability of modern health facilities, most people especially rural people are still forced to practice traditional medicines for their common day disorders. A vast knowledge of how to use plants against different diseases may be expected

2 to have accumulated in areas where the use of plants is still of pronounced significance (Muthu et al. 2006).

Traditional medical knowledge of medicinal plants and their use by indigenous cultures are not only useful for conservation of cultural traditions and biodiversity but also for community healthcare and drug development in the present and future. Medicinal plants are moving from fringe to main stream use with a greater number of people seeking remedies and health approaches free from side effects caused by synthetic chemicals. Recently considerable attention has been paid to utilize ecofriendly plant based products for the prevention and cure of different human diseases. Considering the adverse effects of synthetic drugs, the western population is looking for natural remedies which are safe and effective. Officially, over 3,000 plants have been recognized in India for medicinal value. Also, it has been estimated that over 6,000 plants are in use in traditional folk medicine and herbal medicine representing about 75% of the medicinal needs of the Third world countries. There are very few medicinal herbs of commercial importance such as Allium sativum, Aloe barbedensis and Panax species which are available in India. Moreover, there are about 7,000 firms manufacturing traditional medicines with or without standardization (Dubey et al. 2004).

The medicinal value of the plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these plants bioactive chemical constituents (phytochemicals and infochemicals) are alkaloids, flavonoids, tannins and phenolic compounds (Kwada and Tella 2009).

1.3 Threat to medicinal plants The search for natural products to cure diseases represents an area of great interest in which plants have been the most important source. The unscrupulous collection of plants from wild habitats, biopiracy and overexploitation by traders has threatened the very existence of valuable medicinal plant resources (Dubey et al. 2004). As a result 20-25% of existing plant species in India became endangered. Also, the degree of threat to medicinal plants has been increased since more than 90% of medicinal raw materials used for herbal industries in India and also for export are drawn from natural habitat. Medicinal plants are

3 now under great pressure due to their excessive collection or exploitation. Plant resources are depleting globally at an alarming rate and if the condition remains same a number of economically and medicinally important plant species will soon be extinct. In the last few decades overexploitation of forest resources has led to many species loss.

Continuous utilization of plant species from the natural population and extensive loss of their habitats during the past 15 years have resulted in the population decline of many high value medicinal plants over the years (Kala 2003). There are many other potential causes of scarcity in medicinal plant species, such as habitat specificity, narrow range of distribution, land-use disturbances, introduction of non-natives, habitat alteration, climatic changes, heavy livestock grazing, explosion of human population, fragmentation and degradation of population, population bottleneck and genetic drift (Kala 2000; Weekley and Race 2001; Oostermeijer et al. 2003 and Kala 2005).

1.4 Conservation of plants through in vitro techniques Global concern about the loss of valuable genetic resources has stimulated many new programs for the conservation of plant genetic resources. Within past decade several conservation strategies were developed mainly in the terms of in situ (within natural habitats) and ex situ (outside natural habitats) conservation (Paunescu 2009). Conservation of medicinal plants is possible through either in situ or ex situ or preferably through a combination of both. The major constraints in the conservation of these plants include slow regeneration rates, over exploitation and destruction of their natural habitats. Therefore, alternative techniques need to be developed for their propagation; advances in biotechnology, especially in vitro techniques and molecular biology provide some important tools for multiplication, conservation and management of medicinal plant resources.

During the last three decades, plant biotechnology has grown into a mature area of study and may be defined as a set of biological tools and techniques used in plants for the development of products of commercial value. One of the most important and widely accepted application of plant biotechnology is cell, tissue and organ culture which is used for various purposes including:

4  micropropagation or multiplication of elite plants  production of virus free plants  regeneration of transformed cells or protoplasts or tissues required for development of transgenic crops with novel traits.

The history of plant biotechnology can be traced back to the history of cell and tissue culture, which had its birth with the demonstration of totipotency of plant cells by G. Haberlandt (1902). In the middle of 20th century the production of virus free plants of Dahlia and potato (Morel and Martin 1952; 1955) through shoot meristem culture laid down the initial milestone in large scale production of important plant species using the technique of micropropagation. Further, Skoog and Miller (1957) discovered the role of plant hormones, especially of cytokinins in shoot morphogenesis and in the inhibition of apical dominance. The clarification of the role of cytokinins in apical dominance inhibition, which subsequently results in the release of axillary meristem from dormancy, was the major break-through in plant tissue culture (Sachs and Thimann 1964). The successful application of such fundamental discoveries to the multiplication of plants by micropropagation has been a key factor in the development of this technology, not only for mass propagation of the existing stocks of germplasm for biomass production, but also for the conservation of economically and medicinally important plant species. The conventional propagation practices for propagation of such plants are time consuming and labor intensive. During the past couple of decades, there has been an increased interest in problems related to the large scale plant production as well as in its cost reduction for commercial micropropagation (Donnan 1986 and Andrea-Kodym and Zapata- Arias 2001). At present, being the only commercially exploited tool of plant biotechnology, the micropropagation technique has been applied to about 1000 plant species including crop plants, ornamental plants, medicinal and aromatic plants and trees (Mehrotra et al. 2007).

Plant tissue culture (PTC) techniques offer an integrated approach for the production of standardized quality phytopharmaceutical through mass production of consistent plant material for physiological characterization and analysis of active ingredients (Debnath et al. 2006). Since conventional breeding methods

5 are unable to meet these requirements therefore, the novel PTC technique can be employed for regenerating plants of medicinal value. The recent development in micropropagation of plants through tissue culture techniques has been of great help in the cultivation of medicinal plants by providing planting material of standard quality (George and Sherrington 1984).

Although modern technology in the field of medical service has accomplished miraculous achievements but still plants have their own importance. Nowadays screening of medicinal herbs as potential sources of new bioactive components of therapeutic value has increased. With an ever increasing global inclination towards herbal medicine, there is an obligatory demand for a huge raw material of medicinal plants. In vitro propagated medicinal plants furnish a prepared source of uniform, sterile and compatible plant material for biochemical characterization and identification of active constituents for their vast uses (Banerjee and Shrivastava 2006 and McCoy and O‘Connor 2008).

Micropropagation or in vitro regeneration is a potential biotechnological tool that provides a solution to the problem of medicinal plants decimation. It refers to the in vitro cultivation of plants, seeds or plant parts (organs, tissues, cells or isolated protoplasts) on a nutrient medium under aseptic conditions. It relies on the fact that many plant cells have the ability to regenerate whole plant (totipotency). Micropropagation can be broadly described in four stages:

1. Establishment of aseptic cultures 2. Proliferation and multiplication of shoots in culture 3. Rooting of microshoots and 4. Hardening and acclimatization of tissue culture raised plantlets.

Tissue culture has now become a well established technique for culturing and studying the physiological behavior of isolated plant organs, tissues, cells, protoplasts and even cell organelles under precisely controlled physical and chemical conditions. The importance and application of plant cell and tissue culture in plant science are vast and varied. The last few years of research into plant cell, tissue and organ culture had shown the emergence of technology from technique. The establishment of micropropagation for rapid propagation, the use

6 of shoot tip culture to produce nuclear stock free from parasites, especially viruses, and the application of a variety of procedures including anther and pollen culture to speed up the process of producing better varieties, protoplast culture for hybrid plant production, and genetic manipulation all have contributed to the acceptance of plant tissue culture not only as a valuable tool for plant propagation and multiplication but also for the plant improvement and conservation.

Most of the medicinal plants either do not produce seeds or seeds are too small and do not germinate in soils. Furthermore, the plants raised through seeds are highly heterozygous and show great variations in growth, habit and yield and may have to be discarded because of poor quality of products for their commercial release. Thus, mass multiplication of important medicinal plants is a great problem. In this regard micropropagation is the best alternative way to provide true to type, rapid and mass multiplication under disease free or aseptic conditions. Callus mediated organogenesis exhibit great chances of somaclonal variations that could be exploited for developing superior varieties particularly in vegetatively propagated plant species. Recently, micropropagation protocols have been developed for a wide range of medicinal plants through different developmental pathways using various explants (Pandey et al. 2002; Martin 2002; Sahrawat and Chand 2002; Thomas and Jacob 2004; Thomas and Philip 2005; Karuppusamy et al. 2006; Bouhouche and Ksiksi 2007; Husain et al. 2008; Parveen et al. 2010; Sahai et al. 2010b; Shahzad et al. 2011; Parveen et al. 2012 and Parveen and Shahzad 2012).

1.5 Plant Description Fabaceae (Leguminosae) is the third largest and extremely diverse family of angiosperms with about 18,000 species classified into about 650 genera. The family is further divided into 3 sub families - Papilionoideae, Caesalpinoideae and Mimosoideae, which are sometimes recognized as three separate families i.e. Papilionaceae, Caesalpinaceae and Mimosaceae. In terms of economic importance Fabaceae is the most important family in the Dicotyledoneae and a large number of crude drugs used in Ayurvedic System employ plants of this legume family. Cassia is one of the most important genus of the family Fabaceae

(sub family Caesalpinoideae) and includes about 600 species which are mostly distributed in Tropics and Subtropics. The members of family Caesalpinaceae include trees, shrubs or rarely herbs which exhibit mostly astringent and mucilaginous properties; some have a pectoral and laxative or cathartic action and some are used as tonic (Parveen et al. 2012).

There are considerable economic benefits in the development of indigenous medicines and in the use of medicinal plants for the treatment of various diseases. Keeping in mind the use of in vitro approaches for the conservation of valuable medicinal plants, the present study was focused for in vitro propagation of two medicinally important Cassia spp. i.e. C. angustifolia and C. sophera through various explants via direct regeneration, indirect organogenesis and somatic embryogenesis.

1.5.1 Cassia angustifolia Vahl. Synonyms: C. senna L.; C. acutifolia Delite; C. obovata Baker

Common name: Senna; Alexandrian/Bomabay/Tinnevelly senna

Systemic Position: Kingdom: Planteae Division: Magnoliophyta Class: Magnoliopsida Order: Fabales Family: Fabaceae Subfamily: Caesalpinoideae Genus: Cassia Species: angustifolia

1.5.1.1 Distribution Senna is a shrub native to Egypt, Sudan, Nigeria and North Africa, as well as India, Pakistan and China. In India, it is mainly cultivated in the states of Tamil Nadu, Maharashtra, Gujrat, Rajasthan and Delhi.

The plant has been put in the priority list of National as well as State Medicinal Plant Board for development. It is one of the principal herbal drugs having export potential for developed countries. India is the major supplier of the leaves and pods (shells) as well as senna glycosides to the world market. Approximately 75% of the senna produced in India is exported. Presently the most important markets are Germany, Japan, Czechoslovakia, USA and Hong Kong. Annual demand of the plant in 2001-2002 was reported to be 6462.5 tonnes, which has gone up to 11677.5 tonnes by the year 2006 (Shrivastava et al. 2006).

1.5.1.2 Morphological characters A small shrub about 1.0 to 1.8 m in height with pale subterate or obtusely angled erect or ascending branches, leaves usually 3-9 pairs, leaflets oval, lanceolate, glabrous (Figure 1). Racemes axillary, erect, brilliantly yellow, waxy many flowered. Pods light green when young to dark brown or black when mature, flat, thin, oblong, 3.5-7.0 cm long contains 5-7 obovate dark brown and nearly smooth seeds.

1.5.1.3 Medicinal uses Its medicinal uses were first described in the writings of Arabian physicians Serapion and Mesue as early as the 9th century AD, the name senna itself is Arabic. The leaves and pods are usually used in the Ayurvedic and Unani systems of medicine as infusion and considered a great tonic. In India, several household preparations such as decoction, powder, syrup, infusion and confection are made with senna. Besides being an excellent laxative the senna is used as a febrifuge in splenic enlargemens, anaemia, typhoid, cholera, biliouseness, jaundice, gout, rheumatism, tumours, foul breath and bronchitis and probably in leprosy. It is employed in the treatment of amoebic dysentery, as an anthelmintic and as a mild lever stimulant.

The leaf is one of the constituent of patented drug reported to have protective effects on the lever. The leaf in the form of confection of senna is used for certain skin diseases and the powdered leaves in vinegar are applied to wounds and burns and to remove pimples. The callus cultures and crude alcoholic extract (50%) showed a strong anti-microbial activity against gram positive bacteria (Anonymous 1992a).

Figure 1. Cassia angustifolia

1.5.1.4 Active compounds

Two active anthraquinones, sennosides A (C42H38O20, M.P. 200-240°C) and B

(C42H38O20, M.P. 180-186°C), which are optical isomers, have been isolated from the leaves and pods. Beside these sennosides, presence of sennosides C

(C42H40O19, M.P. 197-205°C decomp.), D (C42H40O19, M.P. 210-220°C decomp),

G (C42H38O20, M.P. 162-176°C decomp), III and A, is also reported. Three sennidins are also present in the leaves. The leaves, pods and roots contain rhein, chrysophanol, emodin and aloe-emodin. Several mono and di-glucosides of anthrone are present in the seedlings, leaves and roots. The leaves also contain mono-β-D glucosides of rhein and aloe-emodin and a water soluble glycoside, which is supposed to be responsible for the synergistic effect.

1.5.1.5 Commercial products Senna fluid extract, senna fruit, compound senna powder (compound licorice powder), senna syrup, senna tablets, senna tea, yashtyadi churna, shataskar churna.

1.5.1.6 Other uses Senna is a potential crop for honey bee culture also, as blooming senna provides food supply for honey bee round the year. C. angustifolia is native of Saudi Arabia and has been naturalized in India. The plant is ecofriendly and has been recommended for developing wastelands and does not require frequent irrigation. Its drought resistance, quick establishment and perennial nature provide permanent green cover on sand in vegetation deficient arid zones (Agrawal and Sardar 2003).

1.5.1.7 Propagation Conventionally it is propagated only through seeds. However poor seed viability and low seed germination frequently restricts its propagation on a large scale. The plant grows well in red soil and black soil; even it grows well in saline and alkaline soil. The pH range for better growth is between 7.0 and 8.5 with adequate drainage.

1.5.2 Cassia sophera Linn. Synonyms: Senna sophera

Common name: Pepper leaved senna, African senna, Kasondi, Baner, Kalkasonji

Systemic Position: Kingdom: Planteae Division: Magnoliophyta Class: Magnoliopsida Order: Fabales Family: Fabaceae Subfamily: Caesalpinoideae Genus: Cassia Species: sophera

1.5.2.1 Distribution Originates from tropical America, but is now pantropical. It occurs throughout tropical Africa, being common in West Africa, but in East Africa and Madagascar it is probably rare. Found throughout India ascending the Himalayas up to an altitude of 750 m. It is common in wastelands, on roadside and in the forest.

1.5.2.2 Morphological characters A diffuse, sub-glabrous, shrubby herb or under shrub, 0.7-3.0 m in height (Figure 2). Leaves foetid, unipinnate 20-30 cm long, leaflets 8-12 pairs, oblong or lanceolate, flowers yellow in short axillary or terminal corymbose racemes, pods slightly falcate, subterete or terete, 6.5 cm x 0.6 cm, seeds dark brown in colour and about 30-40 seeds per pod.

1.5.2.3 Medicinal uses The plant exhibited anticancer activity and is reported to be extensively used in Homeopathy. The leaves possess purgative and antidiuretic properties. It is used as an infusion or decoction or as an expectorant for cough, cold, bronchitis, asthma, hiccups and jaundice and in subacute stages of gonorrhoea. Internally, it is reported to act as a febrifuge in rheumatic and inflammatory fevers and in

Figure 2. Cassia sophera

skin diseases. A paste of the leaves is used for washing syphilitic sores. The seeds are cathartic and also used as a febrifuge (Anonymous 1992b). The chemical analysis of the seeds revealed the presence of the ascorbic acid, dihydroascorbic acid and β- sistosterol (Bilal et al. 2005).

The powdered seeds mixed with honey are used in diabetes. An ointment made from the seeds is used to cure ringworm, sores, scabies and psoriasis. The bark like the leaves and seeds is cathartic in action. Its infusion is considered useful in diabetes and the juice in asthma. Kasondi is described in unani literature to be repulsive of morbid humors (especially phlegm), resolvent, blood purifier, carminative, purgative, digestive, diaphoretic and reported to be useful in epilepsy, ascites, dyscracia of liver, skin disorders, piles, fever, articular pain and palpitation. In ethnobotanical literature, it is reported to be effective in the treatment of pityriasis and convulsions of children (Bilal et al. 2005). The methanol extract of the leaves of C. sophera can be used as a source of natural antioxidants with potential application to reduce oxidative stress with health benefits (Rahman et al. 2008).

1.5.2.4 Propagation Conventionally it is also propagated through seeds. However, hard seed coat and seed dormancy prevent its germination in nature.

1.6 Plant tissue culture studies on C. angustifolia and C. sophera till date Different regeneration pathways such as direct regeneration, indirect organogenesis via callus formation and somatic embryogenesis has been explored in past by several workers in Cassia angustifolia and C. sophera to get optimum multiplication rate utilizing different explants. The work done on the C. angustifolia and C. sophera has been summarized below in table 2:

Table 2. Current status of tissue culture work done on two medicinally valuable Cassia species viz: C. angustifolia and C. sophera.

Species Explants References

C. angustifolia CN, NS, ST Agrawal and Sardar 2003

L, C Agrawal and Sardar 2006

C Agrawal and Sardar 2007

NS Siddique and Anis 2007a

CN Siddique and Anis 2007b

P Siddique et al. 2010

R Parveen and Shahzad 2011

C Parveen et al. 2012

C. sophera CN Parveen and Shahzad 2010

C: Cotyledon, CN: Cotyledonary node, L: Leaf, NS: Nodal segment, P: Petiole, R: Root, ST: Shoot tip

1.7 Objectives The following objectives were carried out to achieve the goals under the broad prospective described above: 1. To germinate seeds of C. angustifolia and C. sophera under aseptic conditions. 2. To establish in vitro cultures from various explants. 3. To select best suited media and culture conditions for direct and indirect organogenesis and to evaluate the specific role of various plant growth regulators. 4. To standardize reliable protocol for induction of somatic embryos. 5. To encapsulate the vegetative tissues for synthetic seed production. 6. To evaluate optimal medium for root induction in microshoots, germination of somatic embryoids and synthetic seeds followed by successful acclimatization.

7. To study the developmental pathways through histology of differentiated tissues. 8. To study various physiological parameters during ex vitro establishment of in vitro raised plantlets.

Chapter 2 Review of Literature

Chapter 2

REVIEW OF LITERATURE

2.1 A brief history of plant tissue culture The foundation of plant tissue culture and modern biotechnology can be drawn back to the “Cell Theory” of Schleiden (1838) and Schwann (1839) which recognised the cell as the primary unit of all living organisms. This theory holds that the cell is the primary unit of structure and function in an organism and therefore capable of autonomy. The concept of ‘totipotency’ itself is inherent in the cell theory, which forms the basis of modern biology. The cell theory received much impetus from the famous aphorism of Virchow (1858), ‘Omnis cellula e cellula’ (All cells arise from a cell), and by the very prescient observation of Vöchting (1878) that the whole plant body can be built up from ever so small fragments of plant organs. However, no sustained attempts were made to test the validity of these observations until the beginning of the 20th century (Gautheret 1985).

The great German botanist Gottlieb Haberlandt (1902), recognised as the father of plant tissue culture, was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf cells of different plant species like palisade cells from leaves of Lamium purpureum, glandular hairs of Pulmonaria and pith cells from petioles of Eicchornia crassipes etc. on Knop’s salt solution (1865) enriched with glucose. In his cultures, cells increased in size, accumulated starch but failed to divide. He failed largely because of the poor choice of experimental materials; insufficient nutrients and infection (Vasil and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to regenerate artificial embryos (somatic embryos) from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells. After Haberlandt’s failure Hanning (1904) choose embryogenic tissue to culture and successfully grew them to maturity on mineral salts and sugar solution. For the next thirty years (up to 1934) there was very little further progress under cell culture research. Within this period an innovative approach to tissue culture using root and stem tips was reported by Kotte (1922) in Germany and Robbins

(1922) in the United States. Further, Robbins and Maneval (1924) reported some improvement in root growth but the first successful report of continuously growing cultures of tomato root tips was made by White in (1934).

The discovery of the naturally occurring auxin indole-3-acetic acid (IAA) by Fritz Went in 1926 and its beneficial effects on plant growth (Went 1928; Kögl et al. 1934 and Thinmann 1935), soon led to its assimilation in plant nutrient media (White 1934 and Gautheret 1985). Several years later, in 1939, three researchers independently used the newly isolated plant growth regulator, IAA (auxin), to establish callus cultures with the potential for indefinite growth. These researchers were RJ Gautheret (Gautheret 1939) and P Nobecourt (Nobecourt 1939) from France working with carrot cells and PR White (White 1939) from the United States, working with tobacco cultures. PR White and A Braun (White and Braun 1941) found that cells isolated from tobacco infected with crown gall would show cell division and growth in cultures without the addition of auxin. Proving one of the predictions of Haberlandt true, in 1941 Van Overbeek and co-workers demonstrated for the first time the stimulatory effect of coconut milk on embryo development and callus formation in Datura (Van Overbeek et al. 1941).

Initially, the explants containing meristematic cells were used to obtain unlimited growth of plant tissues. Continued cell division and bud formation were soon obtained when tobacco pith tissues that contained mature and differentiated cells were cultured on nutrient media containing adenine and high levels of phosphate (Skoog and Tsui 1951). However, cell divisions occurred only when the explant included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts including coconut milk were supplemented to the nutrient medium in an attempt to replace vascular tissues and to identify the factors responsible for their beneficial effect. Among these, yeast extract was found to be most effective and its active component was shown to have purine like properties. This finding led to the addition of DNA to the medium which greatly enhanced the cell division activity (Vasil 1959). These investigations resulted in the isolation of ‘kinetin’ from old samples of herring sperm DNA (Miller et al. 1955) and the understanding of the shoot morphogenesis in plants (Skoog and Miller 1957).

Later studies led to the isolation of naturally occurring as well as many synthetic cytokinins. Since then tissue culture studies revolutionized the improvement in plant species and conservation. The dream of Haberlandt for the cultivation of somatic embryos comes true when the first report on somatic embryogenesis appeared in carrot tissue by Reinert (1958) and Steward et al. (1958).

Besides PGRs, scientists tried to improve culture media by differing essentiality in mineral content. In this direction Murashige and Skoog (1962) prepared a medium by increasing the concentration of some salts by twenty five times higher than Knop’s solution (1865). The principal novel features of the new medium (MS medium) were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol. Even today MS medium is the most widely used culture medium and has immense commercial application in tissue culture.

In 1966, Guha and Maheshwari cultured anthers of Datura and raised embryos which developed into haploid plants initiating androgenesis (Guha and Maheshwari 1964; 1966), which like many great advances in science was a chance discovery (Guha-Mukherjee 1999). The technology was further defined and improved by the work of Nitsch and Nitsch (1969). Vasil and Hildebrandt (1965; 1967) were first to regenerate plantlets from colonies of isolated cells of hybrid N. glutinosa x N. tabaccum. In 1966, the classical work of Steward on induction of somatic embryos from free cells in carrot suspension cultures brought an important breakthrough by finally demonstrating totipotency to somatic cells, thereby, validating the ideas of Haberlandt. Kohlenbach (1966) successfully cultured mature mesophyll cells from Macleaya cordata, the tissue obtained from these cells subsequently differentiated somatic embryos. Until the mid - 1970’s hormonal manipulation in the culture medium remained the main approach to achieve plant regeneration from cultured cells and it proved very successful with many species. However, some very important plants such as cereals and legumes did not respond favourably to this strategy and were, therefore, declared recalcitrant (Bhojwani et al. 1977). In 1972, Saunders and Bingham reported that different cultivars of alfalfa varied considerably in their regeneration potential under a culture regime. More detailed study by Bingham

17 and his associates (Bingham et al. 1975 and Reisch and Bingham 1980) demonstrated that regeneration in tissue cultures is a genetically controlled phenomenon.

In 1974, Murashige defined three major stages of the micropropagation technique. These are:

Stage I: Establishment of aseptic cultures.

Stage II: Multiplication and propagation of propagules.

Stage III: Re-establishment in natural environment.

Subsequently, Debergh and Maene (1981) introduced an additional stage that is Stage 0: care and preparation of stock plant. This is because of the increased recognition of the importance of the genetic make-up of the plant from which explants made. Additionally Murashige’s Stage III is further divided into Stage III: rooting in microshoots and Stage IV: acclimatization of the rooted plantlets. Stage IV is the most important and crucial stage of micropropagation as plants slowly acclimatizes to the external environment of lower relative humidity (Preece and Sutter 1991). However, Stage III and IV can be combined by rooting microshoots under ex vitro conditions, where rooting and acclimatization took place simultaneously, as in Murashige’s original Stage III.

Today, tissue culture technique has been widely accepted as a tool for biotechnology for vegetative propagation of plants of agriculture, horticulture and forestry importance (Chu and Kurtz 1989 and Dave and Purohit 2002). It allows rapid and large scale multiplication of selected plant species under controlled conditions. Micropropagation of various plant species, including many medicinal plants has been reported. This chapter reviews the achievements and advances in the application of tissue culture for the in vitro regeneration of medicinal plants from various explants.

2.2 Micropropagation or in vitro propagation It is referred to the aseptic culture of cells, tissues, organs and their components under defined physical and chemical conditions in vitro (Thorpe 2007). It is also known as ‘micropropagation’ in scientific technology. The technique of

18 micropropagation is based on the concept of totipotency as proposed by Haberlandt (1902), every cell of the plant body is totipotent i.e. capable of giving rise to a new plant under controlled conditions. Callus mediated organogenesis and regeneration through somatic embryogenesis are the usual mode of regeneration by tissue culture. There are three ways or strategies by which micropropagation can be achieved:

i) Axillary bud breaking – direct regeneration ii) Production of adventitious buds directly/indirectly via callus - indirect organogenesis iii) Somatic embryogenesis directly/indirectly on explants

The use of plant tissue culture technology for the vegetative propagation of plants is the most widely used application. Micropropagation has many advantages over conventional method of vegetative propagation, which suffer from several limitations (Debnath et al. 2006). The most outstanding merits offered by tissue culture technique over conventional methods are:

1) In a relatively short time and space large number of plants can be produced, starting from single explant. 2) Unlike the conventional methods of plant propagation, micropropagation of even temperate species may be carried out throughout the year. 3) Regenerated plants are generally free from bacterial and fungal diseases. 4) Virus eradication and maintenance of plants in a virus free state are also readily achieved in tissue culture. 5) The multiplication rate is greatly increased. 6) It also permits the production of pathogen free material.

2.3 Tissue culture studies in some medicinally important Cassia spp. Some of the important medicinal species of genus Cassia are C. alata, C. angustifolia, C. auriculata, C. obtusifolia, C. fistula, C. occidentalis, C. siamea, C. sophera and C. tora. Various regeneration protocols utilizing different strategies of micropropagation have been developed for these medicinal plant species which are summarized in table 3 and have been reviewed by Parveen and 19

Shahzad (2012). A brief account of the micropropagation protocols developed for important Cassia spp. is given below:

2.3.1 Shoot induction and multiplication 2.3.1.1 C. angustifolia Vahl. This is a highly valuable medicinal species of Cassia, in which micropropagation studies have conducted using various explants. Agrawal and Sardar (2003) obtained only 2.4 shoots/explant from cotyledonary nodes of aseptic seedlings of senna on MS medium supplemented with 1.0 µM BA. However, Siddique and Anis (2007a) reported the maximum 17.6 shoots from CN explants when pretreated with 1.0 µM TDZ for 4 weeks and then subsequently transferred to MS basal medium. Furthermore, Siddique and Anis (2007b) improved the shoot regeneration efficiency through nodal segments with the production of maximum 21.7 shoots/explant, when the pretreated explants on MS + TDZ (5.0 µM) + NAA (1.0 µM) for four weeks, were transferred on MS basal medium. Indirect organogenesis using different explants viz. leaflet, cotyledon, petiole and root has also been reported in C. angustifolia through induction of regenerative callus on various hormonal supplements. Agrawal and Sardar (2006) reported regeneration through leaflets and cotyledons derived callus on MS + BA (1.0 µM) + 2,4-D (1.0 µM).

Siddique et al. (2010) reported shoot regeneration through petiole derived callus induced on MS medium supplemented with 2,4-D (5.0 µM) + TDZ (2.5 µM). Optimal response with the production of a maximum of 12.5 shoots was achieved on MS + TDZ (5.0 µM) + IAA (1.5 µM) with an average shoot length of 4.3 cm. However, single TDZ (5.0 µM) treatment was lesser effective and yielded only 8.5 shoots/explant. Besides leaflet, cotyledon and petiole explants another explant, root, had been tried by Parveen and Shahzad (2011) for the induction of regenerative calli and it was found that root explant significantly proved to be the best explant for the induction of organogenic calli in C. angustifolia. Root segments (1-2 cm long) taken from 30 days old aseptic seedlings were cultured on MS medium supplemented with different cytokinins (BA, Kn and TDZ) at various concentrations. The dark green or brown regenerative calli were induced on the MS medium containing TDZ (1.0 µM). This regenerative callus was found

20 to be suitable for shoot induction when transferred to the MS medium containing different concentrations (1.0, 2.5 and 5.0 µM) of cytokinins (BA, Kn and TDZ). Among three cytokinins tested, BA at 2.5 µM produced maximum of 24.5 shoots per explants after 6 weeks of culture. The synergistic effect of cytokinin-auxin combination further enhanced the rate of shoot buds formation from the callus. When the optimal concentration of BA (2.5 µM) was tested with different concentrations of two auxins (IAA & NAA), a further increase in the number of shoot buds per culture was achieved. The medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM) proved to be optimal with the production of a maximum of 35.6 shoots/explant having average shoot length of 5.4 cm.

Parveen et al. 2012 reported enhanced shoot organogenesis in C. angustifolia via callus production through 14 days old cotyledon explants. Callus was induced at various concentrations of 2,4-D and 2,4,5-T. The organogenic callus induced at 5.0 µM 2,4-D, proved to be the best for shoot regeneration when transferred on to MS medium containing cytokinins and auxins singly or in combination. Maximum shoot differentiation with the production of 23.2 ± 1.4 shoots/explant having shoot length of 5.0 ± 0.3 cm was obtained at BA (5.0 µM) + NAA (0.4 µM).

Somatic embryogenesis in C. angustifolia was reported by Agrawal & Sardar (2007) using immature cotyledons dissected from semi mature seeds and inoculated on MS medium containing different auxins (2,4-D, NAA or IAA) alone or in combination with cytokinins such as BA, Kn or 2-iP. A maximum of 75% cotyledons differentiated into somatic embryos on 2,4-D, however, such embryos were failed to form complete plantlets. Addition of BA along with 2,4-D improved this problem. Optimum response was obtained at 2,4-D (5.0 µM) + BA (2.5 µM) where an average of 5.36 somatic embryos were formed along with 2.0 shoots/culture. Some of the embryos showed precocious germination on the same medium. However, a few of them developed into complete plants if transferred to half strength MS basal containing 2% sucrose. Cytokinins alone did not induce somatic embryogenesis but formed multiple shoots. BA at 5.0 µM proved optimum for recurrently inducing shoots in the competent callus with a maximum of 12.0 shoots and an average shoot length of 2.2 cm. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.

Table 3. List of medicinally important Cassia spp. propagated through tissue culture

Species Explants Best Treatment Response References

C. alata CN - - Fett-Neto et Indirect al. 2000 Organogenesis

C. angustifolia CN, NS, MS + BA (1.0 µM) 2.4 shoots/CN Agrawal & ST explant Sardar 2003 Direct Regeneration

C. angustifolia L, C MS + 2,4-D (1.0 µM) 12.0 Agrawal & + BA (1.0 µM) shoots/explant Sardar 2006 Indirect Organogenesis

C. angustifolia C MS + 2,4-D (10.0 µM) 12.0 Agrawal & + BA (2.5 µM) shoots/explant Sardar 2007 Somatic Embryogenesis

C. angustifolia NS MS + TDZ (5.0 µM) + 21.7 Siddique & IAA (1.0 µM) shoots/explant Anis 2007a Direct Regeneration

C. angustifolia CN MS + TDZ (1.0 µM) 17.6 Siddique & shoots/explant Anis 2007b Direct Regeneration

C. angustifolia P MS + 2,4-D (5.0 µM) 12.5 Siddique et + TDZ (2.5 µM) shoots/explant al. 2010 Indirect Organogenesis

C. angustifolia R MS + TDZ (1.0 µM) 35.6 Parveen & shoots/explant Shahzad Indirect 2011 Organogenesis

C. angustifolia C MS + 2,4-D (5.0 µM) 23.2 Parveen et shoots/explant al. 2012 Indirect Organogenesis

C. auriculata CN MS + BA (1.0 mg/l) + >16.0 Negi et al. NAA (1.0 mg/l) + AdS shoots/explant 2011 (25 mg/l) + ascorbic Direct acid (20 mg/l) + regeneration glutamine (150 mg/l)

C. obtusifolia ST MS + 2,4-D (2.0 mg/l) 5.0 Hasan et al. + Kn (0.2 mg/l) shoots/explant 2008 Indirect Organogenesis

E - - Bajaj et al. 1988 C. fistula Gharyal & P, S B5 + BA (1.0 mg/l) + Indirect Maheshwari IAA (0.5 mg/l) Organogenesis 1990

A 2,4-D (2.0 mg/l) + Kn - Gharyal et (0.5 mg/l) + CW Callus induction al. 1983

P, S B5 + BA (1.0 mg/l) + - Gharyal & IAA (0.5 mg/l) Indirect Maheshwari C. siamea Organogenesis 1990

12.2 Parveen et CN MS + BA (1.0 µM) shoots/explant al. 2010 Direct Regeneration

C. sophera CN MS + TDZ (2.5 µM) 14.9 Parveen & shoots/explant Shahzad Direct 2010 Regeneration

C. tora NS MS + BA (2.2 µM) 1.5 Quraishi et shoots/explant al. 2011 Direct Regeneration

A: Anther; C: Cotyledon; CN: Cotyledonary node; E: Embryo; L: Leaf; NS: Nodal segment; P: Petiole; R: Root; S: Stem; ST: Shoot tip

2.3.1.2 C. auriculata L. Negi et al. (2011) developed a micropropagation protocol for C. auriculata using CN explants and also performed anatomical comparison between in vivo and in vitro developed shoots and roots. Explants containing cotyledonary node along with cotyledonary leaves and portion of hypocotyl were cultured on MS media supplemented with different growth regulators in varying concentrations. They found more than 16.0 shoots/explant in MS medium supplemented with BA (1.0 mg/l) + NAA (1.0 mg/l) + AdS (25 mg/l) + ascorbic acid (20 mg/l) + glutamine (150 mg/l). Moderate green to brown, hard and nodular callus was observed originating from cotyledonary leaf margins in MS medium supplemented with BA (3.0 mg/l) + 2,4-D (1.0 mg/l).

2.3.1.3 C. obtusifolia L. The in vitro propagation of this valuable medicinal plant from shoot tips was reported for the first time by Hasan et al. (2008). Different concentrations and combinations of auxin and cytokinin were used in full strength of MS medium to obtain callus, shoot regeneration and root proliferation. The highest percentage of callus (96.6%) was observed on MS medium supplemented with 2.0 mg/l 2,4- D. A maximum of 5.0 shoots/explant was produced on MS medium comprised of 2.0 mg/l 2.4-D + 0.2 mg/l Kn.

2.3.1.4 C. alata L. There is a single report on the micropropagation of C. alata (Fett-Neto et al. 2000), where CN explant was used for the induction of morphogenic calli in 83.0% cultures.

2.3.1.5 C. siamea Lam. Gharyal et al. (1983) reported callus formation from the cultured anthers of C. siamea. Anthers were split open after 1 to 2 weeks of inoculation on B5 medium supplemented with coconut water (CW) (15%) + 2,4-D (2.0 mg/l) + Kn (0.5 mg/l) and produced callus mass. Microscopic examination of the anthers cultured at the late uninucleate or early bi-celled stages, after 7-14 days of culture, revealed many multicellular structures at various stages of development, thus indicating the pollen origin of callus. Gharyal and Maheshwari (1990) also reported the formation of callus from stem and petiole explant of two important Cassia spp.

24 viz: C. fistula and C. siamea. Regenerative calli were induced on B5 medium supplemented with BA (1.0 mg/l) and IAA (0.5 mg/l), although green meristemoids were observed from both types of explants but shoots developed only from stem explants in both the species.

Parveen et al. (2010) for the first time developed an in vitro protocol for the direct shoot regeneration of C. siamea using CN explants taken from aseptically grown seedlings and cultured on MS medium supplemented with different cytokinins (BA, Kn and TDZ) and auxins (IAA, IBA and NAA) either alone or in combination. Among three cytokinins tested, BA at 1.0 µM produced a maximum of 8.2 shoots per explants in 80% cultures. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest number of shoots (12.2 shoots/explant) were obtained at MS + BA (1.0 µM) + NAA (0.5 µM) with 90% regeneration percentage.

2.3.1.6 C. sophera Linn. Parveen and Shahzad (2010) for the first time reported the development of a regeneration protocol for this highly valuable medicinal plant by culturing CN explant on TDZ supplemented MS medium. Explants were collected from 21 days old in vitro raised seedlings and incubated under controlled condition on different concentrations of TDZ. TDZ at 2.5 µM proved to be optimal for the production of maximum number (6.7) of shoots/explant. To avoid adverse effects of prolonged exposure of TDZ in long term establishment, the cultures were transferred to TDZ free MS medium fortified with various concentrations of BA for multiplication, proliferation and elongation of induced shoots. Emergence of new shoot buds and multiplication continued up to second subculture passage and maximum number of 14.9 shoots/explant were obtained on MS + BA (1.0 µM).

2.3.1.7 C. tora L. Micropropagation of this medicinal legume via nodal bud culture method has been first time reported by Quraishi et al. (2011). Nodal explants taken from field grown plants were used to initiate in vitro culture with 90% shoot bud induction producing a maximum of 1.5 ± 0.1 shoots/explant on MS medium supplemented with 2.2 µM BA. Heavy black leaching was observed in the initiation medium

25 from cut ends of the explants, which was effectively checked by incorporation of an absorbent (25 µM PVP-40) and an antioxidant (476 µM citric acid).

2.3.2 Rooting and acclimatization In C. angustifolia rooting has been achieved using different strategies of rooting by different workers. Agrawal and Sardar (2003) reported 80% rooting in C. angustifolia, with the production of 3-5 roots within 20 days on half strength MS medium supplemented with 10.0 µM NAA. Rooted plantlets were taken out of the medium after 1 month, dipped in 0.1% bavistin for half an hour before their transfer to soilrite. Maintained there for 1 week for in vitro hardening and subsequently transferred to soil for further acclimatization. However, in other reports on the same plant species, 95% rooting with 5.4 ± 0.41 roots/shoot (Agrawal and Sardar 2006) and 91.6% rooting with 5.12 0.58 roots/shoot (Agrawal and Sardar 2007) were reported on half strength MS medium containing 10.0 µM IBA. Acclimatization in both these reports was done by adopting the same procedure described by Agrawal and Sardar (2003). Contrary to these reports, Siddique and Anis (2007b) achieved only 29-52% rooting with 3.6 ± 0.29 roots/shoot having root length of 3.9 ± 0.40 cm in C. angustifolia using a two-step rooting procedure by giving a pulse treatment with 60 µM IBA and 1% activated charcoal in MS medium for 1 week and subsequently transferring the shootlets to half strength MS liquid medium without IBA and activated charcoal. The rooted plantlets were hardened off in sterile soilrite for 4 weeks and eventually established in natural soil. The two-step rooting procedure was again tried by Siddique et al. (2010) for the production of maximum 3.3 ± 0.25 roots/shoot having root length of 3.1 ± 0.23 cm in 50.5 ± 3.23% microshoots by giving a pulse treatment with 10 µM IBA for 2 weeks and subsequently transferring the microshoots to MS liquid medium without IBA. In a recent report by Parveen et al. (2012), rooting was best induced (82.0%) on half strength MS medium containing only 1.0 µM IBA along with 5.0 µM PG with the production of 4.8 ± 0.4 roots/shoot having root length of 4.3 ± 0.5 cm after 4 weeks. Regenerated plantlets with well-developed root system were transferred to plastic pots containing sterilized soilrite and hardened off under culture conditions for 4 weeks, after that transferred to earthen pots containing sterilized soilrite and maintained in green house with 85% survival rate.

Ex vitro root induction in C. angustifolia was achieved through pulse treatment with IBA (200 µM) for half an hour followed by their transfer to sterile soilrite (Siddique and Anis 2007a and Parveen and Shahzad 2011). Ex vitro rooting decreased the micropropagation cost and the time from laboratory to field conditions as rooting and acclimatization took place simultaneously. After 6 weeks, the regenerated plantlets were successfully transferred to earthen pots containing sterilized soil and manure (1:1) and maintained in green house with 90% survival rate (Parveen and Shahzad 2011).

In C. auriculata (Negi et al. 2011) rooting was induced from the cut end of the hypocotyls in MS medium supplemented with IBA (1.0 mg/l) or NAA (1.0 mg/l). While, the use of 2.0 mg/l NAA was significant for the production of the highest 5.0 roots/shoot in 80% cultures of C. obtusifolia (Hasan et al. 2008). After 35 days well-rooted plantlets of C. obtusifolia were transferred in plastic pots containing sterile sand, soil and farmyard manure in the ratio of 1:1:1. After proper acclimatization, the plantlets were transplanted in the natural condition with 70% survival.

In Cassia alata (Fett-Neto et al. 2000) rooting was best achieved on IBA supplemented medium and the regenerated plantlets with well-developed root system were successfully transferred to soil.

In C. siamea, successful in vitro rooting was induced from cut end of the microshoots on half strength MS containing 2.5 µM IBA with the production of 3.60 ± 0.24 roots/shoot having root length of 7.88 ± 0.28 cm in 84% microshoots (Parveen et al. 2010). Plantlets with well-developed root and shoot system were hardened off inside the growth room in sterile soilrite for 4 weeks. After successful acclimatization plantlets were transferred to earthen pots containing sterilized garden soil and garden manure and maintained in green house with 85% survival rate.

Similary, in C. sophera (Parveen and Shahzad 2010), rooting was best (93.6%) obtained on half strength MS medium comprised of 1.0 µM IBA with the production of maximum 5.7 ± 0.5 roots/shoot having 5.6 ± 0.5 cm root length after 6 weeks. Regenerated plantlets with well-developed root system were isolated from the rooting medium and hardened off inside the growth room. After

27 acclimatization, plantlets were successfully transferred to greenhouse conditions with 90% survival rate.

In C. tora (Quraishi et al. 2011), healthy and elongated roots with secondary branches were recorded in 100% shoots on half strength MS medium supplemented with 2.5 or 4.9 µM IBA. Plantlets with well-developed root system were acclimatized and transferred to green house with 70% survival rate.

2.4 Different strategies of micropropagation in other plant species 2.4.1 Direct regeneration In vitro regeneration without an intervening callus phase resulted in the direct induction of shoots from various explants and produced genetically identical plants (Hu and Wang 1983). Direct regeneration may occur either through pre- existing meristems (axillary/shoot tip meristems) or from the well differentiated tissues (leaf, stem, cotyledon, petiole, root etc.) lacking the meristem, in that case it is referred as adventitious regeneration.

2.4.1.1 Apical meristem/axillary bud proliferation Shoots of all angiosperms and gymnosperms grow by virtue of their apical meristem. The apical meristem is usually a dome of tissue located at the extreme tip of a shoot and measures approximately 0.1 mm in diameter and approximately 0.25-0.30 mm in length. The apical meristem together with one to three leaves primordial measuring 0.1-0.5 mm constitutes the shoot apex. The first report of apical meristem culture was obtained in 1946 by Ball (Ball 1946). He successfully raised transplantable whole plants of Lupinus and Tropaeoleum by culturing their shoot tips with a couple of leaf primordial. However, Morel together with Martin (Morel and Martin 1952) further refined this technique and for the first time recovered virus free Dahlia plants by culturing shoot tips in vitro. This technique of apical meristem culture since then widely been used with a variety of plant species and has become the most efficient technique for obtaining completely virus free plants (Belkengren and Miller 1962; Mullin et al. 1974 and Boxus et al. 1977). G Morel was the pioneer in applying shoot tip culture for micropropagation of orchid Cymbidium (Morel 1965).

Shoot proliferation from isolated apical or axillary bud under the influence of different PGRs is the most frequently used micropropagation method for commercial mass production of plants as it ensures maximum genetic uniformity of the resulting plants. It involved the stimulation of axillary buds, which are usually present in the axil of leaves, to develop into a shoot. In nature axillary buds remain dormant for various periods depending upon the growth pattern of the plant. In some plants, removal of terminal bud is necessary to break the apical dominance and stimulate the axillary bud to grow into a shoot.

2.4.1.1.1 Effect of cytokinins and auxins on shoot multiplication Wickson and Thimann (1958) showed that the growth of axillary buds, which remain dormant in the presence of terminal buds, can be initiated by the exogenous application of cytokinins. Since then a large number of pants have been successfully micropropagated using cytokinins either singly or in combination with an auxin. Cytokinins were first discovered by F Skoog, C Miller and co-workers during the 1950’s as a factor that encourage cell division. The first cytokinin discovered was an adenine derivative (aminopurine) named kinetin (6-furfurylaminopurine) which was isolated as a DNA degradation product. The first common natural cytokinin recognized was purified from immature maize kernel and named zeatin. Cytokinins are present in all plant tissues. They are abundant in root tips, shoot apex and immature seeds. Their endogenous concentration is in the low nM range (Schmüling 2004). Naturally occurring cytokinins are adenine derivatives with a side chain at the N6-position. An example of synthetic cytokinin is benzyladenine or 6-benzylaminopurine (BA); it is more stable and often used in plant tissue culture. In addition there are the structurally unrelated phenylurea type cytokinins (eg. diphenyl urea, thidiazuron) a class of synthetic cytokinins. In vitro the ratio of cytokinin to auxin regulates the differentiation of cultured plant tissues to either shoots or roots. A high cytokinin to auxin ratio promotes shoot formation, a low ratio, root formation.

Due to their stimulatory effect on plant regeneration cytokinins are extensively used in plant tissue culture. Micropropagation of various plant species, including many medicinal plants, has been accomplished through rapid proliferation of shoot tips and axillary buds in culture during the last few years. Shoot tip explants have been successfully employed in the regeneration of Catharanthus 29 roseus (Bajaj et al. 1988), Picrorhiza kurroa (Upadhyay et al. 1989), Clerodendrum colebrookianum (Mao et al. 1995), Trichopus zeylanicus (Krishnan et al. 1995), Withania somnifera (Kulkarni et al. 2000), Piper longum (Soniya and Das 2002), Decalepis hameltonii (Giridhar et al. 2005), Eclipta alba (Husain and Anis 2006a), Saussurea lappa (Johnson et al. 1997), Swertia chirata (Balaraju et al. 2009), Aloe vera (Gantait et al. 2010) and Clitoria ternatea (Anand et al. 2011). A large number of medicinal plants have been successfully regenerated using axillary meristem either through nodal segment culture or cotyledonary node (derived from aseptic seedlings) culture; a few of these plants are Acacia nilotica (Dewan et al. 1992), Tylophora indica (Sharma and Chandel 1992), Oscimum basilicum (Sahoo et al. 1997), Dalbergia sissoo (Pradhan et al. 1998a), Psoralea corylifolia (Saxena et al. 1998), Gymnema sylvestre (Komalavalli and Rao 2000), Acacia sinuata (Vengadesan et al. 2002), Pterocarpus marsupium (Chand and Singh 2004a; Anis et al. 2005), Mucuna pruriens (Faisal et al. 2006a), Glycyrrhiza glabra (Vadodaria et al. 2007), Acacia senegal (Khalafalla and Daffalla 2008) and Veronica anagallis-aquatica (Shahzad et al. 2011).

Numerous factors are reported to influence the success of in vitro propagation of different medicinal plants and therefore, it is unwise to define any particular reason for the general micropropagation of medicinal plants. The factors that influence the micropropagation of medicinal and aromatic plants have been reviewed by Murashige (1977), Hussey (1980; 1983), Hu and Wang (1983), Bhagyalakshmi and Singh (1988), Short and Roberts (1991), Rout et al. (2000), Chaturvedi et al. (2007), Khan et al. (2009), Sharma et al. (2010) and Krishnan et al. (2011). Amongst all the cytokinins, BA, Kn and 2iP are most frequently used for the micropropagation of different plant species, while, zeatin is rarely used. On the basis of earlier studies it has been well documented that BA is the most potent cytokinin for multiple shoot regeneration and had been successfully used by several workers for the development of efficient micropropagation protocols for various medicinal plants. Generally, a critical level of the hormone is required for the induction of multiple shoots and for this a wide range of concentrations has to be tested to select the best or optimal concentration of the hormone.

Between two cytokinins (BA and Kn) tested by Agrawal and Sardar (2003) for in vitro regeneration of Cassia angustifolia from various explants, 1.0 µM BA was found to be optimal for eliciting best morphogenic response from seedling derived CN explants. An average of 2.4 shoots/CN explant, 1.5 shoots/NS explant and 1.08 shoots/ST explants were obtained at 1.0 µM of BA. The superiority of BA over Kn has also been reported in other medicinal genera of family Fabaceae (Leguminosae) viz: Bauhinia variegata (Mathur and Mukunthakumar 1992), Psoralea corylifolia (Jeyakumar and Jayabalan 2002), Sesbania rostrata (Jha et al. 2004), Acacia senegal (Khalafalla and Daffalla 2008), Acacia catechu (Jain et al. 2009a) and Clitoria ternatea (Pandeya et al. 2010).

In the micropropagation of an endangered medicinal plant Curculigo orchioids (Wala and Jasrai 2003), single BA treatment was found to be suitable for the induction of multiple shoots from meristem tip culture on MS medium containing BA (2.21 µM). Faisal et al. (2006b) reported the development of 23.3 shoots/NS at 5.0 µM of BA in Mucuna pruriens, Raghu et al. (2006a) obtained 6.3 shoots/NS at 8.87 µM of BA in Tinospora cordifolia, while Shahzad et al. (2011) reported the production of a very high number of shoots (43.7 shoots/NS) at very low concentration (0.5 µM) of BA in an amphibious medicinal plant Veronica anagallis-aquatica. The edge of BA over other cytokinins is being well documented in various other medicinal plants including Gymnema sylvestre (Komalavalli and Rao 2000), Cardiospermum halicacabum (Babber et al. 2001), Holostemma ada-kodien (Martin 2002), Ceropegia spp. (Beena et al. 2003 and Nikam et al. 2008), Spilanthes acmella (Deka and Kalita 2005), Eclipta alba (Dhaka and Kothari 2005), Bupleurum kaoi (Chen et al. 2006), Penthorum chinense (Cao et al. 2007), Marsdenia brunoniana (Ugraiah et al. 2010) and Ricinus communis (Alam et al. 2010).

The percentage bud break and multiple shoot induction declined with the increase in BA concentration beyond the optimal level (2.0 mg/l) in Vitex negundo (Sahoo and Chand 1998a) and suppressed the sprouting of dormant axillary buds in the nodal explants. Reduction in the number of regenerated shoots from apical or axillary meristems at a concentration higher than the optimal level has also been reported in many leguminous plants (Gulati and

Jaiwal 1994; Kathiravan and Ignacimithu 1999; Sinha et al. 2000 and Anis et al. 2005) and several other medicinal plants like Kaempferia galanga (Vincent et al. 1992), Pisonia alba (Jagadishchandra et al. 1999), Cunila galioides (Fracaro and Echeverrigaray 2001), Salvia guaranitica (Echeverrigaray et al. 2010) and Veronica anagallis-aquatica (Shahzad et al. 2011).

Kumar (1992) and Nandwani and Ramawat (1993) reported that kinetin was the best cytokinin for in vitro establishment and growth of leguminous trees such as Bauhinia purpurea and Prosopis cinerarea respectively. Lal and Ahuja (1996) reported a rapid proliferation rate in Picrorhiza kurroa using kinetin at 1.0-5.0 mg/l. Similarly, in the micropropagation of a medicinal plant Phyllanthus urinaria (Catapan et al. 2002) a maximum of 20.3 shoots/NS explant were obtained on MS medium supplemented with 5.0 µM Kn. Thangavel et al. (2011) also obtained maximum number (19.7) of shoots per leaf explant on MS medium fortified with 1.5 mg/l Kn with 80% regeneration percentage in the micropropagation of a valuable medicinal herb Plectranthus barbatus. The effect of 2iP on multiple shoot regeneration has been reported by Cellarova and Hocariv (2004) in Digitalis purpurea and by Sujatha and Kumari (2008) in Artemisia vulgaris.

One of the interesting observations was made by Vengadesan et al. (2002) in the study of Acacia sinuata where multiple shoots were produced on MS medium containing a combination of cytokinins (BAP and Kn). They observed that maximum number of shoots was induced from CN explants on medium containing 6.66 µM BA and 4.65 µM Kn. Similarly, Chaudhuri et al. (2007) reported 18 shoots per NS explant in an endangered medicinal herb Swertia chirata on MS medium containing BA (0.44 µM) + Kn (4.65 µM). In another report by Balaraju et al. (2009) in the same plant the highest number of shoots (42.16) per explant was also produced on MS medium containing 1.0 mg/l BA and 0.1 mg/l Kn. BA + Kn combination has proved to be an ideal combination in the micropropagation of Feronia limonia (Hossain et al. 1994) through cotyledon explants excised from aseptic seedlings. However, Joshi and Dhawan (2007a) reported maximum shoot multiplication in S. chirayita on medium containing 4.0 µM BA and 1.5 µM 2iP. Rajeswari and Paliwal (2006) reported the highest frequency for shoot regeneration (82.5%), maximum number of shoots per CN

32 explant (6.9) and maximum shoot length (2.55 cm) in Albizia odoratissima on MS medium containing 10.0 µM BA and 10.0 µM 2iP. Combination of two or more cytokinins also favoured multiple shoot proliferation in Eucalyptus grandis (Teixetra and Da Silva 1990), Fragaria indica (Bhatt and Dhar 2000), Eclipta alba

(Baskaran and Jaybalan 2005), Eucalyptus impensa (Bunn 2005), Ocimum sanctum (Girija et al. 2006), Stevia rebaundiana (Ahmed et al. 2007), Amygdalus communis (Akbas et al. 2009), Withania coagulans (Jain et al. 2009b), Cadaba heterotricha (Abbas and Qaiser 2010) and Streblus asper (Gadidasu et al. 2011).

Generally cytokinin is required in optimal quantity for shoot proliferation in many genotypes but inclusion of low concentration of auxins along with cytokinin triggered the rate of shoot proliferation (Tsay et al. 1989; Roja et al. 1990 and Shasany et al. 1998). Maximum percentage of multiple shoots (85.6%) was observed by Rout (2005) in the medium supplemented with 8.9 µM BA and 1.34 µM NAA in a medicinal legume Clitoria ternatea. Addition of NAA (0.5 µM) along with BA (5.0 µM) significantly enhanced the number of shoots (23.3) in another medicinal plant Mucuna pruriense (Faisal et al. 2006b). Rapid proliferation of shoots was achieved by culturing the in vitro shoots derived from the nodal segments onto MS medium supplemented with 2.0 mg/l BA and 0.5 mg/l NAA in the micropropagation of an important medicinal plant Gynura procumbens (Keng et al. 2009). An average of 18.2 shoots was produced from each shoot explant.

The effective role of NAA in combination with BA for the induction of multiple shoots has been reported in several other medicinal plants like Gomphrena officinalis (Mercier et al. 1992), Gymnema sylvestre (Reddy et al. 1998), Hemidesmus indicus (Sreekumar et al. 2000), Rauvolfia tetraphylla (Faisal and Anis 2002), Ceropegia bulbosa (Britto et al. 2003), Justicia gendarussa (Agastian et al. 2006), Bupleurum distichophyllum (Karuppusamy and Pullaiah 2007), Baliospermum montanum (George et al. 2008b), Boerhaavia diffusa (Biswas et al. 2009), Spilanthes mauritiana (Sharma et al. 2009) and Stevia rebaundiana (Sharma and Shahzad 2011). A rapid in vitro propagation of Abelmoschus moschatus through axillary bud multiplication has been established by Maheshwari and Kumar (2006) using MS basal medium supplemented with different combinations of BA, NAA and IAA. Almost all

33 combinations responded but MS medium with 4.0 mg/l BA + 0.1 mg/l NAA was the best suited for axillary bud proliferation inducing a mean of 15 shoots/node which on further subculture generated more than 25 shoots/node. IAA also favoured shoot induction but to a lesser extent as compared to NAA. Shoot elongation was carried out on MS medium without PGRs. Ahmad and Anis (2007) reported 25 shoots/NS explant in Vitex negundo at 1.0 µM of TDZ, but the optimum shoot multiplication and elongation was achieved when TDZ exposed cultures were subcultured on MS medium containing a combination of BA (1.0 µM) and NAA (0.5 µM).

In contrast to the above mentioned studies some workers reported that the combination of BA and IAA on MS medium favoured multiple shoot buds in Adhatoda beddomei (Sudha and Seeni 1994), Alpinia galanga (Anand and Hariharan 1997), Bupleurum fruticosum (Fratenale et al. 2002), Ocimum gratissimum (Gopi et al. 2006), Acalypha wilkesiana (Sharma et al. 2007), Stevia rebaundiana (Anbazhagan et al. 2010) and Clitoria ternatea (Anand et al. 2011). High frequencies of multiple shoot regeneration were achieved from nodal explants on MS medium fortified with 5.0 mg/l BA and 0.5 mg/l IAA in the micropropagation of a medicinal climber Zehneria scabra (Anand and Jeyachandran 2004). Eight to ten shoots per explant were obtained. An efficient multiplication method from the shoot tips of Centaurium erythraea (Piactczaka et al. 2005) using liquid MS medium supplemented with IAA (0.1 mg/l) and BA (1.0 mg/l) was developed from which a maximum of 60 shoots per explant were produced.

Bohidar et al. (2008) obtained the highest number of shoots from nodal explant of Ruta graveolens on MS medium supplemented with BA (1.0 mg/l) along with IAA (0.25 mg/l). In the micropropagation of a woody medicinal plant (Aegle marmelos), the highest shoot regeneration (86.6%) with an average shoot number of 487.5 shoots per explant in seven week time was obtained on MS medium supplemented with 6.6 µM BA and 1.14 µM IAA (Nayak et al. 2007).

The rate of shoot multiplication was greatly increased in a medium containing BA (4.0-6.0 mg/l), IAA (1.0-1.5 mg/l) and adenine sulphate (AdS) (100 mg/l) in Zingiber officinale (Palai et al. 1997). The addition of AdS at a concentration of

20.0 µM along with BA (4.0 µM) and IAA (0.5 µM) in MS medium improved the rate of shoot multiplication in NS explant of Pterocarpus marsupium (Husain et al. 2008). Not only vigour of shoots but proper leaf expansion and shoot elongation was observed. The combination provided maximum response (85%) with the highest number (8.6) of shoots with 4.8 cm shoot length after 6 weeks of culture.

Beena et al. (2003) described in vitro regeneration protocol for Ceropegia candelabrum through axillary bud multiplication using 8.87 µM BA in combination with 2.46 µM IBA. The MS medium fortified with 1.0 mg/l BA and 0.5 mg/l IBA provided maximum of 20 shoots/NS in a rare medicinal plant Rotula aquatica (Martin 2003b). BA and IBA combination has also been described as the most effective one in the micropropagation of Rheum emodi (Lal and Ahuja 1989), Gardenia jasmenoides (George et al. 1993), Terminalia chebula (Shyamkumar et al. 2003/2004) and Curcuma zedoria (Loc et al. 2005). Sen and Sharma (1991) combined BA with 2,4-D for the multiplication of Withania somnifera. Plantlet regeneration in Prosopis laevigata, a multipurpose leguminous tree, has been achieved from cotyledonary nodes excised from in vitro grown seedlings (Buendía-González et al. 2007). The explants were cultured on MS media containing different concentrations of BA and 2,4-D and a mixture of organic components. The highest number (3.37) of multiple shoots was observed in MS medium containing 2,4-D (9.05 μM) + BA (6.62 μM).

Faster bud break coupled with an enhanced frequency of shoot development (92%) and internode elongation was observed in Vitex negundo (Sahoo and Chand 1998a). It was found to be dependent on the influence of Gibberelic acid

(GA3) when used at an optimal concentration (0.4 mg/l) along with BA (2.0 mg/l).

GA3 at 0.1-0.5 mg/l and AdS at 50-100 mg/l had a promising effect on shoot proliferation and elongation (Rout et al. 2000). The promotive effect of GA3 in combination with BA on shoot bud induction was also reported in Chrysanthemum (Earle and Langhans 1974), Saussarea lappa (Arora and Bhojwani 1989), Ocimum americanum (Pattnaik and Chand 1996), O. basilicum (Sahoo et al. 1997), Tridax procumbens (Sahoo and Chand 1998b) and Tylophora indica (Rani and Rana 2010). In contrast it has been reported to suppress shoot bud differentiation at 0.1-0.3 mg/l in Plumbago indica (Nitsch and

Nitsch 1967), Begonia (Heide 1969), Nicotiana (Thorpe and Murashige 1970) and Duboisia myoporoides (Kukreja and Mathur 1985). Thus, the role of GA3 in shoot bud induction in plant species is controversial.

2.4.1.1.2 Effect of TDZ on shoot multiplication Thidiazuron (TDZ) a substituted phenyl urea (N – phenyl-N-1,2,3 – thiadiazol – 5 –ylurea ) is one of the most active cytokinin-like substances that have been used for high rate of axillary shoot proliferation in many plant species (Fiola et al. 1990; Malik and Saxena 1992a; Huettman and Preece 1993; Kim et al. 1997 and Thomas 2003). TDZ was developed originally by AG Shering for use as a defoliant for Gossypium hirsutum (Arndt et al. 1976). TDZ directly helps growth due to its own biological activities in a manner similar to that of an N – substituted cytokinin or it may induce the synthesis and accumulation of an endogenous cytokinin (Capelle et al. 1983). In woody plant species, low levels of TDZ induce the axillary shoot proliferation but higher levels may inhibit it. Higher levels on the other hand encourage caulogenesis and somatic embryogenesis (Huettman and Preece 1993; Lu 1993; Murthy et al. 1998; Shibli et al. 2001 and Fengyun and Liying 2002). TDZ has been revealed to stimulate accumulation of endogenous cytokinins (Murthy et al. 1995 and Hutchinson et al. 1996). In addition to the cytokinin like activity, Hutchinson et al. (1996) observed that TDZ promoted auxin accumulation. Other studies established that TDZ affected auxin transport in hypocotyl tissues of Pelargonium x hortorum Bailey (Murch and Saxena 2001) and encouraged the regeneration frequency by varying the levels of abscisic acid (Li and Yang 1988), ethylene (Yip and Yang 1986) and proline (Murch and Saxena 1997).

TDZ can be replaced for auxins or auxin-cytokinin required to stimulate somatic embryogenesis. This is perhaps due to the association of TDZ in the modulation of endogenous plant growth regulators especially auxins and cytokinins (Murthy et al. 1995 and Chhabra et al. 2008). TDZ has been used in the range of 0.5- 10.0 µM to induce somatic embryogenesis from cotyledon explants of white ash (Preece and Bates 1990 and Bates et al. 1992), eastern black walnut (Neuman et al. 1993), Rubus (Fiola et al. 1990) and Vitis vinifera (Matsuta and Hirabayashi 1989). TDZ induced somatic embryogenesis has also been reported in several other plant species like - geranium (Visser et al. 1992), Malus × domestica 36

(Daigny et al. 1996), Pelargonium x hortorum (Hutchinson et al. 1996), Prunus avium × P. pseudocerasus (Pesce and Rugini 2004), Cicer arietenum (Kiran et al. 2005) and Mangifera indica (Kidwai et al. 2009).

The mode of action of TDZ may be attributed to its ability to induce cytokinin accumulation (Victor et al. 1999) and/or to enhance the accumulation and translocation of auxin (Murch and Saxena 2001). The morphogenetic response in which TDZ has been found to mimic cytokinin like activity was 20 times more effective in dormancy breaking (Wang et al. 1986) and used successfully in plant regeneration system of many plant species including several medicinal plants viz: Hypericum perforatum (Murch et al. 2000), Bacopa monniera (Tiwari et al. 2001), Artemisia judaica (Liu et al. 2003), Arachis correntia (Mronginski et al. 2004), Curcuma longa (Prathanturarug et al. 2005), Hyoscyamus niger (Uranbey 2005), Sterculia urens (Hussain et al. 2007) and Andrographis neesiana (Karuppusamy and Kalimuthu 2010), Similarly, TDZ has been proved to be effective in regeneration of recalcitrant system such as grain legume (Malik and Saxena 1992a). During last two decades it has been extensively used for the high frequency shoot regeneration in various leguminous plants such as: Albizia julibrissin (Sankhla et al. 1994), Cajanus cajan (Dolendro et al. 2003), Cicer arietinum (Rizvi and Singh 2000 and Jayanand et al. 2003), Acacia sinuata (Vengadesan et al. 2002), Robinia pseudoacacia (Hosseini-Nasr and Rashid 2003/4), Sesbania drummondii (Cheepala et al. 2004), Psoralea corylifolia (Faisal and Anis 2006), Pterocarpus marsupium (Husain et al. 2007a) and Leucaena leucocephala (Shaik et al. 2009).

The higher concentrations of TDZ has been proved to be inhibitory for multiple shoot regeneration and decreased the shoot number in a number of plant species like Phaseolus spp. (Malik and Saxena 1992a), Arachis hypogea (Saxena et al. 1992) and Murraya koenigii (Bhuyan et al. 1997). Moreover, the continuous or prolonged exposure of TDZ resulted in the distortion, stunting and fasciation of shoots. Deleterious effects of TDZ have been well documented in several plant species (Van Nieuwkerk et al. 1986; Preece et al. 1987; Yusnita et al. 1990; Sankhla et al. 1994; Pradhan et al. 1998a; Ket et al. 2004; Khurana et al. 2005 and Ahmad and Anis 2007). Addition of low concentration of an auxin or a second cytokinin to the proliferation media containing TDZ significantly

37 enhanced shoot proliferation. Chalupa (1987) observed increased shoot proliferation and elongation when BA + IBA or NAA were added to a TDZ containing medium in Robinia pseudoacacia, Sorbus aucuparia and Tilia cordata. Enhanced axillary shoot proliferation has also been reported in other plant species when TDZ was added to a BA containing medium like: Acer x fremanii (Kerns and Meyer 1987), Fraxinus americana (Navarrete et al. 1989), Vitis rotundifolia (Sudarsono and Goldy 1991) and Hylocereus undatus (Mohamed-Yasseen 2002).

2.4.1.1.3 Effect of subculture passages A rapid rate of shoot multiplication and proliferation depends on the frequent sub culturing of the regenerative shoot cultures. In case of prolonged cultures, the nutrients in the medium are gradually exhausted and the same time relative humidity in the vessels decreases leading to drying of the developing shoots for lack of nutrients. Upadhyay et al. (1989) reported a propagation profile for Picrorhiza kurroa and observed that the shoot multiplication rate gradually improved as the number of sub culture passages increased. They proposed the adaptation of the explants to the in vitro conditions, which was essentially completed during the first few subcultures. Saxena et al. (1998) reported an average of 3-5 fold multiplication in Psoralea corylifolia when axillary shoots were allowed to continue in primary cultures for 8 weeks. Similarly, Shirin et al. (2000) reported in vitro plantlet production in Kaempferia galanga on MS medium containing 12.0 µM BA and 3.0 µM NAA and they observed 13 fold rate of plantlet production every 4 weeks. Sub culturing of nodal segments harvested from in vitro derived axenic shoots on the multiplication medium enable continuous production of shoots in Centella asiatica (Tiwari et al. 2000). Repeated sub culturing of shoot tips and single nodes of Cunila galoides at 4 weeks interval for eight months on the primary medium enable mass multiplication of shoots without any evidence of decline (Fracaro and Echeverrigaray 2001). In contrast Arya et al. (2003) demonstrated significant shoot improvement in Leptadenia reticulata through repeated transfer of mother explant to fresh medium. After three or four sub cultures, basal clump with shoot bases was divided into three or four sub-clumps and multiplied on fresh medium. They reported 15-20 shoots from each clump within 15 days.

2.4.1.2 Adventitious shoot regeneration The induction of shoot buds from any site on the plant other than primary meristem is termed as adventitious shoot regeneration. Buds are generally induced directly from the explant without any intervening callus phase. Various explants viz.: leaf, stem, internode, petiole, epicotyl, hypocotyl, cotyledon and root are used as the source of adventitious shoot regeneration.

In Achras sapota (Purohit et al. 2004) direct shoot bud differentiation was achieved through leaf segments on Schenk and Hildebrandt’s medium (SH medium) supplemented with BA (8.88 µM) and TDZ (5.0 µM). Middle portion of the leaves showed highest potential for shoot buds regeneration. Histological observations also confirmed their de novo regeneration with clear vascular connection with the mother tissues. The promotory effect of BA with IAA in inducing direct shoot buds from leaf segments of Eryngium has been reported by Arockiasamy et al. (2002). Similar results were also provided by Zhang et al. (2005) in Perilla frutescens, where a maximum of 91.06% cotyledons induced adventitious shoot buds on BA (4.44 µM) supplemented media while a combination of BA (2.22 µM) and IAA (2.85 µM) was proved to be effective for the regeneration of shoot buds from hypocotyl explant with 76.4% regeneration in the same plant. 100% shoot regeneration was achieved in Verbena officinalis - a medicinal plant (Turker et al. 2010) from stem internode explants, when BA (13.32 µM) was combined with IAA (5.71 µM). While, BA along with AdS has also found to be beneficial for shoot buds differentiation in Cajanus (Mishra 2002). A simple and efficient in vitro protocol was developed by Barik et al. (2005) for high frequency shoot regeneration in Lathyrus sativus using epicotyl segments. Highest shoot regeneration frequency (80%) was achieved on MS medium containing BA (17.76 µM) and NAA (10.74 µM) with maximum shoot regeneration (8.2 shoots/explant). Shoots were directly induced from the explant. BA along with NAA has also been used by Burdyn et al. (2006) and Seetharam et al. (2007) for direct shoot buds induction from leaf explants of Aloysia polystachia and Vernonia cineria respectively.

However, in contrast to the above reports, Rao and Purohit (2006) suggested that BA (4.44 µM) alone was effective in inducing direct shoot buds from internode explants of Celastrus paniculatus. Incorporation of IAA and NAA did 39 not improve regeneration, rather promoted callusing. Similar results were also obtained in different plant species (Espino et al. 2004; Purohit et al. 2004; Abdi and Khosh-Khui 2007 and Sahai and Shahzad 2010). Bouhouche and Ksiksi (2007) suggested the effective role of Kn (3.0 mg/l) in combination with IAA (0.5 mg/l) for the production of multiple shoots directly from hypocotyl explants of Teucrium stocksianum, compared to other combination of PGRs. In Populus deltoides, Yadav et al. (2009) also revealed that 0.25 mg/l Kn + 0.25 mg/l IAA combination was most responsive for differentiation of direct adventitious shoot buds from all along the explant surface in leaf, internode and root explants.

The effect of TDZ on adventitious shoot regeneration has been reported by Debnath (2009) in a two-step procedure on excised leaves of lowbush blueberry. TDZ induced cultures were transferred to medium containing 2.3-4.6 µM zeatin and produced usable shoots after one additional subculture. Malik et al. (2010) demonstrated the effective role of TDZ for direct shoot regeneration from intact in vitro leaves (attached to shoots) of Arnebia euchroma. Shoot buds proliferated to form multiple shoots on MS medium supplemented with Kn (5.0 µM). Direct shoot regeneration was achieved when shoots were initially precultured for 40 days on higher concentrations of TDZ (20.0 µM) and then transferred to lower concentration (5.0 µM). TDZ has also been used effectively to induce adventitious shoot buds from leaf explant of many plant species (Huettman and Preece 1993; Mithila et al. 2003; Gu and Zhang 2005 and Deore and Jhonson 2008).

2.4.2 Indirect organogenesis The regeneration of plants via intermediate callus phase is termed as “indirecct regeneration” or “indirect organogenesis”. The explants first dedifferentiate to form an unorganised mass of cells called ‘callus’, the callus cells reorganize to form ‘meristemoids’ which again redifferentiate to shoot buds, then finally developed into shoots and regenerate plantlets. Various explants such as leaf, stem, petiole, node, inter node, hypocotyl, root, cotyledon etc. can be used to induce callus (Khurana et al. 2005). The induction of callus following differentiation and organogenesis is accomplished by the differential use of growth regulators and the control of conditions in the culture medium. With the stimulus of endogenous growth substances or by addition of exogenous growth 40 regulators to the nutrient medium, cell division, cell growth and tissue differentiation are induced (Tripathi and Tripathi 2003). Indirect organogenesis often results in somaclonal variations, making the strategy less suitable for large scale clonal propagation but at the same time the variants, thus, produced may be utilized for the genetic improvement of species.

Callus mediated organogenesis has been reported in several medicinal plants including Dioscorea deltoidea (Ravishankar and Grewal 1991), Datura innoxia (Missaleva et al. 1993), Digitalis lantana (Pradel et al. 1997), Bacopa monnieri (Shrivastava and Rajani 1999), Solanum nigrum (Shahzad et al. 1999), Ocimum sanctum (Shahzad and Siddiqui 2000), Withania somnifera (Rani et al. 2003), Tylophora indica (Faisal et al. 2005a), Ruta graveolens (Faisal et al. 2006c) and Hypericum perforatum (Wojcik and Podstolski 2007). In Ceropegia candelabrum (Beena and Martin 2003) and Decalepis hameltonii (Giridhar et al. 2004) callus was induced from leaf and internodal explants and later the same callus produced somatic embryos.

Saxena et al. (1997) regenerated plantlets of Psoralea corylifolia via callus formation through mature leaves, stem, petioles and roots of young seedlings. The callus differentiated into green nodular structure which developed into dark green shoot buds in the medium comprised of MS + BA (2.5-3.0 mg/l) and NAA (1.0 mg/l). Augmentation of AdS (5.0 mg/l) in the culture medium resulted in quick growth of shoot buds within 4 weeks of culture. Pradhan et al. (1998b) and Pattnaik et al. (2000) reported regeneration of plants from cell susupension derived callus of Dalbergia latifolia and D. sissoo respectively. Vengadesan et al. (2003a) and Shahzad et al. (2006) described efficient plant regeneration in a medicinally valuable leguminous tree species Acacia sinuata from cotyledon derived callus. Regeneration via callus phase has also been reported in other members of Fabaceae family such as Sesbania bispinosa (Sinha and Mallick 1991), Lathyrus sativus (Roy et al. 1991; 1992), Dalbergia lanceolaria (Dwari and Chand 1996), Acacia sinuata (Vengadesan et al. 2000), Leucaena leucocephala (Saafi and Borthakur 2002 and Maity et al. 2005) and Clitoria ternatea (Shahzad et al. 2007).

Generally auxins are considered as the best hormone for callus production. Hu and Wang (1983) proved the superiority of 2,4-D over other auxins for induction of callus and strongly antagonize any organized development. Shahzad et al. (1999) suggested that 2,4-D at 2.0 mg/l was better than NAA at the same concentration for the production of compact, nodular callus from the leaf explants of Solanum nigrum. Similar findings were reported in Rauwolfia serpentina (Perveen and Elahi 1987) and Plumbago rosea (Harikrishnan and Hariharan 1996). Similarly, 2,4-D was considered as the best auxin to induce organogenic callus in case of Ceropegia bulbosa var lushii (Patil 1998) and Gymnema sylvestris (Gopi and Vatsala 2006).

Addition of a cytokinin along with 2,4-D has found to be significant for callus production, as in case of Acacia sinuata, Vengadesan et al. (2000) reported the production of compact and nodular calli from hypocotyl explants on MS medium comprised of 2,4-D and BA. Maximum number of shoot buds (12-15 per explant) were differentiated on medium containing BA (13.3 µM) and IAA (3.42 µM) after 25-30 days of culture. The regenerated buds developed into shoots in less than 2 weeks after initiation. Faisal et al. (2006c) also suggested the production of organogenic callus from stem explant of Ruta graveolens on MS medium augmented with 2,4-D (10.0 µM) and BA (2.5 µM). Similarly, in Cerpegia juncea (Nikam and Savant 2009), best organogenic callus was induced from nodal segments on MS medium containing 2,4-D (1.0 µM) and BA (5.0 µM). However, in Tylophora indica (Faisal et al. 2005a) the highest frequency (100%) of light yellow organogenic callus was obtained from petiole explants by addition of TDZ along with 2,4-D i.e. on medium comprised of MS + 2,4-D (10.0 µM) and TDZ (2.5 µM). Inclusion of 2iP along with 2,4-D was also effective for callus production in Pergularia daemia (Kiranmai et al. 2008). Baskaran and Jayabalan (2009a) suggested the production of organogenic calli from leaf and petiole explants in Melothria maderaspatana on MS medium containing 2,4-D (6.0 µM) + TDZ (0.5 µM) and 2,4-D (6.0 µM) + BA (1.0 µM) combinations respectively. From these studies it has been proved that combination of 2,4-D and cytokinins was efficient for the induction of callus and subsequent proliferation and differentiation of shoots from such calli.

Inclusion of a cytokinin along with an auxin to induce callus has also been reported in several other plant species (Gharyal et al. 1993; Sarasan et al. 1994; Roy et al. 2000 and Hariharan et al. 2002). Rout et al. (1992) suggested the callus production and subsequent plant regeneration in Cephalis ipecacuanha from leaf explants cultured on MS medium containing Kn (4.5 mg/l) and NAA (0.1 mg/l). Basu and Chand (1996) reported shoot buds differentiation from root derived callus of Hyoscyamus muticus on MS medium comprised of BA (0.5 mg/l) and NAA (0.05 mg/l). Similarly, Irvani et al. (2010) achieved the best callus production (100%) from root explants of Doreum ammoniacum on MS medium supplemented with BA (2.0 mg/l) and NAA (1.0 mg/l).

In contrast to the above stated studies, Manjula et al. (2000) was of the opinion that cytokinin alone was sufficient for the induction and subsequent growth of callus in Tylophora indica and the inclusion of an auxin along with cytokinin inhibited the growth of callus. Reddy et al. (2001) suggested that Kn alone showed prominent growth of callus from leaf explants of Coleus forskohlii. Addition of 2,4-D did not enhance the callus formation and the callus developed on this medium subsequently turned brown within 4 weeks of culture. Shahzad et al. (2006) also supported the above studies and produced callus from cotyledon explants of Acacia sinuata on TDZ supplemented MS medium. Efficient shoot buds production was observed when TDZ induced calli were subcultured at different concentrations of BA.

Sood and Chauhan (2009) reported that addition of another auxin (IBA) along with 2,4-D proved to be beneficial to induce callus cultures from different explants such as leaf discs, nodal and root segments of Picrorhiza kurroa. There are several other studies where inclusion of two auxins was proved to be optimal for the induction of organogenic callus (Rani et al. 2003; Shahzad et al. 2009 and Ahmad et al. 2010).

2.4.3 Somatic embryogenesis Somatic embryogenesis may be defined as a unique developmental pathway that includes a number of characteristic events like: differentiation of cells, activation of cell division and reprogramming of their physiology, metabolism and give expression patterns. The somatic cells under suitable induction conditions

43 undergo restructuring through embryogenic pathway to develop embryogenic cells. These cells then undergo a series of morphological and biochemical changes that result in the formation of somatic embryo and generation of new plants (Schmidt et al. 1997; Komamine et al. 2005 and Yang and Zhang 2010). Somatic embryos resemble zygotic embryos and undergo almost the same developmental stages (Dodeman et al. 1997). Regeneration via somatic embryogenesis coupled with genetic engineering provides an opportunity of producing a large number of elite or transgenic plants (Jin et al. 2005 and Li et al. 2006). Sánchez et al. (2005) provided the first report on genetic transformation of Quercus suber using somatic embryos through Agrobacterium tumefaciens LBA4404/p35S GUS INT/pCAMBIA 1301 strain.

The first report of somatic embryogenesis in the history of plant tissue culture was documented by Steward et al. (1958) and Reinert (1959) in carrot cell suspension cultures. Since then somatic embryogenesis has been reported in a wide range of plant species of dicot and monocot plants (Krishnaraj and Vasil 1995; Merkle et al. 1995; Quiroz-Figueroa et al. 2006 and Mathieu et al. 2006). Somatic embryogenesis may involve the development of embryos either directly from the explant without an intermediate callus phase or indirectly after a callus phase and thus, referred as direct somatic embryogenesis (DSE) and indirect somatic embryogenesis (ISE) respectively (Sharp et al. 1980). Chung et al. (2007) documented plant regeneration through direct embryogenesis from leaf explants of Dendrobium on MS medium containing 1.0 mg/l TDZ. Somatic embryos were mostly found on cut ends near the leaf surface and occasionally occurred near the leaf tips. DSE proved to be advantageous for the production of true clones of the plants due to the minimal chances of changes in the genotype (Peshke and Phillips 1992), however, reports of DSE are relatively rare (Gill and Saxena 1992; Raghvan 1997; Chen et al. 1999; Chen and Chang 2006; Sudha and Seeni 2006; You et al. 2007 and Varshney et al. 2009). Various explants have been exploited for the induction of embryogenic calli but generally immature, meristematic tissues proved to be the most suitable explant for somatic embryogenesis. For instance immature zygotic embryos and cotyledons have been used for the induction of somatic embryogenesis in majority of legumes (Parrott et al. 1991; 1992; Neuman et al. 1993; Sagare et al. 1995; Rout

44 and Samantaray 1995; Ahmed et al. 1996 and Gairi and Rashid 2005). Choi et al. (1999) reported the production of somatic embryos directly from the cotyledon explants of Panax ginseng on growth regulator free medium.

Somatic embryogenesis results in the production of large number of plantlets within short span of time. Regeneration of complete plantlets via somatic embryogenesis has been successfully reported in a large number of medicinal plant species (Ghosh and Sen 1991; Fuentes et al. 1993; Sehgal and Abbas 1994; Basu and Chand 1996; Kunitake and Mii 1997; Das et al. 1999; Jayanthi and Mandal 2001; Tawfik and Noga 2002; Martin 2004a; Sudha and Seeni 2006 and Sahai et al. 2010a) including many leguminous plants of economic as well as medicinal importance (Tetu et al. 1990; Durham and Parrott 1992; Arrilaga et al. 1994; Dineshkumar et al. 1994; Garg et al. 1996; Rao and Lakshmisita 1996; Mary and Jayabalan 1997; Luo and Jia 1998; Girija et al. 2000; Chand and Singh 2001; Chand and Sahrawat 2002; Faisal et al. 2008 and Husain et al. 2010).

PGRs played an important role in the induction of embryogenic callus and then subsequent conversion and development of somatic embryos to produce complete plantlets. There are a number of species which showed embryogenesis on medium supplemented with various cytokinins either singly or in combinations. In an endangered medicinal plant Psoralea corylifolia regeneration has been achieved via somatic embryogenesis through root segments (Chand and Sahrawat 2002) on MS medium supplemented with NAA (10.74 µM) and BA (2.2 µM) with highest frequency (95.2%) of embryogenic calli. While Faisal et al. (2008) reported somatic embryogenesis and plant regeneration in the same species from the nodal explants on MS medium containing TDZ (16.0 µM). Further development of embryos to heart, torpedo and cotyledonary stages was achieved on transferring to PGR free MS basal medium within 2 weeks. Complete plantlets were regenerated from the somatic embryos on half strength MS augmented with 1.0 µM GA3. Choi et al. (1999) suggested that the requirement of GA3 for the germination of somatic embryos was due to their dormant nature. The promotory effect of GA3 in somatic embryo germination has also been reported in other plant species including Santalum album (Rai and McComb 2002), Gossypium hirsutum (Kumaria et al. 2003) and

Panax japonicus (You et al. 2007). The high concentration of TDZ (7.5 µM) was found to induce embryogenesis in Catharanthus roseus from mature zygotic embryos, while the hypocotyl and cotyledon explants of the same species failed to induce such response on TDZ supplemented media (Dhandapani et al. 2008). Higher concentrations of TDZ have also been reported to induce somatic embryogenesis in other plant species (Liu et al. 2003 and Mithila et al. 2003). Contrary to these reports Gairi and Rashid (2005) observed high frequency (up to 100%) direct somatic embryos regeneration at very low concentration of TDZ (0.5 µM) from immature cotyledons of a highly medicinal trees species Azadirachta indica. However, an increase in the concentration of TDZ to 1.0 µM, further improved the regeneration potential.

Similarly, BA has also been reported to induce embryogenesis through various explants in a number of other plants like Carthamus tinctorius (Mandal and Gupta 2003) and Quassia amara (Martin and Madassery 2005). Inclusion of an auxin along with BA found to enhance the rate of embryogenesis in Leptadenia reticulata (Martin 2004a), wherein, somatic embryos at the highest frequency was induced from ST and NS explants on MS medium supplemented with BA (8.87 µM) and IBA (2.46 µM). While, differentiation of direct somatic embryos from the nodal explant of a medicinal plant Hygrophila spinosa (Varshney et al. 2009) was observed on medium containing BA (1.0 µM) and NAA (0.5 µM). Similarly, various cytokinins along with auxin found to facilitate induction of embryogenic callus in a number of plant species (Samantaray et al. 1997; Hussein and Batra 1998; Venkatachalam et al. 1999a; Nikam et al. 2003 and Emek and Erdag 2007).

In general, auxins, particularly 2,4-D are considered as the most effective growth regulators for the induction of embryogenic callus. 2,4-D has been proved to play signalling role in induction of somatic embryos in many plant studies (Nomura and Komamine 1995). The efficacy of 2,4-D in the induction of somatic embryos and subsequent transferring of these embryos for further conversion and development into plantlets on media comprised of different PGRs has been observed in many reports (Choi et al. 1997; 2002; Martin 2003a; Chithra et al. 2005; Hu et al. 2008; Simões et al. 2010 and Naik and Murthy 2010). While, there are reports available, in different species of plants where both the stages

46 i.e. induction and further development were achieved on the same medium (Inamdar et al. 1990; Anbazhagan and Ganapathi 1999; PremAnand et al. 2000; Magioli et al. 2001 and Park et al. 2005). But there are several reports available in the literature, where 2,4-D was failed or proved to be insignificant for the induction of embryogenesis ( Zhou et al. 1992; Chen et al. 1999; Kuo et al. 2005; Sudha and Seeni 2006 and Varshney et al. 2009). However, a rapid and reliable protocol for high frequency regeneration via somatic embryogenesis has been developed in Mucuna pruriens, an important medicinal legume by Vibha et al. (2009). They reported that embryogenic callus was induced from cotyledon explants excised from axenic seedlings on MS medium containing 6.7 µM 2,4-D, and a maximum of 60.5 cotyledonary staged embryos were obtained after 10 weeks of transfer on medium supplemented with 2.3 µM Kn and 5.4 µM NAA along with 13.6 µM AdS. Mature embryos converted into complete plantlets on half strength MS basal medium and exhibited 90% survival in field conditions.

Amoo and Ayisire (2005) reported successful callus production from the cut ends of the cotyledon explant on 2,4-D supplemented medium. Calli turned friable and nodular with small protuberances when transferred on media containing Kn along with 2,4-D, while, further development of embryos was observed on auxin free suspension culture medium. Similarly, augmentation of Kn along with 2,4-D for efficient somatic embryogenesis has been studied in many plant species (Toth and Lacy 1992; Hunault and Du Manoir 1992; Xie and Hong 2001a; Jayanthi and Mandal 2001 and Naik and Murthy 2010). While, Husain et al. (2010) achieved somatic embryogenesis in Pterocarpus marsupium from hypocotyl explants cultured on MS medium augmented with 2,4-D (5.0 µM) and BA (1.0 µM).

Maturation of somatic embryo and germination to produce complete plantlets is the most crucial step of somatic embryogenesis. In some plant species MS basal medium without any PGR was found to be sufficient for somatic embryo germination and conversion into plantlets (Murthy and Saxena 1994 and Kim et al. 2007). However, in many plant species reduction in MS salt composition (half strength MS) proved to be beneficial for maturation of somatic embryos (Borad et al. 2001; Yan et al. 2010a; Sahai et al. 2010a and 2010b). Augmentation of BA to the germination medium also facilitates embryos maturation and

47 conversion as in case of Arachis hypogea (Venkatachalam et al. 1999a) and Coriandrum sativum (Stephan and Jayabalan 2001). Addition of certain additives like abscisic acid (ABA) along with BA also influenced the maturation of somatic embryos in Vigna radiata (Girija et al. 2000). Mauri and Manzanera (2004) also evaluated the effect of ABA and stratification during the maturation and germination of holm oak (Quercus ilex). Stratification also promoted somatic embryo germination in many other plant species like Rosa (Marchant et al. 1996) and Quercus suber (Manzanera et al. 1993 and González-Benito et al. 2002). In Medicago truncatula, BA along with NAA proved to be beneficial for somatic embryo maturation and germination (Nolan et al. 1989). Nevertheless, García- Martín et al. (2001) reported the effect of sucrose concentration, chilling treatment and incubation condition on germination and conversion of somatic embryos of Quercus suber.

2.5 Rooting in microshoots Induction of roots in the regenerated microshoots is essential for the development of complete plantlets and to make any regeneration protocol a success. Various strategies have been applied by different workers to induce healthy root system either under controlled conditions (in vitro) or in the external environment (ex vitro) through the application of different auxins singly, in combination or with different additives. A brief survey of literature showing rooting in microshoots is described in the following heads:

2.5.1 In vitro rooting Isolation of elongated shoots from the cultures and transferring to rooting media comprised of different auxins facilitated in vitro root induction. However, the microshoots of various medicinal plants have been successfully rooted on PGR free MS basal medium (Cristina et al. 1990; Saxena et al. 1998 and Faisal and Anis 2003). Reducing the strength of MS salts to half or one fourth or three fourth also helped in the induction of roots. Half strength MS medium devoid of any hormone induced rooting in Potentilla potaninii (He et al. 2006). Borthakur et al. (2000) also reported that half strength MS medium was suitable for the multiplication and growth of shoots with simultaneous rooting in Eclipta alba and Eupatorium adenophorum. Half strength MS medium was found to be superior to

48 full strength MS medium for root induction and development in Mucuna pruriens (Faisal et al. 2006a). Incorporation of IBA (2.0 µM) to the rooting medium facilitated better rhizogenesis and maximum rooting percentage (92%) was observed with fairly good length (5.5 cm) and number (7.8) of roots/shoot.

Presence of IBA in the medium found to induce rooting in many medicinal plants including Sesbania acculeata (Bensal and Pandey 1993), Cajanus cajan (Sivaprakash et al. 1994), Swaisona formosa (Jusaitis 1997), Gymnema sylvestre (Komalavalli and Rao 2000), Hemidesmus indicus (Sreekumar et al. 2000), Cunila galoides (Fracaro and Echeverrigaray 2001), Holostemma ada- kodien (Martin 2002), Tylophora indica (Faisal and Anis 2003), Sesbania drummondii (Cheepala et al. 2004), Eclipta alba (Baskaran and Jayabalan 2005), Psoralea corylifolia (Faisal and Anis 2006), Clitoria ternatea (Barik et al. 2007), Nolina recurvata (Bettaieb et al. 2008) and Vigna unguiculata (Aasim et al. 2009). A combination of two auxins IBA (9.84 µM) and NAA (5.37 µM) in half strength MS induced rooting in microshoots of Baliospermum montanum (Johnson and Manickam 2003). While, Singh et al. (2003) suggested that full strength MS basal medium was sufficient for root development in B. axillare.

In Citrus species (Thirumalai and Thumburaj 1996) rooting of regenerated shoots was achieved on three fourth strength MS with NAA (3.0 mg/l). NAA was found to be more effective than IBA when used singly for rooting in Scoparia dulcis (Rashid et al. 2009). Efficient root induction was achieved on half strength MS containing 0.5 mg/l NAA. In an earlier report, half strength MS fortified with 0.5 mg/l NAA induced a mean of 5.2 roots/shoot and the roots were well branched with hairs in Rotula aquatica (Martin 2003b). Similar results were also observed in other plants like Centella asiatica (Amin et al. 2003) and Tectona grandis (Shirin et al. 2005). Peeters et al. (1991) concluded that NAA was taken up six times faster than IAA and Van der Krieken et al. (1993) suggested that IBA was taken up four times faster than IAA. Consequently the efficacy of NAA may be due to its faster uptake (Martin 2003b). Higher rooting percentage (90%) was obtained in Liquidamber styraciflua shoots cultivated in half strength WPM with 0.5 mg/l NAA (Durkovic and Lux 2010). The efficacy of NAA at lower concentration in rooting has also been reported in other medicinal plants like Lippia alba (Gupta et al. 2001), Verbascum thapsus (Turker et al. 2001),

Safflower (Mandal and Gupta 2001), Santolina canescens (Casado et al. 2002) and Ruta graveolens (Bohidar et al. 2008).

Swamy et al. (1992) observed the induction of roots in Dalbergia latifolia on IAA supplemented medium. Similarly, Peternal et al. (2009) observed 60% rooting in Populus tremula in half strength MS supplemented with IAA (1.0 µM) without activated charcoal (AC). However, the effect of AC on root induction has been reported in different plant species such as Rollina mucosa (Figueiredo et al. 2001), Curcuma zedory (Loc et al. 2005) and Swertia chirayitia (Joshi and Dhawan 2007a and 2007b). But, the results of Balaraju et al. (2009) indicated that no AC is required for rooting in S. chirata and the most effective rooting (83%) was achieved on media comprised of MS + NAA (0.1 mg/l) with maximum 22.48 roots/shoot within 40 days.

To improve rooting percentage in Albizia odoratissima (Rajeswari and Paliwal 2006), a two-step method was adopted. Microshoots were treated with 25 µM IBA for 24 hrs and then transferred to PGR free MS medium. Such shoot exhibited highest 85% rooting and produced maximum number (6.15) of roots. Similar results were also obtained in an earlier study by Sinha et al. (2000) in Albizia chinensis, where microshoots were treated with 2.0 mg/l IBA in MS medium and then subsequently subcultured in IBA free medium for the development of efficient rooting. Two step rooting procedure was also found to be effective in Quercus floribunda (Purohit et al. 2002). In another leguminous tree species Pterocarpus marsupium (Anis et al. 2005), a pulse treatment with an auxin IBA (200 µM) together with a phenolic acid for 5 days and subsequently transferring to lower concentration of IBA (0.5 µM) on half strength MS was proved to be efficient for 40-50% rooting. Husain et al. (2008) further suggested the incorporation of (Phloroglucinol) PG along with IBA to facilitate better rhizogenesis in the same plant in a two-step rooting procedure. Two-step rooting procedure has also been used in several other studies (Shekhawat et al. 1993; Choi et al. 2001; Romano et al. 2002; Husain and Anis 2004 and Husain et al. 2007a).

2.5.2 Ex vitro rooting Ex vitro rooting provides is an alternative approach to induce rooting in microshoots, it is economical, saves time and requires less labour, chemicals and equipments. Ex vitro rooted plantlets did not require any additional step for hardening and acclimatization prior to transplanting to the green house or field conditions (Pruski et al. 2000). Like in vitro rooting, ex vitro rooting is also affected by the chemical nature and concentration of auxins and the explant source (Yan et al. 2010b) and it has been successfully applied in a variety of plant species (Stapfer et al. 1985; Economou and Spanoudaki 1985; Zhang and Davis Jr 1986; Shibli and Smith 1996; Kim et al. 1997 and Liu and Li 2001). For the induction of ex vitro rooting different workers have emphasized the careful selection of planting substrate and rooting treatment. The cut ends of the microshoots were first dipped in different concentrations of rooting media comprised of different auxins (IAA, IBA and NAA) followed by subsequent transferring to sterile planting substrate. Successful ex vitro rooting has been achieved in Siratia grosvenorii (Yan et al. 2010a) by NAA treatment. NAA has also been reported to induce ex vitro rooting in Rotula aquatica (Martin 2003b). But there are certain reports which suggested that IBA is more effective for ex vitro root induction (Bhatia et al. 2002; Siddique and Anis 2006 and Ahmad and Anis 2007). Contrary to all the above reports Feyissa et al. (2007) observed that IAA is more effective for the induction of ex vitro roots in Hagenia abyssinica.

Martin et al. (2006) obtained 90% rooting in Celastrus paniculatus with 2-3 roots of 2-4 cm in length through pulse treatment in a solution of 100 mg/l each of IBA and NOA for 2 h and then for 3 min in 10 mg/l chlogengenic acid. Further they reduced the cost and time involved in rooting by directly planting the in vitro grown shoot tips of Celastrus paniculatus in polythene bags filled with river sand and coir pith compost (1:1) and kept in humid chamber in green house. Within 9 days a maximum of 98-99% shoots were successfully rooted, hence reduced the number of separate steps and the time for root induction and hardening.

2.6 Synthetic seeds The encapsulation of in vitro derived propagules in a nutrient gel matrix leads to the production of synthetic seeds or artificial seeds. Synthetic seeds also known

51 as ‘syn seeds’ may be defined as the artificially encapsulated somatic embryos, shoot buds, cell aggregates or any other tissue that can be used for sowing as a seed and possess the ability to convert into a plant under in vitro or ex vitro conditions, and that retain this potential also after storage (Capuano et al. 1998 and Ara et al. 2000). Encapsulation provides protection and facilitates conversion of in vitro derived propagules, therefore, encapsulation matrix must contain nutrients, growth regulators and other components necessary for germination and conversion of synthetic seeds (Ara et al. 2000). A number of substances like potassium alginate, sodium alginate, carrageenan, agar, gelrite, sodium pectate etc. have been used as encapsulation matrix but sodium alginate

(Na2-alginate) obtained from brown algae is the most suitable and is being widely used at present (Redenbaugh 1993).

Encapsulation technology provides an effective means for in vitro germplasm conservation, easy handling, exchange of genetic material between laboratories, short or long term storage and direct transfer of in vitro material to ex vitro conditions (Standardi and Picconi 1998; Ara et al. 2000; Chand and Singh 2004b; Rai et al. 2009 and Germaná et al. 2011). During the last 25 years intensive researches have been made in the field of synthetic seed technology. This technology provides a new dimension for future plant production and can be applied to reduce the need for transplanting and subculturing during off season periods. During cold storage, encapsulated nodal segments requires no transfer to fresh medium, thus reduces the cost of maintaining germplasm cultures (West et al. 2006). The first report of synseed production was made by Kitto and Janick (1982), describing the encapsulation of carrot somatic embryos followed by their desiccation. Since then synthetic seeds have been widely utilized for the mass multiplication and conservation of a large number of plant species including many medicinal plants such as Ocimum species (Mandal et al. 2000), Adhatoda vasica (Anand and Bansal 2002), Rauvolfia tetraphylla (Faisal et al. 2006d) Phyllanthus amarus (Singh et al. 2006a), Withania somnifera (Singh et al. 2006b), Rauvolfia serpentina (Ray and Bhattacharya 2008) and Cannabis sativa (Lata et al. 2009a).

Somatic embryos are largely favoured for the production of synthetic seeds as these structures possess both radicle and plumule that are able to develop into a

52 root and shoot in one step, without any pre-treatment (Redenbaugh 1993). Encapsulation of somatic embryos for the production of artificial seeds has been reported in many plants including Arachis hypogea (Padmaja et al. 1995), Camellia japonica (Janeiro et al. 1997), Carica papaya (Castillo et al. 1998), Asparagus cooperi (Ghosh and Sen 1994a), Arnebia euchroma (Manjkhola et al. 2005), Rotula aquatica (Chithra et al. 2005) and Quercus suber (Pintos et al. 2008). Various types of synseeds have been prepared using somatic embryos which have been either dried (Gray 1987 and Senaratna et al. 1995) or maintained fully hydrated (Redenbaugh 1990; Ghosh and Sen 1994a; Padmaja et al. 1995; Onay et al. 1996 and Ara et al. 1999), these may or may not be encapsulated (Kitto and Janick 1985a; 1985b; Redenbaugh et al. 1987 and Bapat and Rao 1992).

In many plant species the unipolar structures such as hairy roots (Uozumi et al. 1992 and Nakashimada et al. 1995), axillary shoot buds (Ahmad and Anis 2010 and Singh et al. 2010), apical shoot tips (Rai et al. 2008 and Singh et al. 2009) and protocorm like bodies (Sarmah et al. 2010) have also been encapsulated to produce synthetic seeds. Since, axillary buds and shoot tips do not have root meristems they should be induced to regenerate roots before encapsulation. Piccioni (1997) and Capuano et al. (1998) described the conversion of shoot buds of apple clonal root stocks, encapsulated after an appropriate root induction treatment with IBA (24.6 µM) for 3-6 days. However, Bapat and Rao (1990) and Ganapathi et al. (1992) described the conversion of encapsulated shoots buds of Mulberry and Banana into plantlets without any specific root induction treatments. A number of woody plant species have been successfully propagated from artificial seeds containing in vitro shoot tips or nodal segments, like Cedrela odorata (Maruyama et al. 1997a; 1997b), Coffea arabica (Nassar 2003), Dalbergia sissoo (Chand and Singh 2004b), Hibiscus moschetus (West et al. 2006), Olea europea (Micheli et al. 2007 and Ikhlaq et al. 2010) and Corymbia torelliana x C. citriodora (Hung and Trueman 2012).

Potential advantages of synthetic seeds include their designation as ‘genetically identical materials’ ease of handling and transportation, along with increased efficiency of in vitro propagation in terms of space, time and labour and overall cost (Nyende et al. 2003). Faisal and Anis (2007) reported the regeneration of

Tylophora indica from encapsulated nodal segments collected from two years old plant. Ideal beads were produced using 3% sodium alginate and 100 mM calcium chloride. Maximum of 91% beads were converted into plantlets on MS medium containing 2.5 µM BA and 0.5 µM NAA. Similar combination of 3% sodium alginate and 100 mM calcium chloride was also tried earlier by Faisal et al. (2006d) for the encapsulation of NS of Rauvolfia tetraphylla to produce ideal and uniform beads. Lower concentrations of Na2-alginate resulted in the production of soft and fragile beads which were difficult to handle while the higher concentrations produced harder beads which retarded the regeneration frequency. Maximum conversion (75.3%) of beads into multiple shoots was recorded on MS medium containing BA (10.0 µM) and NAA (0.5 µM). An encapsulation matrix of 5% Na2-alginate with 50 mM CaCl2.2H2O with ion exchange duration of 30 min was found to be suitable for the formation of ideal beads in Cannabis sativa (Lata et al. 2009a). However, in Picrorhiza kurroa (Mishra et al. 2011), in vitro grown microshoots (ST and NS) were encapsulated in the alginate beads containing 3% Na2-alginate and 3% CaCl2.2H2O. After 3 months of storage at 25 ± 2ºC in moist conditions, the encapsulated explants were capable of regrowth within 2 weeks of following cultures, the frequency of regrowth was 89.33% after 4 weeks of culture on PGR free MS medium.

Synseed technology offers an effective tool for the germplasm conservation and short term storage for important medicinal plant species. Therefore, storage of propagules at appropriate temperature and duration is essential to maintain the viability of germplasm during exchange of material between laboratories and also for future germination of synseeds. The percent conversion of encapsulated NS into complete plantlets was quite high in comparison to non-encapsulated explants after 4 weeks of storage at 4ºC as reported in Tylophora indica (Faisal and Anis 2007). This report was in consonance with several earlier reports (Mandal et al. 2000; Chand and Singh 2004b; Faisal et al. 2006d; Singh et al. 2006a and 2006b) which also supported the fact that cold storage of encapsulated explants was more beneficial than storing at room temperature. In most of the studies, 4ºC temperature was found to be the best for synseeds storage (Saiprasad and Polisetty 2003; Kavyashree et al. 2006; Singh et al. 2007; Pintos et al. 2008; Sharma et al. 2009; Ikhlaq et al. 2010 and Tabassum et

54 al. 2010). However, Hung and Trueman (2012) suggested that storage of synthetic seeds of Corymbia torelliana x C. citridora at 25ºC was much more effective than refrigeration at 4ºC. Direct transfer of synseeds to the external environment or ex vitro sowing in the sterile potting media (compost/potting mix/vermiperlite) helped in the elimination of in vitro culture passages but its application is limited due to its high cost of sterilising potting media (Hung and Trueman 2012). However, ex vitro conversion of synthetic seeds has been reported in many plant species (Mandal et al. 2000; Pattnaik and Chand 2000; Soneji et al. 2002 and Naik and Chand 2006). Earlier, Bapat and Rao (1990) reported 60% conversion of encapsulated axillary buds of mulberry under non sterile conditions in autoclaved alginate matrix MS medium without sucrose. Similarly, Lata et al. (2009a) obtained 100% conversion of synseeds of Cannabis sativa on 1:1 potting mix composed of fermilome and coco natural medium.

2.7 Acclimatization The ultimate success of micropropagation protocols depend on the ability to transfer plants from in vitro to ex vitro or external environment with high survival rates (Saxena and Dhawan 1999). Direct transfer of in vitro raised plantlets to the field conditions is not possible due to high mortality rate as the plantlets developed within the culture vessels were under low level of light, aseptic conditions on a nutrient medium containing ample sugar to allow for heterotrophic growth and in an atmosphere with high level of humidity. These culture conditions resulted in plantlets with abnormal morphology, anatomy and physiology (Pospíšilová et al. 1992; Desjardins 1995; Hazarika 2003 and Chandra et al. 2010). The concentration of sucrose and agar in the medium also affected the subsequent acclimatization to ex vitro conditions (Synková 1997 and Lavanya et al. 2009). Regenerated plantlets on transferring to field or green house conditions faced various abiotic (altered temperature, light intensity and humidity conditions) and biotic (soil microflora) stress conditions. Direct transfer to sunlight also causes charring of leaves and wilting of the plants. Thus, a period of acclimatization or more specifically a period of transitional development is required in which both anatomical characters and physiological performances overcome the influence of in vitro culture conditions for the successful

55 establishment and survival of the plantlets (Donnelly et al. 1986; Hiren et al. 2004; Lavanya et al. 2009 and Deb and Imchen 2010).

The acclimatization period may vary from 15 days to 1 month depending upon the nature and hardness of the plant species (Ziv et al. 1987; Donnelly and Tisdall 1993; Nowak and Shulaev 2003 and Hazarika 2003). The in vitro raised plantlets have non-functional stomata, weak root system and poorly developed cuticle which resulted in high mortality of regenerants upon transfer to ex vitro conditions (Mathur et al. 2008). Stomata are generally large with changed shape and structure having guard cells with thinner cell wall and contain more starch and chloroplast (Martin et al. 1988). During acclimatization to ex vitro conditions leaf thickness gradually increases, leaf mesophylls differentiated into palisade and spongy parenchyma, stomatal density decreased and became elliptical from circular shape. Development of cuticle, epicuticular waxes and effective stomatal regulation of transpiration occur leading to stabilization of water potential of field transferred plantlets (Pospíšilová et al. 1999 and Chandra et al. 2010). Several growth retardants can be used in micropropagation to reduce damage due to wilting without deleterious side effects. Use of paclobutrazol (0.5-4.0 mg/l) in the rooting medium is reported to reduce stomatal apertures, increase epicuticular wax, short stem and thick roots (Smith et al. 1990a; 1990b; 1991 and Chandra et al. 2010). Abscisic acid (ABA) a naturally occurring plant hormone played an important role in plant water balance and in the adaptation of plantlets to stress environments including low temperature (Hetherington 2001 and Finkelstein and Gibson 2002). Various stresses induce ABA synthesis and considered as a plant stress hormone (Tuteja 2007). Aguilar et al. (2000) studied the role of ABA on tolerance to abiotic stress in Tagetes erecta in controlling leaf water loss, survival and growth of microshoots when transferred directly to the field.

Various strategies have been applied by different workers for the successful acclimatization and establishment of regenerated plants in field conditions. Bhuyan et al. (1997) reported the acclimatization of plantlets of Murraya koenigii in vermicompost inside the plant growth chamber for 3 weeks and then subsequently established the plantlets in soil with about 85% survival rate. The rooted plantlets of Yucca aloifolia (Atta-Alla and van Staden 1997) were treated with 0.2% Benlate (Active ingredient, Benzimidazole) for 10 min to reduce fungal

56 contamination. These plantlets were then hardened off under mist culture media containing loam soil, Sphagnum peat and pine bark in 1:1:1 ratio. After 3 weeks of acclimatization plantlets were transferred to greenhouse conditions. Komalavalli and Rao (2000) reported 80-85% survival of plantlets of Gymnema sylvestre in the soil. While, Anitha and Pullaiah (2002a) observed only 40% plant survival in Sterculia foetida in the field conditions after adopting a series of hardening and acclimatization procedure. Rooted plantlets of S. foetida were placed in liquid quarter strength MS basal medium and then transferred to pots containing sterilized sand, soil and manure mixture (1:1:1). Plantlets were irrigated with half strength MS basal medium and acclimatized to room temperature for 14 days and later shifted to green house and kept under shade for 30 days, then finally planted in soil with 40% survival. Rooted plantlets of Tinospora cordifolia (Raghu et al. 2006a) were acclimatized in thermocol cups containing sand and soil (1:1) for 14 days. After that plantlets were transferred to nursery for 2 months and then transplanted in the soil with 80% plant survival. Direct transfer of plantlets from culture vessels to pots showed a high rate of mortality in Pterocarpus santalinus (Prakash et al. 2006). Thus, plantlets were first acclimatized in the plant growth chambers for 2 weeks with high humidity and other incubation conditions. Plantlets were then transferred to pots containing a mixture of soil and farmyard manure (4:1) and kept under shade area of forest nursery for about 4 weeks and exhibited 70% survival rate. About 90% of the micropropagated plants of Mucuna pruriens (Faisal et al. 2006b) survived following transfer from soilrite to natural soil and did not show any detectable variation in respect to morphology or growth characteristics. Khatun et al. (2010) gradually shifted the micropropagated plants of Citrullus lanatus from culture room to field conditions. Firstly, the plantlets were kept in growth room for two days then transferred from growth room to open room and kept there for four days. Plantlets were regularly sprayed with water and covered with polythene bags to maintain higher humidity. Finally, the plants were gradually acclimatized to outdoor conditions with 80% survival rate and satisfactory growth.

2.8 Physiological studies The transferring of in vitro raised plantlets from low light intensity (1200-3000 lux) and controlled temperature (25 ± 2ºC) to broad spectrum sunlight (4000-12000 lux) and high temperature (26-36ºC) of external environment often resulted in the charring of leaves and wilting of the plantlets. It also might lead to transient decrease in photosynthetic parameters. Therefore, it is necessary to hardened or acclimatized the plants under greenhouse or shade conditions before being exposed to field environment (Lavanya et al. 2009 and Chandra et al. 2010). In many plant species, the leaves formed in vitro are unable to develop further under ex vitro conditions and are replaced by newly formed leaves (Preece and Sutter 1991 and Diettrich et al. 1992). However, if the ex vitro transplantation of plantlets is successful, the increase in their growth can be enormous for example, total dry mass of Nicotiana tabacum plants was several times higher than that of plantlets grown in vitro, the transplanted plants were taller, had higher dry mass of leaves, stem and roots and larger leaf area and leaf thickness (Kadleček et al. 1998). After transplantation of tissue culture plants to ex vitro conditions, most of the plants develop a functional photosynthetic apparatus and the contents of photosynthetic pigments increased, although the increase in light intensity is not linearly translated in an increase in photosynthesis (Kozai 1991; Trillas et al. 1995; Rival et al. 1997; Synková 1997; Pospíšilová et al. 1998 and Amâncio et al. 1999).

In Calathea louisae the in vitro formed leaves were unable to photosynthesize during the first days after transplantation but in Spathiphyllum floribundum the in vitro formed leaves were photosynthetically capable and normal source-sink relations were observed. Nevertheless, in both plant species, considerable photosynthetic activities were measured when new leaves were fully developed (Van Huylenbroeck et al. 1998). Further, Van Huylenbroeck et al. (2000) reported three times higher contents of chlorophyll and carotenoids in ex vitro formed leaves in comparison to in vitro ones and they observed an inverse relation between PPFD and the chlorophyll/carotenoids ratio at the end of acclimatization. In Solanum tuberosum and Spathiphyllum floribundum net photosynthetic rate (PN) decreased in the first week of transplantation and increased thereafter (Baroja et al. 1995; Van Huylenbroeck and Debergh 1996

58 and Pospíšilová et al. 1999). Transfer of in vitro raised plants to ex vitro conditions under direct sunlight might cause photoinhibition and chlorophyll 14 photobleaching. After transplantation, the CO2 uptake by persistent leaves of Fragaria and Rubus idaeus was similar to that in plantlets grown in vitro or was 14 slightly increased, and a significantly increased CO2 uptake was found only in newly formed leaves (Short et al. 1984 and Deng and Donnelly 1993). The above mention result suggests that photoinhibition might be the cause of the transient decrease in photosynthesis after transplantation (Chandra et al. 2010).

2.9 Different factors affecting in vitro regeneration 2.9.1 Nature of explant: source, type and age of the explant Khalafallah and Daffala (2008) observed that the CN explants of a leguminous tree species Acaica senegal derived from 7 days old axenic seedlings provided better response as compared to the NS explants collected from 12 months old greenhouse grown plants. CN explants gave the highest number and longest in vitro regenerated shoots compared to those induced from NS on the same regeneration medium. Thus, the study suggested that young and juvenile explants (CN) responded better than the mature explants (NS) and it appeared that multiplication rate is feasible by using defined PGRs and supplements when combined with appropriate physiological state of the explant. The rate of shoot regeneration in Sterculia urens (Sunnichan et al. 1998) is relatively quite low (6.0 shoots/explant) when the explants (NS) were collected from mature tree compared to the (11.24 shoots/CN) explants derived from in vitro grown seedlings (Devi et al. 2011). The source and the type of explants had a great influence on the induction and multiplication of shoots in vitro in different plant species including many woody legumes such as Dalbergia sissoo (Pradhan et al. 1998a), Albizia lebbeck (Mamun et al. 2004), Pterocarpus marsupium (Anis et al. 2005) and Albizia odoratissima (Rajeswari and Paliwal 2006).

The rate of regeneration and morphogenetic response varied to a great extent according to the type of explants. The differences in culture requirements exist among different parts of the same plant may be attributed to the various levels of endogenous plant growth regulators of explants from different positions (Ghosh and Sen 1994b; Yucesan et al. 2007 and Lisowska and Wysokinska 2000).

According to Thengane et al. (2001) leaf explant was proved to be more significant for the regeneration of adventitious shoots in Nothapodytes foetida followed by hypocotyl and cotyledons. Vengadesan et al. (2002) suggested that the percentage response varied with the type of growth regulators used, its concentration and the type of explants. Between CN and ST explants derived from axenic seedlings, CN explants provided better morphogenic potential for multiple shoot development in Acacia sinuata. Sivanesan and Jeong (2007) reported more number of shoots from NS explants as compared to ST explants in Pentanema indicum. However, maximum shoot regeneration in Carlina acaulis (Trejgell et al. 2009) and Senecio macrophyllus (Trejgell et al. 2010) was reported from seedling derived ST explants in comparison to the hypocotyl, cotyledon and root explants. In Stevia rebaudiana also, ST explants were proved to be the most effective explant for maximum shoot regeneration than NS and L explants when cultured on MS medium supplemented with 1.0 mg/l BA and 0.5 mg/l IAA (Anbazhagan et al. 2010). However, Gonçalves et al. (2010) reported maximum shoot proliferation from NS than apical ST explants in Tuberaria major.

Age of the different explants collected from in vitro grown seedlings had differential response on multiple shoot regeneration in different plant species and it varied according to the type of growth regulators used, their concentrations and type of the explants. CN explants collected from 20 days old axenic seedlings of Psoralea corylifolia (Jeyakumar and Jayabalan 2002) produced multiple shoots on MS medium supplemented with different PGRs. A significantly greater number of shoots were produced from cotyledons of 14 days old embryos in mulberry on MS medium containing TDZ and BA compared to 7 and 21 days old explants (Thomas 2003). In Pterocarpus marsupium also, among various explants used for the induction of multiple shoots, CN explants excised from 18 days old seedlings provided excellent response in terms of highest number of shoots/explant as well as maximum shoot length (Anis et al. 2005 and Husain et al. 2007a). However, Chand and Singh (2004a) excised CN explants of P. marsupium from 20 days old seedlings for multiple shoot regeneration. Alam et al. (2010) used CN explants derived from 5-7 days old aseptic seedlings of Ricinus communis for multiple shoot regeneration. Karuppusamy and

Kalimuthu (2010) developed an efficient regeneration protocol for an endemic medicinal plant Andrographis neesiana through NS derived from 30 days old seedlings. Husain et al. (2008) used 18 days old nodal segments excised from axenic seedling in Pterocarpus marsupium. Different explants like leaf segments, nodal segment and root segments derived from 5 weeks old seedlings were used for the induction of callus in Citrus jambhiri by Savita et al. (2010). Similarly, Prakash and Gurumurthi (2010) induced callus in Eucalyptus camaldulensis using mature zygotic embryos and cotyledons explants collected from 10, 15, 25 and 30 days old aseptic seedlings. The frequency of callus induction decreased with the increasing age of the explants and the highest frequency was obtained in 10 day old explants (60%) followed by 15 day old explants (43%).

2.9.2 Media composition The rate of regeneration often depends not only on the selection of the most suitable explants but also on the composition of the basal medium. The nutritional requirement varies according to the cell, tissues and organ and also with respect to particular plant species (Basu and Chand 1996). A variety of media and salt concentrations have been used, but MS medium still remain the most widely used formulation for the multiplication of different plant species. The

B5 and WPM media are also used most frequently for the micropropagation of several plants. Several researchers have used the modification of these media or other formulations like White’s medium (White 1963), RWM (Risser’s and White’s Medium 1964), LS (Linsmaier and Skoog Medium 1965), NN (Nitsch and

Nitsch 1969), SH (Schenk and Hildebrandt’s Medium 1972), L2 (Philips and

Collins 1979), AE (Von Arnold and Eriksson 1981) and UM (Litvay et al. 1985) for axillary, adventitious as well as somatic embryogenesis etc. various mineral salt formulations have been used for in vitro culture, however, full strength mineral salts are not always optimum and different formulations may work better at different stages (Thorpe et al. 1991). These combinations are used either at full strength or with modifications based on experimental results. Modification in the MS such as MS salts reduced to one half, one third, one fourth, one fifth or three fourth have been found effective in different reports (Badji et al. 1993; Hung et al. 1994; Das et al. 1996a and Faisal et al. 2007).

B5 medium has been used by several workers in the micropropagation of important plant species and proved to be effective in the regeneration of Pterocarpus marsupium (Lakshmi Sita et al. 1992), Acacia nilotica (Dewan et al. 1992 and Samake et al. 2011), P. santalinus (Anuradha and Pullaiah 1999), Gardenia jasmonoides (Chuenboonngarm et al. 2001) and Jatropha curcas (Warakagoda and Subasinghe 2009). Joshi et al. (2003) concluded that for the establishment and elongation of shoots in a leguminous tree species, Dalbergia sissoo, B5 medium is better than MS medium, while, the maximum number of shoots were obtained on MS medium. Similar results were also obtained by Berger and Schaffner (1995) in another leguminous tree species Swartzia madagascariensis. However, Abbas et al. (2010) proved that MS medium was more effective than B5 medium in A. nilotica for the production of maximum number of shoots. Bhatt and Dhar (2004) suggested that WPM provided better response than B5, MS and half strength MS in Myrica esculanta. Moreover, WPM has also been used in a number of plant species as the regeneration medium such as Ixora coccinea (Lakshmanan et al. 1997), Acacia catechu (Das et al. 1996b), Cinnamomum camphora (Nirmal Babu et al. 2003), Tinospora cordifolia (Raghu et al. 2006a) and Salix tetrasperma (Khan et al. 2011). Wang et al. (2005) were of the opinion that WPM and B5 had better effects for shoot regeneration than MS medium in Camptotheca acuminata and relatively higher shoot buds were produced. Douglas and McNamara (2000) obtained adventitious shoot regeneration in Acacia mangium by using Juglans medium

(DKW). Baskaran and Jayabalan (2008 and 2009b) used L2 medium for the micropropagation of Psoralea corylifolia.

Tiwari et al. (2004) tested three different media (MS, B5 and White’s) with or without different PGRs for the in vitro propagation of Pterocarpus marsupium using NS explants. Amongst all the three media tested, MS medium was proved to be the best for highest shoot regeneration. Among three different media (MS,

B5 and SH) tested by Baskaran and Jayabalan (2005) for the micropropagation of Eclipta alba, MS medium was found to support better shoot regeneration than

B5 and SH media. MS medium has also found more effective than other media in several other medicinal plants also (Gao et al. 1999; Komalavalli and Rao 2000; Wondyifraw and Surawit 2004; Wang et al. 2008 and Perveen et al. 2011).

2.9.3 Sources of carbohydrate and their concentration In vitro culture of plant cells, tissue and organ generally requires the addition of a carbon or energy source to the culture medium (George 1993 and Karhu 1997). Sucrose has been used as the most common source of carbon in plant tissue culture probably, because it is the most common carbohydrate in the phloem sap of many plants (Lemos and Baker 1998 and Fuentes et al. 2000). However, besides sucrose, some additional carbohydrate sources like glucose, fructose, maltose and sorbitol etc. has also been used by different workers as an alternative source (Lou and Sako 1995; Hisachi and Murai 1996; Mamiya and Sakamoto 2000; Vijaya Chitra and Padmaja 2001; Mosaleeyanon et al. 2004; Anwar et al. 2005 and Mohamed and Alsadon 2010).

In most of the cases, optimum concentration (3%) of sucrose than 2 and 4% was used for shoot multiplication and proliferation as recommended by Murashige and Skoog (1962). Baskaran and Jayabalan (2005) reported that among three different carbohydrate sources (glucose, fructose and sucrose), 3% sucrose was found to be better for shoot regeneration than fructose and glucose. Augmentation of 3% sucrose in the culture medium has been found to provide best response in cork oak (Romano et al. 1995) and Kaempferia (Shirin et al. 2000). While, sucrose and glucose induced the highest frequency of shoot regeneration in Bixa orellana (De Paiva Neto et al. 2003). There are many researchers who used the various concentrations of sucrose either higher or lower for shoot proliferation. Effect of different concentrations of sucrose was studied by Gürel and Gülşen (1998) on in vitro shoot production of Amygdalus communis during three successive stages of development. During the initiation and transplantation stage, 5 and 6% sucrose gave the best results with respect to shoot production and growth. During the multiplication stage, the highest rate of shoot production was achieved with 3 and 4% sucrose. Beck et al. (1998) reported that an increase in sucrose concentration increased the shoot production in Acacia mearnsii. While, in some cases of somatic embryogenesis, lower and higher concentrations of sucrose has been tested for specific purposes as reported by Xie and Hong (2001a). Karami et al. (2006) suggested that higher concentrations (9 to 15%) of sucrose improved maturation of somatic embryos, while regeneration of complete plantlets from these embryos was

63 achieved on half strength MS medium without any PGR containing 3% sucrose only. Higher concentrations of sucrose have also influenced somatic embryogenesis in other plant species (Tremblay and Tremblay 1991; Ricci et al. 2002 and Biahoua and Bonneau 1999).

Tyagi et al. (2007) recommended the use of LR grade sucrose and sugar cubes in Curcuma longa cultures. Joshi et al. (2009) tried to develop a micropropagation protocol for Wrightia tomentosa using different carbon sources, gelling agents and type of culture vessels in an effort to reduce the cost of micropropagation. Observations revealed that the best regeneration was achieved on the medium containing sugar cubes (3%) as the carbon source. The promontory effect of sugar cubes in shoot multiplication has been previously reported in Leucaena leucocephala (Dhawan and Bhojwani 1984). Sridhar and Naidu (2011) observed that the highest number of shoots in Solanum nigrum were obtained on MS medium supplemented with 4% fructose, while, maximum shoot length was achieved on medium containing 4% sucrose.

2.9.4 pH of the medium The pH of the medium has a promotory effect on nutrient uptake as well as enzymatic and hormonal activities in plants. Plant cells and tissues require an optimum pH for growth and development (Hussey 1986; Gürel and Gülşen 1998 and Bhatia and Ashwath 2005). Changes in the pH of the medium influenced the performance and development of explants (George et al. 2008a). The optimum pH level regulates the cytoplasmic activity that affects cell division and growth of shoots and it does not interrupt the function of the cell membrane and the buffered pH of the cytoplasm (Brown et al. 1979). Lazzeri et al. (1987) concluded that the pH value had no significant effect on the efficiency of shoot regeneration in soybean. But in most of the studies, pH value 5.8 was found to be the best for shoot morphogenesis (Gautam et al. 1993; Nair and Seeni 2003 and Perveen et al. 2011). However, certain plants require acidic pH for maximum regeneration (Naik et al. 2010).

Sanavy and Moeini (2003) reported that the pH level of 5.5 was the best for the overall growth of the plants in potato meristem culture. Low and high levels of pH than 5.5 were found to reduce the growth and rooting. The reduction was more

64 pronounced at low levels than high levels of pH. Similarly, Huda et al. (2009) also reported that the highest rate of shoot regeneration was achieved in a medium comprised of 0.5 mg/l IAA and 3.0 mg/l BA at pH 5.5 in tossa jute. A range of pH level from 5.8 to 6.6 was found to be effective for shoot regeneration of Camptotheca acuminata (Wang et al. 2005), while, the best response (90%) and a high shoot number was found on the medium at pH 5.8. At pH 7.0 and below 5.4 the regeneration was low; moreover on the medium with pH value below 5.4, regenerated shoots show vitrification. In chickpea (Barna and Wakhlu 1993) pH 6.5 was proved to be the optimum for embryo maturation which was adversely affected by the pH above 7.0 and below 4.0.

Chapter 3 Materials & Methods

Chapter 3

MATERIALS AND METHODS

3.1 Plant material and explant source The present study deals with the in vitro morphogenetic studies in selected medicinally important Cassia spp. namely C. angustifolia Vahl. and C. sophera Linn. The certified seeds of both species were obtained from Prem Nursery and Seed Store, Dehradun, India and used to raise aseptic seedlings for the collection of various explants like cotyledonary node (CN), nodal segment (NS), shoot tip (ST), cotyledonary leaf (CL), leaf (L) and root (R).

3.2 Culture media Growth and morphogenesis of plant tissues in vitro are largely governed by the composition of culture media. A number of media have been devised for specific tissues and organs but the most notable one, which served as a basic medium for wide spectrum of plant tissues for morphogenetic studies is that formulated by Murashige and Skoog (1962) i.e., MS medium or Murashige and Skoog’s medium. MS medium is the most suitable and commonly used medium for plant regeneration from tissues and callus. This is a high salt medium due to its content of potassium and nitrogen salts. The medium comprised of three basic components:

i) Inorganic nutrients

ii) Organic supplements

iii) Carbon source

3.2.1 Inorganic nutrients: Mineral elements are very important in the life of a plant. For example, magnesium is a part of chlorophyll molecules, calcium is a constituent of the cell wall and nitrogen is an important part of amino acids, vitamins, proteins and nucleic acids. These are classified as macronutrients and micronutrients:

a) Macronutrients: Those elements which are required in concentration greater than 0.5 mM/l are classified as macronutrients or major elements (De Fossard 1976). These include nitrogen (N), potassium (K), phosphorus (P), calcium (Ca), magnesium (Mg) and sulphur(S) in the form of salts in media. + Nitrogen is usually supplied in the form of ammonium (NH4 ) and nitrate - (NO3 ) ions. Nitrate is superior to ammonium as the sole nitrogen source but use of NH4+ checks the increase of pH towards alkalinity. b) Micronutrients: Those elements which are required in concentration less than 0.05 mM/l are classified as micronutrients or minor elements (De Fossard 1976). These include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu) and molybdenum (Mo). These inorganic elements although required in small quantity are essential for plant growth, most critical of them being iron, which is provided as Fe-EDTA complex.

3.2.2 Organic supplements: To achieve the best growth of the tissue it is often essential to supplement the medium with one or more vitamins and amino acids.

a) Vitamins: These are organic substances required for metabolic processes as cofactors or parts of enzymes. The most widely used

vitamins in plant tissue culture are thiamine (vitamin B1), nicotinic acid

(vitamin B3), pyridoxine (vitamin B6) and myo-inositol (a member of the vitamin B complex).

b) Amino acids: Amino acids may be directly utilized by the plant cells or may serve as a nitrogen source. The most often used amino acid is L- glycine.

In the present investigation, MS medium was used as primary basal medium for in vitro studies in both species. Other media like B5 (Gamborg et al. 1968), L2 (Philips and Collins 1979) and WPM (Lloyd and McCown 1980) were also tested for shoot differentiation in both species. Different constituents of MS, B5, L2 and WPM medium along with their concentrations used are listed in Table 4.

Table 4. Nutritional composition of MS, B5, L2 and WPM medium in mg/l.

Components MS B5 L2 WPM (1962) (1968) (1979) (1980)

Macronutrients

MgSO4·7H2O 370 250 435 370

KH2PO4 170 - 325 170

KNO3 1900 2500 2100 -

NH4NO3 1650 - 1000 400

CaCl2·2H2O 440 150 600 96

Ca(NO3)2·4H2O - - - 556

K2SO4 - - - 990

NaH2PO4.H2O - 150 85 -

(NH4)2SO4 - 134 - - Micronutrients

H3BO3 6.2 3.0 5.0 6.2

MnSO4·4H2O 22.3 - 19.8 22.3

MnSO4·H2O - 10.0 - -

ZnSO4·7H2O 8.6 2.0 5.0 8.6

Na2MoO4·2H2O 0.25 0.25 0.4 0.25

CuSO4·5H2O 0.025 0.025 0.1 0.25

CoCl2·6H2O 0.025 0.025 0.1 - KI 0.83 0.75 1.0 -

FeSO4·7H2O 27.8 27.8 25.0 27.8

Na2EDTA·2H2O 37.3 37.3 33.5 37.3

Organic supplements

Vitamins Thiamine HCl 0.1 10.0 2.0 1.0 Pyridoxine HCl 0.5 1.0 0.5 0.5 Nicotinic acid 0.5 1.0 - 0.5 Myo-inositol 100 100 250 100 Amino acid Glycine 2.0 - - 2.0

3.2.3 Preparation of stock solutions of different media

The constituents of MS, B5, L2 and WPM depicted in Table 4 were prepared in the form of four different stock solutions (Table 5). MS stocks consisting of Stock I- Major Salts (20X Concentrated), Stock II- Minor salts (200X Concentrated),

Stock III- FeSo4·7H2O and Na2EDTA·2H2O (100X Concentrated), Stock IV - Organic nutrients except sucrose (100X Concentrated). The stock solutions of

B5, and L2 were prepared hundred times stronger than the final medium and diluted according to the need (Gamborg and Phillips 1996), while, WPM was used as a readymade preparation. All stock solutions were prepared by dissolving the required amount of solute in double distilled water DDW). The reasons for preparing different stock solutions is that certain kind of chemicals, when mixed together will precipitate and does not remain in solutions. To prepare one litre 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 taken.

3.3 Plant Growth Regulators (PGRs) or plant hormones In addition to the nutrients, it is generally necessary to add one or more growth substances, such as auxins, cytokinins and gibberellins, to support good growth of tissues and organs. The growth regulators are required in very minute quantities (µmol/l values).

i) Auxins: These hormones induce cell division, cell elongation, apical dominance, adventitious root formation and somatic embryogenesis. The commonly used auxins are indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). IBA and IAA are widely used for rooting and in combination with a cytokinin, for shoot proliferation. 2,4-D and 2,4,5-T are very effective for the induction and growth of callus. 2,4-D is also an important factor for the induction of somatic embryogenesis. In the present study different auxins were used at concentrations ranging from 1.0 to 10.0 µM for organogenesis and embryogenesis. Auxins are generally dissolved either in ethanol or dilute NaOH.

Table 5. Stock solutions for MS, B5 and L2 medium in mg/l.

Components MS B5 L2

Stock solution I (20X)

MgSO4·7H2O 7400 5000 8700

KH2PO4 3400 - 6500

KNO3 38000 50000 42000

NH4NO3 33000 - 20000

CaCl2·2H2O 8800 3000 12000

NaH2PO4·H2O - 3000 1700

(NH4)2SO4 - 2680 - Stock solution II (200X)

H3BO3 1240 600 1000

MnSO4·4H2O 4460 - 3960

MnSO4·H2O - 2000 -

ZnSO4·7H2O 1720 400 1000

Na2MoO4·2H2O 50 50 80

CuSO4·5H2O 5.0 5.0 20

CoCl2·6H2O 5.0 5.0 20 KI 166 150 200 Stock Solution III (100x)

FeSO4·7H2O 2780 2780 2500

Na2EDTA·2H2O 3730 3730 3350 Stock Solution IV (100x)

Thiamine HCl 10 1000 200 Pyridoxine HCl 50 100 50 Nicotinic acid 50 100 - Myo inositol 10000 10000 25000 Glycine 200 - -

ii) Cytokinins: These hormones are concerned with cell division, modification of apical dominance and stimulate initiation and growth of shoots in vitro. 6-Benzyladenine (BA), 6-furfurylaminopurine (Kn), 2- isopentanyladenine (2-iP) and thidiazuron (TDZ) are commonly used cytokinins. Compared to other cytokinins, TDZ is generally used at very low concentrations and in the present study a wide range of TDZ concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) was tested. While other cytokinins were used at concentrations of 1.0, 2.5, 5.0, 7.5 and 10.0 µM. Cytokinins are generally dissolved in dilute HCl or NaOH.

iii) Gibberellins: There are over 20 known gibberellins. Of these, generally,

GA3 is used for internode elongation, seed germination and meristem

growth. GA3 is readily soluble in cold water up to 1000 mg/l.

The MS basal medium supplemented with various plant growth regulators such as cytokinins and auxins at different concentrations either singly or in combination was used in the present study.

3.3.1 Preparation of stocks of different PGRs Separate stock solutions were prepared for each plant growth regulator (PGR) by dissolving it in a minimal quantity of appropriate solvent (1.0 N NaOH or absolute alcohol) and then diluted with DDW to the desired volume to make a clear solution of 1.0 mM strength. All the stock solutions were stored in a refrigerator at 4°C and regularly checked for visible contamination. The different concentrations of PGRs used in the present study were prepared from stock solutions by using the following formula:

S1V1 = S2V2

Where,

S1 = Strength of stock solution

V1 = Volume of the stock solution required

S2 = Strength of desired solution

V2 = Volume of desired solution

3.4 Carbon and energy source Sugar is a very important part of nutrient medium since most plant cultures are unable to photosynthesize effectively owing to inadequately developed cellular and tissue development, lack of chlorophyll, limited gas exchange and carbon dioxide in tissue culture vessels etc. Hence they lack autotrophic ability and need external carbon for energy. The most commonly used carbon or energy source is sucrose, at a concentration of 2-5%. In the present study 3% (w/v) sucrose was used as a sole carbon source in all the experiments. However, the effect of different concentrations of sucrose i.e. 1, 2, 3, 4 and 5% was also tested on optimized concentrations of PGRs to examine the regeneration potential of the explants. While autoclaving the medium, sucrose is hydrolysed to glucose and fructose which are then used up for growth.

3.5 Adjustment of pH and gelling of the medium

The pH of the MS, WPM, and L2 medium were adjusted to 5.8 and B5 to 5.7 using 1N NaOH or HCl using a pH meter (Elico Pvt. Ltd., India) prior to autoclaving. However, the effect of different pH levels (5.0, 5.4, 5.8, 6.2 and 6.6) was also tested with different explants on optimized concentrations of PGRs. The medium was solidified with 0.8% (w/v) agar or 0.25% (w/v) phytagel by dissolving it in a microwave until a clear gel is formed.

3.6 Filling of the medium The media was dispensed in 25 × 150 mm Borosil test tubes each containing 15- 20 ml of medium while 50 ml in a 100 ml capacity Erlenmeyer flasks (Borosil, India) and closed with cotton plugs (single layered cheese cloth stuffed with non- absorbent cotton).

3.7 Sterilization 3.7.1 Sterilization of the medium All the culture tubes containing medium were placed in a plastic cage, wrapped with butter paper or cellophane sheet and sterilized in an autoclave at 1.06 72

Kgcm-2 (121°C) for about 15-20 min. After autoclaving plastic cages were placed in a tilted position to allow the medium in culture tubes to set as slants and leaved overnight for solidification.

3.7.2 Sterilization of glasswares and instruments All the glasswares, instruments (wrapped in aluminium foil) and DDW etc. were sterilized by autoclaving at 1.06 kgcm-2 for 25-30 min. The forceps, scalpel, etc. made of stainless steel were also sterilized at the time of inoculation under laminar air flow by dipping them in ethanol (95%) followed by flaming and cooling before inoculation.

3.7.3 Sterilization of plant material (seeds) The certified seeds of both species were thoroughly washed under running tap water for about 30 min to remove adherent particles. Washed seeds were dipped in 1% Bavistin (Carbendazim Powder, BASF India Ltd.), a broad spectrum systemic fungicide, for 25-30 min followed by thorough washing with 5% (v/v) Teepol, a liquid detergent, by gentle shaking for 15 min. Subsequent washing with sterile double distilled water (DDW) was done 3-4 times under the laminar air flow hood followed by a short treatment of 30-40 s with ethanol (70%). The seeds were then surface sterilized with 0.1% (w/v) mercuric chloride (HgCl2) (Qualigens, Mumbai, India) for 5-6 min followed by repeated washing (4-5 times) with sterile DDW to remove any traces of sterilant. Sterilized seeds were germinated under controlled aseptic conditions on seed germination media.

3.7.4 Sterilization of laminar air flow hood The laminar air flow cabinet (NSW, Delhi, India) was sterilized by switching on ultraviolet (UV) light for 30 min followed by wiping the working surface with ethanol (70%) before any operation inside the cabinet

3.8 Inoculation of seeds and establishment of aseptic seedlings Inoculation was performed under aseptic conditions of laminar air flow cabinet by using sterilized culture media, instruments and distilled water. The instruments were re-sterilized (time-to-time) during inoculation by dipping them in 95% ethanol followed by their flaming and cooling. The surface sterilized seeds of C.

angustifolia and C. sophera were transferred to Petri dishes and inoculated using sterilized forceps in culture vials containing different strength (full and half) of MS medium (Murashige and Skoog, 1962) with or without Gibberellic acid (GA3) at various concentrations (0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) to raise aseptic seedlings. All the cultures were maintained at 24 ± 2ºC under 16/8 h photoperiod with a photosynthetic photon flux density (PPFD) of 50 µmolm-2s-1 provided by cool white fluorescent tubes (40 W, Philips, India) with 55-60% of the relative humidity.

3.9 Collection of explants and establishment of cultures Cotyledonary node explants (1-1.5 cm), nodal segment explants (1-1.5 cm) and shoot tips (1-1.5 cm) were excised from 7, 14, 21 and 28 days old aseptic seedlings and used for direct shoot regeneration in both the species. However, for callus mediated organogenesis, cotyledons were excised from 7, 14 and 21 days old seedlings while leaf and root explants from 20, 30 and 40 days old aseptic seedlings. For somatic embryogenesis, immature cotyledons (IC) were excised from semi mature seeds of green pods of field grown plants. Single explant was inoculated in each culture tube containing medium and each treatment has 10 replicates. The MS basal medium with or without different cytokinins (BA, Kn, 2-iP and TDZ) and auxins (IAA, IBA, NAA, 2,4-D and 2,4,5-T) at various concentrations either singly or in combination were tested to assess the regeneration potential of various explants. Data for percent regeneration, number of shoots/explant and shoot length were recorded after 6 weeks of culture.

3.10 Rooting in microshoots Rooting was attempted in vitro and ex vitro in microshoots measuring about 3-5 cm with 2-3 pairs of leaves. For in vitro root induction, the isolated shoots were transferred on MS basal and half medium MS medium with or without auxins namely IAA, IBA or NAA at various concentrations (0.5, 1.0, 2.0 and 2.5 µM) as mentioned in results. The rooting medium was gelled with 0.8% (w/v) agar or 0.25% (w/v) phytagel, while liquid medium was prepared without addition of any gelling agent. For ex vitro rooting, the basal end of the healthy shoots (3-4 cm) 74

were dipped in IBA (50, 100, 150, 200, 250 and 300 µM) for 30 min and then planted in small thermocol cups containing sterile soilrite (Keltech Energies Pvt. Ltd.) and covered with transparent polythene bags, having a few perforations for gaseous exchange and to maintain high relative humidity. The shoots were irrigated with one-fourth strength of MS salt solution (without vitamins) for 2 weeks followed by tap water. Hardening and acclimatization of ex vitro rooted shoots was done under controlled conditions. Polythene bags were removed gradually upon emergence of new leaves in order to acclimatize the plants. The rooted plantlets were then transferred to earthen pots containing sterilized soil and manure (1:1) and maintained in greenhouse under natural environment. Data on percentage rooting, number of roots and root length were recorded after 4 and 6 weeks of in vitro and ex vitro rooting respectively.

3.11 Hardening and acclimatization Plantlets with well-developed roots and shoots were removed from the culture medium and washed gently under running tap water to remove any adherent gel from the roots and transferred to thermocups containing sterile soilrite. These were kept under diffuse light conditions (16:8 h photoperiod) covered with transparent polythene bags to ensure high humidity, irrigated after every three days with quarter strength MS salt solution (without vitamins) for two weeks. Polythene membranes were removed after 2 weeks in order to acclimatize plantlets and after 4 weeks they are transferred to pots containing garden soil or a mixture of garden soil and manure (1:1) and maintained in green house under normal day length conditions.

3.12 Synthetic seeds 3.12.1 Explant source Nodal segments approximately 0.5 cm long excised from in vitro shoots of C. angustifolia and C. sophera were used as explants for the preparation of synseeds.

3.12.2 Encapsulation matrix

Sodium alginate (Na2-alginate) (Qualigens, India) was used as encapsulation matrix and prepared in liquid MS medium (with 3% sucrose) at different 75

concentrations i.e. 1, 2, 3, 4 and 5% (w/v). For complexion, 25, 50, 75, 100 and

200 mM calcium chloride (CaCl2·2H2O) solution was prepared using liquid MS medium. The pH of the gel matrix and the complexing agent was adjusted to 5.8 prior to autoclaving at 121°C for 20 min.

3.12.2.1 Encapsulation of explants

Encapsulation was accomplished by mixing the nodal segments with Na2 alginate solution and dropping them in CaCl2·2H2O solution using a pipette. The droplets containing the explants were held at least for 25-30 min to achieve polymerization. The alginate beads containing the nodal segments were retrieved from the solution and rinsed twice with sterilized DDW to remove the traces of CaCl2·2H2O and transferred to sterile filter paper in Petri dishes for 5 min under the laminar airflow cabinet to eliminate the excess of water and thereafter transferred to culture vials containing nutrient medium.

3.12.3 Planting media and culture conditions The encapsulated nodal segments (alginate beads) were transferred to wide mouth culture flask (Borosil, India) containing MS basal and MS medium supplemented with various plant growth regulators either singly or in combination. The culture medium was gelled with 0.8% (w/v) agar and pH was adjusted to 5.8 prior to autoclaving at 121ºC for 20 min. Cultures were maintained at 24 ± 2ºC under 16/8 h light-dark conditions with a PPFD of 50 µmol m-2s-1 provided by cool white fluorescent tubes.

3.12.4 Low temperature storage A set of encapsulated nodal segments and non-encapsulated nodal segments was kept in sterile beakers properly covered with aluminium foil and stored in refrigerator at 4ºC. Six different low temperature exposure times (0, 1, 2, 4, 6 and 8 weeks) were evaluated for regeneration of synseeds into plantlets. After each storage period, synseeds and non-encapsulated nodal segments were transferred to MS medium containing optimal concentration of PGRs for conversion into plantlets. The percentage conversion of encapsulated nodal segments as well as non-encapsulated nodal segments was recorded after 6

weeks of culture. The plantlets developed from encapsulated nodal segments were hardened off and acclimatized as specified above.

3.12.5 Ex vitro conversion of synthetic seeds into plantlets Encapsulated nodal segments were also transferred to sterile soilrite for ex vitro germination and recovery of complete plantlets. The soilrite was regularly moistened with quarter strength MS salt solution (without vitamins) after every 4 days. The conversion response (%) was recorded after 6 weeks of sowing.

3.13 Physiological studies A set of in vitro regenerated plantlets with well developed shoot and roots were transplanted in sterile soilrite and placed in culture room at 25 ± 2ºC and 16/8 h photoperiod at 70-80% RH for 30 days under controlled conditions at 50 µmol m-2s-1 PPFD. Light was provided by 40 W Philips (India) lamps. Leaf samples were taken at transplantation day (day 0, control) and after 1, 2, 3 and 4 weeks and used for physiological studies.

3.13.1 Chlorophyll and carotenoids estimation The chlorophyll (Chl a and b) and carotenoids content from leaf tissue was estimated by the method of MacKinney (1941) and MacLachan and Zalik (1963) respectively. About 100 mg fresh tissues from interveinal areas of leaves were grind in 5 ml acetone (80%) with the help of a mortar and pestle. The suspension was filtered with Whatman filter paper no. 1, if necessary the supernatant was again washed and filtered, the total filtrate was taken in graduated test tubes and final volume was made up to 10 ml with 80% acetone. The optical densities (O.D.) of chlorophyll solution was read at 645 nm and 663 nm wavelengths and for carotenoids, the O.D. was read at 480 nm and 510 nm wavelengths with the help of spectrophotometer (UV-Pharma Spec 1600, Shimadzu, Japan). The chlorophyll and carotenoids content was calculated using the following formulae:

Chlorophyll a (mg/g fresh tissue)

12.7O.D.663 2.69O.D.645 = V 1000W

Chlorophyll b (mg/g fresh tissue)

22.9O.D.645 4.68O.D.663 = V 1000W

Total Chlorophyll (mg/g fresh tissue)

20.2O.D.645 8.02O.D.663 = V 1000W

Carotenoids (mg/g fresh tissue)

7.6O.D.4801.49O.D.510 = V D1000W where,

V = Final volume of chlorophyll extract in 80% acetone.

W = Fresh weight of leaf tissue.

O.D = Optical density at the given wavelength.

D = Length of light path.

3.13.2. Leaf gas exchange measurements

The net photosynthetic rate (PN) of in vitro regenerated plants was measured during different stages (0, 1, 2, 3 and 4 weeks) of acclimatization on fully expanded leaves using portable Infra-Red Gas Analyzer (IRGA, LICOR 6400,

Lincoln, USA) 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 concentrations changed over a short time intervals (10-20 sec). -2 -1 The net photosynthetic rate was expressed as µmol CO2 m s .

3.14 Histological studies 3.14.1 Fixation and storage The regenerative tissues at different developmental stages were fixed in FAA solution consisting of Formalin : Glacial Acetic Acid : Alcohol (70%) in the ratio of 5 : 5 : 90 (v/v) for about 24 hours and then stored in 70% alcohol. 78

3.14.2 Embedding, sectioning and staining The tissues were dehydrated through graded ethanol-xylol series and then embedded in paraffin wax as described by Johansen (1940). The embedded tissues were cut using a Spencer 820 rotary microtome (American Optical Corporation, Buffalo, New York) at 10 µm thickness and resulting paraffin ribbons were mounted on clean glass slides. After dewaxing in xylol-ethanol series sections were stained with safranin and fast green followed by permanent mounting in Canada balsam. The sections were observed under Olympus Compound Microscope (Olympus CH20i, Japan) and photographs were shot with Cannon Power Shot 640.

3.15 Chemicals and glasswares used

PGRs (BA, Kn, 2-iP, TDZ, IAA, IBA, NAA, 2,4-D, 2,4,5-T and GA3), WPM, vitamins (Thiamine HCl, Pyridoxine HCl, Nicotinic acid, Myo-inositol) and amino acids (Glycine etc.) were obtained from Duchefa Biochemie, Netherland. 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.

3.16 Statistical analysis All the experiments were conducted with ten replicates per treatment and repeated three times. Each treatment represents one explant per culture vial. Data were analyzed statistically through one-way ANOVA using SPSS ver. 16 (SPSS Inc., Chicago, USA). The significance of difference among means was carried out by Duncan’s Multiple Range Test (DMRT) at P = 0.05. The results are expressed as the mean ± SE of three repeated experiments.

Chapter 4 Observations & Results

Chapter 4

OBSERVATIONS AND RESULTS

4.1 C. angustifolia Vahl. 4.1.1 In vitro seed germination To raise aseptic seedlings mature and dried seeds of C. angustifolia (Figure 3 A & B) were germinated under controlled conditions on full and half strength MS media with or without GA3 for the collection of different explants. The full and half strength MS medium without GA3 (control treatments) did not help in seed germination and seeds remain dormant even after 2 weeks of inoculation.

Addition of GA3 at different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) helped in breaking seed dormancy. Rate of seed germination increased with an increase in the concentration of GA3 from 1.0 µM to 5.0 µM and beyond that a decline was observed (Figure 4). Half strength MS medium with GA3 at various concentrations provided better response than the corresponding full strength MS medium. The maximum percent (77.00 ± 2.31%) of seed germination was obtained at half strength MS + 5.0 µM GA3, which took a minimum of 3.20 ± 0.24 days for the emergence of radicle, while full strength MS medium containing the same concentration of GA3 showed only 40.00 ± 4.21% seed germination and required 7.60 ± 0.37 days for the start of seed germination (Table 6). Higher concentration of GA3 was inhibitory for seed germination, reducing the percent germination to 17.00 ± 2.60% on half strength MS + GA3 (10.0 µM), similar trend was observed on full strength MS medium. Thus, half strength MS + GA3 (5.0 µM) proved to be the optimal seed germination medium for C. angustifolia and was used throughout the study for seed germination to collect different explants.

4.1.2 Direct shoot regeneration 4.1.2.1 Cotyledonary node (CN) explant 4.1.2.1.1 Effect of explant age on multiple shoot regeneration In a preliminary experiment, the effect of seedling age on regeneration response was examined by excising the cotyledonary nodes from axenic seedlings of different age group (7, 14 and 21 days old) followed by their culture on MS basal

Explanation of Figure 3 Source of explants for shoot organogenesis in C. angustifolia. A. Mature and dried pods. (Bar = 1.01 cm)

B. Mature seeds. (Bar = 0.75 cm)

Figure 3

Table 6. Effect of GA3 on seed germination of C. angustifolia in MS basal and half strength MS media.

Germination Mean number of Germination medium days to germination rate (%)

MS 00.00  0.00f 00.00  0.00f a ef MS + GA3 (1.0 µM) 14.90  0.31 8.00  2.49 b de MS + GA3 (2.5 µM) 12.30  0.47 10.00  2.58 d b MS + GA3 (5.0 µM) 7.60  0.37 40.00  4.21 c c MS + GA3 (7.5 µM) 9.80  0.44 25.00  3.41 a cd MS + GA3 (10.0 µM) 15.50  0.30 17.00  2.60

½ MS 00.00  0.00f 00.00  0.00f bc de ½ MS + GA3 (1.0 µM) 10.80  0.29 10.00  2.58 d c ½ MS + GA3 (2.5 µM) 7.90  0.37 20.00  2.58 e a ½ MS + GA3 (5.0 µM) 3.20  0.24 77.00  2.31 d b ½ MS + GA3 (7.5 µM) 7.30  0.42 36.00  3.71 b cd ½ MS + GA3 (10.0 µM) 12.50  0.45 17.00  2.60

-Data recorded after 4 weeks. -Values represent Mean  SE of three repeated experiments with 10 replicates each. Means followed by the same letter within columns are not significantly different (P = 0.05) using Duncan’s multiple range test (DMRT).

Explanation of Figure 4 In vitro seed germination in C. angustifolia. Aseptic seedlings (28 days old) on half strength MS medium supplemented with 5.0 µM GA3. (Bar = 1.0 cm)

Figure 4

medium as well as MS medium containing different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA. Explants remained dormant on control medium and died without eliciting any response. The induction response varied with the age of the explant. The cotyledonary nodes taken from 14 days old seedlings induced multiple shoot buds after 10 days of inoculation and produced maximum 25.33 ± 0.20 shoots/explant on MS + BA (5.0 µM) after 6 weeks of culture with the highest regeneration potential of 75.40 ± 0.30%. Explants collected from 7 days old seedlings were quite young and some of the explants died without showing any regeneration response, thus, only 60.00 ± 1.54% cultures showed shoot regeneration at 5.0 µM of BA. Induction of shoot buds from 7 days old explants delayed and started after 15 days of culture wherein an average of 14.23 ± 0.43 shoots/explant was produced at the end of 6 weeks on the medium comprised of MS + BA (5.0 µM). On the other hand, CN explants collected from 21 days old seedlings were harder and some of the seedlings dropped their cotyledonary leaves, such explants further delayed the response, induction of shoot buds started after 18 days of incubation in 52.00 ± 1.15% cultures at 5.0 µM of BA, producing only 12.10 ± 0.77 shoots/explant after 6 weeks of culture (Table 7).

4.1.2.1.2 Effect of cytokinins on multiple shoot regeneration CN explants excised from 14 days old aseptic seedlings of C. angustifolia were cultured on MS medium supplemented with three different cytokinins (BA, Kn and 2iP) for the induction of multiple shoot buds through direct regeneration. Augmentation of various cytokinins at different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) as depicted in table 8, proved to be effective for direct shoot regeneration. All the three cytokinins produced multiple shoots, however, the percent response and the number of shoots/explant along with shoot length varied with the type and concentration of the cytokinins tried. Among three cytokinins tested, BA found to be the best in providing maximum number of shoots/explant with highest regeneration potential, while Kn and 2iP were proved to be lesser effective. Slight callusing was observed at the base of the regenerative tissues in all the three cytokinins tested which hampered the growth and development of new shoot buds. Thus, the callus was regularly removed from the base of the tissue by frequent transferring of the cultures onto the fresh medium of same composition after every 2 weeks.

Table 7. Effect of age of cotyledonary nodes on direct shoot regeneration in C. angustifolia cultured on MS medium containing different concentrations of BA.

BA (µM) 7 days old explant 14 days old explant 21 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00d 00.00  0.00d 00.00  0.00d 00.00  0.00e 00.00  0.00e 00.00  0.00d 1.0 34.66  1.76c 8.56  0.23c 48.13  0.32c 10.90  0.20d 27.33  1.45d 5.90  0.37c 2.5 40.33  1.76b 11.30  0.55b 62.26  0.24b 13.76  0.37c 35.66  1.76c 7.13  0.24c 5.0 60.00  1.54a 14.23  0.43a 75.40  0.30a 25.33  0.20a 52.00  1.15a 12.10  0.77a 7.5 56.66  1.76a 13.23  0.67a 60.00  0.30b 18.80  0.11b 42.33  1.45b 9.33  0.61b 10.0 41.33  2.02b 11.43  0.29b 51.10  0.20c 14.80  0.11c 38.00  1.15c 6.70  0.36c

-Data recorded after 6 weeks. -Values represent Mean  SE of three repeated experiments with 10 replicates each. Means followed by the same letter within columns are not significantly different (P = 0.05) using Duncan’s multiple range test (DMRT).

Table 8. Effect of different cytokinins on direct shoot regeneration from cotyledonary nodes of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 48.13  0.32f 10.90  0.20e 2.90  0.20cd 2.5 - - 62.26  0.24b 13.76  0.37d 3.23  0.14c 5.0 - - 75.40  0.30a 25.33  0.20a 4.33  0.20a 7.5 - - 60.00  0.30c 18.80  0.11b 3.86  0.14b 10.0 - - 51.10  0.20e 14.80  0.11c 2.73  0.14d - 1.0 - 31.03  0.27j 2.56  0.23j 1.90  0.11e - 2.5 - 34.56  0.23i 5.40  0.30h 2.93  0.17cd - 5.0 - 43.76  0.14g 6.33  0.14g 3.33  0.17c - 7.5 - 54.60  0.23d 8.96  0.20f 4.23  0.14ab - 10.0 - 44.23  0.14g 5.46  0.26h 3.03  0.14cd - - 1.0 00.00  0.00l 0.00  0.00k 0.00  0.00f - - 2.5 27.40  0.30k 2.33  0.35j 2.73  0.14d - - 5.0 34.40  0.30h 3.56  0.23i 3.10  0.11cd - - 7.5 42.40  0.30h 6.10  0.20g 3.26  0.17c - - 10.0 31.33  0.35j 3.76  0.14i 2.70  0.15d

Initial response was shown as enlargement and swelling of the explant within 4-6 days of incubation followed by multiple shoot buds differentiation after 10 days of culture (Figure 5 A & B). The MS medium comprised of BA (1.0 µM) produced 10.90 ± 0.20 shoots/explant with an average shoot length of 2.90 ± 0.20 cm in 48.13 ± 0.32% cultures after 6 weeks of culture. The number of shoots/explant further increased with an increase in the concentration of BA to 5.0 µM, and beyond that a decline in percent response and number of shoots was observed. The explants swelled within 4 days and produced multiple shoot buds after 10 days of culture on MS + 5.0 µM of BA producing a maximum of 25.33 ± 0.20 shoots/explant having shoot length of 4.33 ± 0.20 cm with highest 75.40 ± 0.30% regeneration potential after 6 weeks of culture (Figure 5 C, D & E). At 10.0 µM of BA, the number of shoots/explant was reduced to 14.80 ± 0.11 with an average shoot length of 2.73 ± 0.14 cm in only 51.10 ± 0.20% cultures after 6 weeks.

Between Kn and 2iP, Kn exhibited better response than 2iP although it was much less than BA supplemented media. In contrast to BA, Kn and 2iP were effective at higher concentration (7.5 µM). On 7.5 µM of Kn, induction of shoot buds took place after 14 days of inoculation and produced a maximum of 8.96 ± 0.20 shoots/explant having shoot length of 4.23 ± 0.14 cm with a regeneration potential of 54.60 ± 0.23% at the end of 6 weeks of culture (Figure 6 A). While 2iP at the same concentration produced 6.10 ± 0.20 shoots/explant with 3.26 ± 0.17 cm of shoot length in 42.40 ± 0.30% cultures after 6 weeks of culture (Figure 6 B, Table 8).

4.1.2.1.3 Effect of cytokinin-auxin combinations For further proliferation of multiple shoots, the CN explants were also cultured on MS medium containing the optimal concentrations of each cytokinin with different concentrations (0.4, 0.6 and 0.8 µM) of auxins (IAA, IBA and NAA). Among various combination treatments, BA + NAA was proved much efficient for inducing multiple shoot buds which later on converted into healthy shoots. The MS medium containing BA (5.0 µM) + NAA (0.4 µM) produced 32.30 ± 0.17 shoots/explant with shoot length of 4.66 ± 0.35 cm in 78.26 ± 0.14% cultures after 6 weeks of culture. Further increase in the concentration of NAA enhanced the regeneration efficiency and multiple shoot buds were initiated within 8 days of incubation (Figure 7 A), these shoot buds enlarged in size and differentiated

Explanation of Figure 5 In vitro morphogenic responses of cotyledonary nodes of C. angustifolia. A. Basal swelling and enlargement along with differentiation of multiple shoots on medium comprised of MS + BA (5.0 µM) - 1 week old culture. (Bar = 0.49 cm) B. Basal callusing and multiple shoots differentiation on medium comprised of MS + BA (10.0 µM) - 1 week old culture. (Bar = 0.41 cm) C. Multiple shoots development on MS medium supplemented with 5.0 µM BA - 3 weeks old culture. (Bar = 0.34 cm)

D. Multiplication and proliferation of shoots on MS medium containing 5.0 µM BA - 5 weeks old culture. (Bar = 0.66 cm)

E. Elongation of shoots on the same medium - 6 weeks old culture. (Bar = 0.73 cm)

Figure 5

B C

D E E

Explanation of Figure 6 In vitro morphogenic responses of cotyledonary nodes of C. angustifolia. A. Multiplication and proliferation of shoots on MS medium containing 7.5 µM Kn - 6 weeks old culture. (Bar = 0.78 cm)

B. Multiplication and proliferation of shoots on MS medium containing 7.5 µM 2iP - 6 weeks old culture. (Bar = 0.70 cm)

Figure 6

A B

into healthy shoots. On MS medium containing BA (5.0 µM) + NAA (0.6 µM) the maximum 39.16 ± 0.14 shoots/explant having highest shoot length of 5.63 ± 0.20 cm were produced with regeneration potential of 85.26 ± 0.17% after 6 weeks of culture (Figure 7 B). However, the higher concentration of NAA (0.8 µM) resulted in heavy callusing which restricted the growth and development of new shoots reducing the mean number of shoots/explant (35.20 ± 0.11) as well as percent response (80.10 ± 0.17%). Between other two auxins IBA and IAA, IBA at various levels provided better responses than IAA. On MS + BA (5.0 µM) + IBA (0.6 µM) shoot buds were differentiated after 12 days of inoculation producing a maximum of 33.23 ± 0.14 shoots/explants having 5.40 ± 0.23 cm of shoot length in 79.23 ± 0.14% cultures after 6 weeks. While, IAA at the same concentration provided 28.60 ± 0.11 shoots/explant having shoot length of 5.00 ± 0.17 cm with 77.40 ± 0.11% response in 6 weeks of incubation (Table 9).

Among various combinations treatments of Kn (7.5 µM) and auxins, Kn + NAA at various concentrations found to be effective for the enhanced regeneration of multiple shoots than the single Kn treatments. The MS medium containing Kn (7.5 µM) + NAA (0.4 µM) produced 9.60 ± 0.64 shoots/explant having shoot length of 4.90 ± 0.15 cm with regeneration response of 61.66 ± 3.75% after 6 weeks of culture. The number of shoots further increased to 15.20 ± 4.32 shoots/explant with maximum shoot length of 5.40 ± 0.30 cm at 0.6 µM NAA and the percent response was also enhanced to 70.00 ± 2.88% (Figure 7 C). Beyond this optimal level of NAA, a decrease in response was observed at 0.8 µM because of heavy callusing. IBA and IAA were found to be less effective than NAA with Kn (7.5 µM). At an optimal concentration of 0.6 µM, an average of 10.36 ± 2.02 and 9.86 ± 1.69 shoots/explant were produced with IBA and IAA respectively (Table 10).

Similarly, 2iP also found to improve the induction of multiple shoots with NAA but exhibited delayed response, shoot buds were initiated after 14 days of incubation. An average of 13.33 ± 0.12 shoots/explant having shoot length of 4.10 ± 0.20 cm in 53.83 ± 0.37% cultures were obtained on medium comprised of MS + 2iP (7.5 µM) + NAA (0.6 µM) (Figure 7 D). IBA and IAA at an optimal

Table 9. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from cotyledonary nodes of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.4 - - 78.26  0.10d 32.30  0.17d 4.66  0.35bc 5.0 0.6 - - 85.26  0.17a 39.16  0.14a 5.63  0.20a 5.0 0.8 - - 80.10  0.17b 35.20  0.11b 4.60  0.23bc 5.0 - 0.4 - 76.03  0.14f 25.56  0.14f 4.23  0.14c 5.0 - 0.6 - 79.23  0.14c 33.23  0.14c 5.40  0.23a 5.0 - 0.8 - 76.43  0.17f 28.46  0.14e 4.66  0.17bc 5.0 - - 0.4 75.33  0.24g 21.20  0.11h 3.96  0.14c 5.0 - - 0.6 77.40  0.11e 28.60  0.11e 5.00  0.17ab 5.0 - - 0.8 74.06  0.17h 25.06  0.14g 4.40  0.20b

Explanation of Figure 7 In vitro morphogenic responses of cotyledonary nodes of C. angustifolia. A. Multiple shoots differentiation on MS medium containing BA (5.0 µM) and NAA (0.6 µM) - 2 weeks old culture. (Bar = 0.53 cm) B. Multiplication and proliferation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) - 6 weeks old culture. (Bar = 0.92 cm) C. Multiplication and proliferation of shoots on medium comprised of MS + Kn (7.5 µM) + NAA (0.6 µM) - 6 weeks old culture. (Bar = 0.83 cm) D. Multiplication and proliferation of shoots on medium comprised of MS + 2iP (7.5 µM) + NAA (0.6 µM) - 6 weeks old culture. (Bar = 0.80 cm)

Figure 7

C D

Table 10. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from cotyledonary nodes of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.4 - - 61.66  3.75ab 9.60  0.64ab 4.90  0.15ab 7.5 0.6 - - 70.00  2.88a 15.20  4.32a 5.40  0.30a 7.5 0.8 - - 64.66  4.37ab 10.00  1.78ab 4.60  0.70ab 7.5 - 0.4 - 60.66  4.66ab 9.06  2.33ab 4.30  0.25ab 7.5 - 0.6 - 65.33  3.71ab 10.36  2.02ab 4.66  0.33ab 7.5 - 0.8 - 57.33  3.71ab 7.30  1.21b 4.13  0.17b 7.5 - - 0.4 57.00  4.72ab 7.70  2.15b 4.06  0.24b 7.5 - - 0.6 63.33  4.80ab 9.86  1.69a 4.33  0.26ab 7.5 - - 0.8 54.00  3.05b 7.60  1.41b 3.86  0.35b

concentration of 0.6 µM along with 7.5 µM 2iP produced 8.36 ± 0.20 and 7.80 ± 0.15 shoots/explant respectively after 6 weeks of culture (Table 11).

4.1.2.1.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration The MS medium containing TDZ at a wide range of concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) showed good morphogenic changes and the induction of multiple shoot buds occurred within 6 days of incubation. The explants swelled and enlarged in size along with the differentiation of multiple shoot buds (Figure 8 A), slight basal callusing was also observed which was regularly removed by frequent transfer on to the fresh medium of same composition at every 2 weeks interval. All the treatments of TDZ found to be effective for inducing multiple shoots and the number of shoots/explant increased linearly with an increase in the concentration up to 2.5 µM. Beyond the optimal concentration a decrease in the percent response as well as number of shoots/explant was observed. The medium comprised of MS + TDZ (2.5 µM) produced a maximum of 28.53 ± 0.99 shoots/explant with shoot length of 4.03 ± 0.14 cm in 94.33 ± 2.33% cultures after 6 weeks of inoculation (Figure 8 B, C & D). The higher concentrations (5.0 to 10.0 µM) of TDZ resulted in the formation of heavy callus at the base of the explant (Figure 8 E) and thus reduced the regeneration potential and number of shoots/explant. At 10.0 µM of TDZ only 11.43 ± 0.40 shoots/explant were obtained having an average shoot length of 2.23 ± 0.14 cm in 62.33 ± 1.45% cultures (Table 12).

4.1.2.1.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Repeated transferring of the regenerative tissue on the medium containing same concentration of TDZ resulted in distortion, fasciation and stunting of regenerated shoots. Thus, to avoid these deleterious effects of TDZ, the regenerative cultures after 6 weeks of incubation on TDZ were transferred to TDZ free MS basal medium and MS medium containing lower concentrations (0.5. 1.0, 2.5 and 5.0 µM) of BA. The MS basal medium devoid of BA did not show any remarkable change in the number of shoots/explant and shoot length. However, the media supplemented with BA enhanced the number as well as improved the growth of regenerated microshoots. The best response was

Table 11. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from cotyledonary nodes of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.4 - - 50.96  0.31b 7.53  0.24d 3.36  0.10bc 7.5 0.6 - - 53.83  0.37a 13.33  0.12a 4.10  0.20a 7.5 0.8 - - 50.03  0.27bc 9.26  0.14b 3.80  0.11ab 7.5 - 0.4 - 42.76  0.12g 7.00  0.11f 3.60  0.11bc 7.5 - 0.6 - 48.13  0.17d 8.36  0.20c 3.80  0.11ab 7.5 - 0.8 - 43.90  0.20f 6.16  0.26g 3.23  0.14c 7.5 - - 0.4 42.66  0.16g 6.86  0.13f 3.40  0.11bc 7.5 - - 0.6 45.26  0.17e 7.80  0.15de 3.46  0.17bc 7.5 - - 0.8 43.16  0.12g 6.66  0.20fg 3.26  0.17c

Explanation of Figure 8 In vitro morphogenic responses of cotyledonary nodes of C. angustifolia. A. Swelling and enlargement along with differentiation of large number of shoot buds on MS medium supplemented with 2.5 µM TDZ - 1 week old culture. (Bar = 0.38 cm) B. Multiple shoots development on medium comprised of MS + TDZ (2.5 µM) - 3 weeks old culture. (Bar = 0.54 cm) C. Multiplication of shoots along with production of deformed and stunted shoots on medium comprised of MS + TDZ (2.5 µM) - 4 weeks old culture. (Bar = 0.52 cm) D. -do- 6 weeks old culture. (Bar = 0.92 cm) E. Differentiation of shoots on medium containing 10.0 µM TDZ along with production of heavy basal callusing - 1 week old culture. (Bar = 0.52 cm) E. Multiplication and elongation of TDZ (2.5 µM) induced cultures on BA (2.5 µM) supplemented medium - 6 weeks old culture. (Bar = 0.96 cm)

Figure 8

E F

Table 12. Effect of various concentrations of TDZ on direct shoot regeneration from cotyledonary nodes of C. angustifolia.

TDZ (M) Regeneration Mean number of Mean shoot (%) shoots/explants length (cm)

0.1 53.33  1.76d 7.93  0.17g 2.40  0.20de 0.25 64.00  2.30c 11.40  0.52f 2.76  0.14cde 0.5 65.66  2.33c 13.23  0.14e 2.96  0.14bcd 1.0 80.66  2.33b 25.00  0.23b 3.40  0.11b 2.5 94.33  2.33a 28.53  0.99a 4.03  0.14a 5.0 77.33  3.71b 21.03  0.26c 3.03  0.31bc 7.5 67.66  1.45c 15.10  0.47d 2.66  0.17cde 10.0 62.33  1.45c 11.43  0.40f 2.23  0.14e

Table 13. Effect of various concentrations of BA on TDZ (2.5 M) induced cultures from cotyledonary nodes of C. angustifolia for further multiplication and proliferation.

PGR (M) Mean number of Mean shoot shoots/explant length (cm)

MS 28.53  0.99c 4.60  0.30c MS + BA (0.5) 31.20  0.52c 5.00  0.11bc MS + BA (1.0) 36.93  0.75b 5.33  0.17b MS + BA (2.5) 45.50  0.51a 6.20  0.11a MS + BA (5.0) 38.00  1.28b 5.43  0.23b

observed at MS + BA (2.5 µM) where the number of shoots increased to the maximum 45.50 ± 0.51 shoots/explant from initial 28.53 ± 0.99 shoots/explant and shoots attained an average shoot length of 6.20 ± 0.11 cm after 6 weeks of transfer (Figure 8 F). The higher concentration (5.0 µM) of BA suppressed the growth and development of shoots and their number was reduced to 38.00 ± 1.28 shoots/explant with mean shoot length of 5.43 ± 0.23 cm (Table 13).

4.1.2.1.5 Effect of different media

The four different media (B5, L2, MS and WPM) were tested with the optimized treatment i.e. BA (5.0 µM) and NAA (0.6 µM) to study the effect of different composition of nutrients on growth and development of shoots. Different media provided differential response because of their differences in nutrient composition. The explants cultured on MS medium with BA (5.0 µM) and NAA (0.6 µM) induced multiple shoot buds within 8 days of inoculation and by the end of 6 weeks, a maximum of 39.16 ± 0.14 shoots/CN explant were harvested attaining maximum shoot length of 5.63 ± 0.20 cm. WPM was found less effective than MS but better than L2 and B5. Induction of shoot buds started after 11 days of inoculation on WPM and an average of 24.20 ± 1.85 shoots/explant having 4.43 ± 0.34 cm of shoot length were obtained after 6 weeks of culture.

The explants cultured on L2 and B5 medium showed late response that is after 14 and 16 days of inoculation respectively. An average of 14.53 ± 0.95 shoots/explant was produced on L2 medium, while, the number was further reduced to 10.60 ± 0.72 shoots/explant on B5 medium (Figure 9).

4.1.2.1.6 Effect of pH The effect of different pH levels (5.0, 5.4. 5.8, 6.2 and 6.6) of the medium was tested on MS medium with the optimum concentration of BA (5.0 µM) and NAA (0.6 µM). The optimum value of pH was observed at 5.8 wherein a maximum of 39.16 ± 0.14 shoots/CN explant were produced after 6 weeks of culture. The regeneration potential as well as number of shoots/explant was drastically decreased when pH of the medium was decreased (5.4 and 5.0) or increased (6.2 and 6.6) from the optimum value of 5.8. The low pH of the medium increased its acidic nature and resulted in the formation of loose or watery medium which hampered the growth of the shoots and the number of

85 shoots/explant was reduced to 30.83 ± 1.75 and 25.86 ± 1.16 at pH 5.4 and 5.0 respectively. High pH values increased the alkaline/basic nature of the medium and it became comparatively hard in texture which adversely affected the regeneration potential of the explant. The number of shoots reduced to 30.13 ± 1.73 and 23.90 ± 0.73 shoots/explant at pH 6.2 and 6.6 respectively (Figure 10).

4.1.2.1.7 Effect of sucrose concentrations Sucrose as the only source of carbon and energy was used throughout the study but different concentrations (1, 2, 3, 4 and 5%) of sucrose were tested with the optimal medium i.e. MS + BA (5.0 µM) + NAA (0.6 µM) to observe the effect on growth and morphogenesis of the regenerative tissue. The explants responded best on the medium containing 3% sucrose where a maximum of 39.16 ± 0.14 shoots/CN explant were produced having maximum shoot length of 5.63 ± 0.20 cm after 6 weeks of culture. However, the lower concentrations (2 or 1%) were less effective and produced only 26.26 ± 2.25 and 20.43 ± 1.15 shoots/explant at 2% and 1% sucrose respectively. Moreover, increase in the concentration of sucrose from optimal 3% also reduced the growth and development therefore; the number of shoots decreased to 27.80 ± 2.22 and 19.76 ± 2.22 shoots/explant at 4% and 5% of sucrose respectively (Figure 11).

4.1.2.2 Nodal segment (NS) explant 4.1.2.2.1 Effect of explant age on multiple shoot regeneration Nodal segments excised from seedlings of three different age group (14, 21 and 28 days old) were cultured on MS medium containing different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA to select the best responsive age of the explant for maximum shoot regeneration. The MS medium without BA was served as control medium on which all the explants failed to respond. However, at 5.0 µM BA, the explants from 21 days old seedlings get swelled within 8 days of incubation and provided best response; axillary bud breaking was observed from the 10th day and gradually multiple shoot buds were regenerated by the end of 13th day of incubation. A maximum of 20.20 ± 0.11 shoots/explant were produced with highest regeneration rate (66.00 ± 1.15%) from NS of 21 days old seedlings on MS + BA (5.0 µM) after 6 weeks of culture. The explants from 14 and 28 days old seedlings proved to be less responsive than 21 days old as they

Mean number of shoots/explant Mean shoot length (cm) 50 10

a 40 8

30 a 6 b

bc bc 20 c 4 c

d length (cm)Mean shoot 10 2 Mean shoots/explant number of

0 0 MS WPM L2 B5

Different media

Figure 9. Effect of different culture media supplemented with BA (5.0 µM) and NAA (0.6 µM) on shoot regeneration from cotyledonary nodes of C. angustifolia after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 50 7

a 6 40 a

b b 5 30 c b b c 4

c 3

c (cm) length shoot Mean 10 Mean number of shoots/explant of number Mean 2

0 1 5.0 5.4 5.8 6.2 6.6 Different pH of medium

Figure 10. Effect of different pH values on shoot regeneration from cotyledonary nodes of C. angustifolia on MS medium containing optimal concentration of BA (5.0 µM) and NAA (0.6 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 50 10

40 a 8

b 30 b a 6

c c 20 4 b b

Mean shootlength (cm) 10 Mean numbershoots/explant of c c 2 0 1 2 3 4 5 Concentration of sucrose (%)

Figure 11. Effect of different concentrations of sucrose on shoot regeneration from cotyledonary nodes of C. angustifolia on MS + BA (5.0 µM) + NAA (0.6 µM) after 6 weeks of culture.

provided less number of shoots at the same treatment i.e. 8.93 ± 0.76 and 12.23 ± 0.43 shoots/explant respectively after 6 weeks of culture (Table 14).

4.1.2.2.2 Effect of cytokinins on multiple shoot regeneration The effect of three different cytokinins (BA, Kn and 2iP) at various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 µM) was evaluated with 21 days old nodal segments. Addition of different cytokinins in MS medium resulted in swelling and enlargement of explants which stimulated the axillary buds to induce multiple shoot buds. Different cytokinins showed differential response and accordingly the number of shoots/explant varied with the type and concentration of the cytokinin. Among three cytokinins tested, BA produced maximum number of shoots/explant with maximum shoot length and highest regeneration response. The number of shoots increased linearly with an increase in the concentration of BA from 1.0 µM up to 5.0 µM (Table 15). The MS medium containing 1.0 µM BA produced 7.06 ± 0.17 shoots/explant having shoot length of 2.83 ± 0.20 cm with 30.66 ± 0.66% regeneration response (Figure 12 A). The best response was obtained on MS + BA (5.0 µM) wherein differentiation of multiple shoot buds initiated within 13 days of incubation (Figure 12 B & C). On this medium a maximum of 20.20 ± 0.11 shoots/explant were harvested in 66.0 ± 1.15% cultures, attaining an average shoot length of 4.20 ± 0.11 cm after 6 weeks of inoculation (Figure 12 D). The higher concentrations (7.5 µM and 10.0 µM) of BA displayed inhibitory effects on explants and delayed the induction of shoot buds (15-17 days after inoculation). Rate of shoot multiplication reduced at 10.0 µM of BA with the production of an average 11.20 ± 0.17 shoots/explant having shoot length of 2.73 ± 0.13 cm in 39.00 ± 1.73% cultures. Kn and 2iP also influenced the induction of multiple shoots from the nodal explants but the number of shoots/explant and regeneration potential was comparatively low than the BA augmented medium. The explants on Kn supplemented medium exhibited moderate response and the induction of shoot buds started after 17 days of incubation. The lower concentration (1.0 µM) of both Kn and 2iP failed to induce any morphogenic response and explants died without showing any regeneration. The maximum regeneration potential (40.00 ± 1.15%) was obtained on MS medium containing 7.5 µM Kn wherein the maximum of 7.00 ± 0.11 shoots/explant were produced with shoot length of 4.03 ± 0.14 cm after 6 weeks

Table 14. Effect of age of nodal segments on direct shoot regeneration in C. angustifolia cultured on MS medium containing different concentrations of BA.

BA (µM) 14 days old explant 21 days old explant 28 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00d 0.00  0.00e 00.00  0.00e 00.00  0.00f 00.00  0.00d 00.00  0.00e 1.0 23.00  2.08c 3.16  0.49d 30.66  0.66d 7.06  0.17e 27.00  1.73c 6.56  0.34c 2.5 31.33  2.40b 7.13  0.52b 39.66  1.20c 15.20  0.23c 32.33  1.45b 8.33  0.35c 5.0 39.33  1.76a 8.93  0.76a 66.00  1.15a 20.20  0.11a 49.00  2.08a 12.23  0.43a 7.5 26.66  2.02c 5.53  0.35c 45.66  2.33b 18.23  0.14b 33.33  1.45b 6.90  0.20c 10.0 30.00  1.15b 5.20  0.45c 39.00  1.73c 11.20  0.17d 30.33  1.45bc 5.03  0.26d

Table 15. Effect of different cytokinins on direct shoot regeneration from nodal segments of C .angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 30.66  0.66d 7.06  0.17e 2.83  0.20cd 2.5 - - 39.66  1.20c 15.20  0.23c 3.46  0.24b 5.0 - - 66.00  1.15a 20.20  0.11a 4.20  0.11a 7.5 - - 45.66  2.33b 18.23  0.14b 3.03  0.14bcd 10.0 - - 39.00  1.73c 11.20  0.17d 2.73  0.13de - 1.0 - 00.00  0.00f 0.00  0.00j 0.00  0.00h - 2.5 - 22.33  1.45e 2.23  0.14h 2.76  0.17de - 5.0 - 27.66  1.45d 5.33  0.17f 3.26  0.17bc - 7.5 - 40.00  1.15c 7.00  0.11e 4.03  0.14a - 10.0 - 29.66  1.45d 5.26  0.17f 2.96  0.14cd - - 1.0 00.00  0.00f 0.00  0.00j 0.00  0.00h - - 2.5 19.66  1.45e 1.23  0.14i 1.80  0.20g - - 5.0 22.66  1.76e 2.30  0.17h 2.33  0.17ef - - 7.5 30.66  1.76d 4.23  0.14g 2.53  0.17de - - 10.0 20.00  1.15e 2.20  0.11h 2.00  0.17fg

Explanation of Figure 12 In vitro morphogenic responses of nodal segments of C. angustifolia. A. Induction of multiple shoots on medium comprised of MS + BA (1.0 µM) - 1 week old culture. (Bar = 0.69 cm)

B. Induction of multiple shoots on medium comprised of MS + BA (5.0 µM) - 1 week old culture. (Bar = 0.65 cm)

C. Multiplication of shoots on MS medium containing 5.0 µM BA - 2 weeks old culture. (Bar = 0.54 cm)

D. Proliferation and elongation of shoots on MS medium containing 5.0 µM BA - 4 weeks old culture. (Bar = 0.86 cm)

E. Production of multiple shoots on MS medium containing 7.5 µM Kn - 4 weeks old culture. (Bar = 0.71 cm)

F. Production of multiple shoots on MS medium containing 7.5 µM 2iP - 4 weeks old culture. (Bar = 0.69 cm)

Figure 12

A B C

D E F

of culture (Figure 12 E, Table 15). On this medium the regenerated shoots were thin and leaves were smaller in size as compared to those on BA supplemented medium. 2iP found to provide least response and the regenerated shoots were thin and delicate with reduced lamina. The MS medium supplemented with 7.5 µM 2iP produced only 4.23 ± 0.14 shoots/explant having 2.53 ± 0.17 cm of shoot length in 30.66 ± 1.76% cultures (Figure 12 F).

4.1.2.2.3 Effect of cytokinin-auxin combinations The NS were also cultured on cytokinin-auxin combination treatments to observe the combined effect of PGRs on direct shoot regeneration. In this experiment the optimal concentrations of each cytokinin (BA, Kn and 2iP) were tested with different concentrations (0.4, 0.6 and 0.8 µM) of three auxins (IAA, IBA and NAA). Explants swelled and the induction of shoot buds started within 10 days of incubation (Figure 13 A). Addition of NAA enhanced the induction of shoot buds and helped in better growth and proliferation of multiple shoots than the single BA treatment. The MS medium containing BA (5.0 µM) + NAA (0.4 µM) produced 27.13 ± 0.40 shoots/explant with 68.00 ± 0.15% regeneration response having an average shoot length of 4.96 ± 0.14 cm. When the concentration of NAA was increased to 0.6 µM, the regeneration response was also increased to 79.33 ± 1.76% and produced the maximum 30.33 ± 0.24 shoots/explant having shoot length of 5.26 ± 0.14 cm after 6 weeks of culture (Figure 13 B). Further increase in the concentration of NAA from optimal level exhibited inhibitory effects and resulted in the formation of callus which hampered the multiplication of shoots and thus, the number of shoots was reduced to 24.86 ± 0.17 shoots/explant in 72.33 ± 1.45% cultures at 0.8 µM NAA. Incorporation of IBA and IAA exhibited lesser response as compared to NAA, the MS medium containing BA (5.0 µM) + IBA (0.6 µM) produced a maximum of 25.00 ± 0.46 shoots/explant with shoot length of 4.83 ± 0.20 cm in 72.00 ± 0.15% cultures while IAA at the same concentration produced 21.66 ± 0.24 shoots/explant having an average shoot length of 4.73 ± 0.14 cm in 68.66 ± 2.02% cultures. At higher concentration (0.8 µM), both the auxins (IBA and IAA) proved to be inhibitory and reduced the rate of shoot multiplication due to basal callusing (Table 16).

Explanation of Figure 13 In vitro morphogenic responses of nodal segments of C. angustifolia. A. Induction of multiple shoots along with basal swelling and enlargement of explant on cytokinin-auxin combination medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) - 2 weeks old culture. (Bar = 0.35 cm)

B. Multiplication and proliferation of shoots on MS medium containing BA (5.0 µM) and NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.88 cm)

C. Multiple shoots production on medium comprised of MS + Kn (7.5 µM) + NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.95 cm)

D. Multiple shoots production on medium comprised of MS + 2iP (7.5 µM) + NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.98 cm)

Figure 13

A B

C D

Table 16. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from nodal segments of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.4 - - 68.00  0.15bc 27.13  0.40b 4.96  0.14ab 5.0 0.6 - - 79.33  1.76a 30.33  0.24a 5.26  0.14a 5.0 0.8 - - 72.33  1.45b 24.86  0.17c 4.76  0.14bcd 5.0 - 0.4 - 67.33  1.45c 23.43  0.23d 4.40  0.11cdef 5.0 - 0.6 - 72.00  0.15b 25.00  0.46c 4.83  0.20abc 5.0 - 0.8 - 69.00  1.15bc 21.46  0.29e 4.33  0.14def 5.0 - - 0.4 66.00  1.15cd 21.20  0.11e 4.20  0.11f 5.0 - - 0.6 68.66  2.02bc 21.66  0.24de 4.73  0.14bcde 5.0 - - 0.8 62.00  1.15d 20.33  0.17f 4.26  0.14ef

Among various Kn and NAA combinations treatments, the MS medium containing Kn (7.5 µM) + NAA (0.6 µM) produced an optimal 15.6 ± 0.23 shoots/explants in 52.66 ± 1.45% cultures having shoot length of 4.80 ± 0.11 cm after 6 weeks of culture (Figure 13 C). Higher concentration of NAA (0.8 µM) was proved inhibitory leading to reduction in the number of shoots (10.90 ± 0.26) as well as shoot length (4.16 ± 0.12 cm). IBA and IAA were found to be less effective than NAA as only 11.93 ± 0.17 and 10.86 ± 0.17 shoots/explant were obtained on optimal concentration of 0.6 µM IBA and IAA respectively (Table 17).

Cultures on 2iP amended media exhibited lest response and the regenerated shoots were thin having reduced leaf lamina. The medium containing optimized concentrations of 2iP (7.5 µM) and NAA (0.6 µM) produced only 10.46 ± 0.35 shoots/explants with an average shoot length of 4.10 ± 0.20 cm in 42.33 ± 1.45% cultures. On further increasing the concentration of NAA beyond optimal 0.6 µM, a reduction in percent response (35.33 ± 1.76%) as well as in number of shoots/explant (5.53 ± 0.24) was observed (Figure 13 D). Incorporation of IBA and IAA along with optimal 7.5 µM of 2iP showed low regeneration potential. The medium comprised of MS + 2iP (7.5 µM) + IBA (0.6 µM) produced 8.20 ± 0.17 shoots/explant while IAA at the same concentration produced 7.06 ± 0.17 shoots/explant after 6 weeks of culture (Table 18).

4.1.2.2.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration The comparison of TDZ activity with other cytokinins like BA, Kn and 2iP was not possible because TDZ is effective at much lower concentrations. Therefore, another experiment was set in which 21 days old aseptic NS of C. angustifolia were cultured at various concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) of TDZ. Induction of multiple shoot buds started within 7 days of incubation on TDZ supplemented media and the number of shoots/explant increased with an increase in the concentration of TDZ from 0.1 µM up to an optimal level of 2.5 µM. The explants started swelling within 4-5 days of incubation along with slight basal callusing. The medium containing TDZ (0.1 µM) produced 5.10 ± 0.20 shoots/explant having an average shoot length of 2.20 ± 0.11 cm in 44.66 ± 1.40% cultures. At 2.5 µM TDZ a maximum of 21.70 ± 0.62 shoots/explant was

Table 17. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from nodal segments of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.4 - - 45.00  1.15bc 12.96  0.26b 4.46  0.14ab 7.5 0.6 - - 52.66  1.45a 15.60  0.23a 4.80  0.11a 7.5 0.8 - - 44.33  2.33bcd 10.90  0.26d 4.16  0.12bc 7.5 - 0.4 - 42.00  1.15cde 9.36  0.23e 4.10  0.11bc 7.5 - 0.6 - 49.00  1.73ab 11.93  0.17c 4.23  0.14bc 7.5 - 0.8 - 40.00  1.15de 7.43  0.23g 4.03  0.14bc 7.5 - - 0.4 42.00  1.15cde 8.23  0.14f 4.00  0.11c 7.5 - - 0.6 48.00  1.15ab 10.86  0.17d 4.16  0.12bc 7.5 - - 0.8 38.66  1.76e 7.26  0.17g 3.86  0.17c

Table 18. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from nodal segments of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.4 - - 37.66  1.45b 6.03  0.26d 3.83  0.20ab 7.5 0.6 - - 42.33  1.45a 10.46  0.35a 4.10  0.20a 7.5 0.8 - - 35.33  1.76bc 5.53  0.24de 3.23  0.14cd 7.5 - 0.4 - 34.33  1.20bc 5.20  0.11ef 3.50  0.17bcd 7.5 - 0.6 - 38.00  1.15b 8.20  0.17b 3.76  0.14abc 7.5 - 0.8 - 32.00  1.15d 5.23  0.14ef 3.46  0.17bcd 7.5 - - 0.4 32.66  1.45d 5.03  0.14ef 3.26  0.17cd 7.5 - - 0.6 34.00  1.15bc 7.06  0.17c 3.53  0.14bcd 7.5 - - 0.8 32.00  1.15d 4.73  0.14f 3.16  0.14d

obtained in 86.00 ± 2.30% cultures, attaining an average shoot length of 3.80 ± 0.17 cm after 6 weeks of culture (Figure 14 A & B). It was also observed that the higher concentrations of TDZ produced heavy callusing, thereby, adversely affected the regeneration potential and growth of the shoots. Beyond the optimal level of TDZ (2.5 µM), a decline in percent response as well as in number of shoots was observed; the number of shoots was considerably reduced to 8.16 ± 0.40 shoots/explant with a regeneration response of 61.00 ± 2.08% on medium containing 10.0 µM TDZ (Table 19).

4.1.2.2.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Prolonged exposure of TDZ containing media resulted in distortion and stunting of microshoots, thus, to avoid these deleterious effects, the cultures were transferred to TDZ free MS basal medium after 6 weeks of incubation. The MS basal medium without any PGR did not improve the growth and development of shoots. Therefore, MS medium was amended with different concentrations (0.5, 1.0, 2.5 and 5.0 µM) of BA for further improvement in the regeneration of multiple shoots. The number of shoots increased to 23.66 ± 0.24 shoots/explant with an enhancement in shoot length (4.73 ± 0.17 cm) at 0.1 µM of BA (Figure 14 C). On further increasing the concentration of BA, the number of shoots increased to 32.03 ± 0.31 shoots/explant at MS + BA (2.5 µM) having average shoot length of 5.66 ± 0.17 cm. However, beyond this optimal level, a decline in the number of shoots (25.00 ± 0.41) and shoot length (5.26 ± 0.17 cm) was observed (Table 20).

4.1.2.2.5 Effect of different media To evaluate the effect of different composition of nutrients on multiple shoots regeneration, four different media (B5, L2, MS and WPM) were tested with the optimized concentration of PGRs i.e. BA (5.0 µM) and NAA (0.6 µM). Among four media tested, MS medium provided the best response for multiplication and proliferation of shoots, differentiation of shoot buds started within 10 days of inoculation and produced a maximum of 30.33 ± 0.24 shoots/explant with maximum shoot length of 5.26 ± 0.14 cm after 6 weeks of culture. Explants were less responsive in WPM as compared to MS medium and produced lesser

Explanation of Figure 14 In vitro morphogenic responses of nodal segments of C. angustifolia. A. Induction of multiple shoots on MS medium supplemented with 2.5 µM TDZ - 2 weeks old culture. (Bar = 0.53 cm)

B. Production of deformed and stunted shoots on prolonged exposure to TDZ (2.5 µM) containing medium - 6 weeks old culture. (Bar = 0.70 cm)

C. Proliferation and elongation of TDZ (2.5 µM) induced cultures on BA (2.5 µM) supplemented medium, after 4 weeks of transfer. (Bar = 0.39 cm)

Figure 14

B C

Table 19. Effect of various concentrations of TDZ on direct shoot regeneration from nodal segments of C. angustifolia.

TDZ (M) Regeneration Mean number of Mean shoot (%) shoots/explants length (cm)

0.1 44.66  1.40g 5.10  0.20f 2.20  0.11d 0.25 53.66  2.02f 6.10  0.26f 2.60  0.23bc 0.5 69.00  2.08cd 7.76  0.14e 2.90  0.26b 1.0 77.66  1.45b 14.33  0.35c 3.23  0.14ab 2.5 86.00  2.30a 21.70  0.62a 3.80  0.17a 5.0 72.33  1.45bc 16.76  0.53b 2.96  0.32b 7.5 65.66  2.33de 12.10  0.20d 2.63  0.20bc 10.0 61.00  2.08e 8.16  0.40f 2.20  0.11d

Table 20. Effect of various concentrations of BA on TDZ (2.5 M) induced cultures from nodal segments of C. angustifolia for further multiplication and proliferation.

PGR (M) Mean number of Mean shoot shoots/explant length (cm)

MS 21.70  0.62d 3.93  0.17c MS + BA (0.5) 23.66  0.24c 4.73  0.17b MS + BA (1.0) 29.70  0.65b 5.20  0.11ab MS + BA (2.5) 32.03  0.31a 5.66  0.17a MS + BA (5.0) 25.00  0.41c 5.26  0.17ab

shoots (20.73 ± 0.26) with reduced shoot length (4.16 ± 0.20 cm). The number of shoots further reduced to 13.9 ± 0.20 shoots/explant having 3.76 ± 0.14 cm of shoot length in L2 medium. B5 medium proved to be the least effective among four tested media. The explants on B5 medium showed very late response that is after 20 days of incubation and produced only 8.56 ± 0.23 shoots/explant after 6 weeks of culture (Figure 15).

4.1.2.2.6 Effect of pH pH of the medium played an important role in nutrients uptake and shoot multiplication, therefore, the effect of different pH (5.0, 5.4. 5.8, 6.2 and 6.6) of the medium was tested on NS explants in MS medium with the optimized concentrations of BA (5.0 µM) and NAA (0.6 µM). The lower pH values (5.0 and 5.4) resulted in the formation of slightly liquid medium and produced 13.13 ± 0.23 and 21.93 ± 0.23 shoots/explant at pH 5.0 and 5.4 respectively after 6 weeks of culture. The explants showed best response with pH 5.8 producing a maximum of 30.33 ± 0.24 shoots/explant with 5.26 ± 0.14 cm shoot length after the same time period. High pH values also adversely affected the shoot regeneration capacity of the explant as medium became hard in texture. The number of shoots reduced to 19.10 ± 0.37 and 9.56 ± 0.34 shoots/explant at pH 6.2 and 6.6 respectively (Figure 16).

4.1.2.2.7 Effect of sucrose concentrations Concentration of sucrose in the medium affected the shoot multiplication and proliferation, therefore, different concentrations (1, 2, 3, 4 and 5%) of sucrose were tested with the optimal medium i.e. MS + BA (5.0 µM) + NAA (0.6 µM). The explants responded best with 3% sucrose producing a maximum of 30.33 ± 0.24 shoots/explant having maximum shoot length of 5.26 ± 0.14 cm after 6 weeks of culture. However, explants on the lower concentrations (1 and 2%) of sucrose exhibited less response and produced 10.90 ± 0.37 and 19.26 ± 0.43 shoots/explant at 1% and 2% sucrose respectively. Furthermore, increase in the concentration of sucrose from optimal 3% also reduced the regeneration potential and 22.56 ± 0.23 shoots/explant having shoot length of 4.53 ± 0.24 cm were obtained in medium containing 4% sucrose. The explants exhibited least

Mean number of shoots/explant Mean shoot length (cm) 35 6.0

a 30 a 5.5

25 5.0 b 20 4.5 b 15 c

bc 4.0 10

d length (cm)Mean shoot

Mean number of shoots/explant Mean number of c 3.5 5

0 3.0 MS WPM L2 B5

Different media Figure 15. Effect of different culture media supplemented with optimal concentration of BA (5.0 µM) and NAA (0.6 µM) on shoot regeneration from nodal segments of C. angustifolia after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 35 6.0 a 30 a 5.5

25 b 5.0

20 b c

4.5

15 d bc 4.0 bc 10 e

Mean shoot length (cm) length shoot Mean d Mean number of shoots/explant of number Mean 3.5 5

0 3.0 5.0 5.4 5.8 6.2 6.6 Different pH of medium

Figure 16. Effect of different pH of the medium on shoot regeneration from nodal explants of C. angustifolia on optimized medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 35 6.0

a 30 5.5 a

25 5.0 b

20 c b 4.5

d 15 4.0 d e c 10 3.5

Mean shoot length (cm) length shoot Mean e

Mean number of shoots/explant of number Mean 5 3.0

0 2.5 1 2 3 4 5 Concentration of sucrose (%)

Figure 17. Effect of different concentrations of sucrose on shoot regeneration from nodal segments of C. angustifolia on MS medium containing optimal concentration of BA (5.0 µM) and NAA (0.6 µM) after 6 weeks of culture.

response at 5% sucrose producing an average of 14.63 ± 0.23 shoots/explant with shoot length of 3.73 ± 0.14 cm (Figure 17).

4.1.2.3 Shoot tip (ST) explant 4.1.2.3.1 Effect of explant age on multiple shoot regeneration To select the best responsive age of the ST explants for multiple shoot regeneration, explants were taken from axenic seedlings of three different age group (14, 21 and 28 days) and cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA. ST explants on hormone free MS (control) medium did not induce any response and died within 2 weeks of culture. Addition of BA influenced the morphogenic changes and induced multiple shoots. On medium comprised of MS + BA (5.0 µM), best response was exhibited by the explants excised from 21 days old seedlings where multiple shoot buds were differentiated after 16 days of incubation and a maximum of 12.56 ± 0.34 shoots/explant with highest regeneration response (60.00 ± 3.46%) was obtained by the end of 6 weeks of culture. The explants excised from 14 and 28 days old seedlings were comparatively less responsive at the same concentration and produced 8.60 ± 0.20 and 5.96 ± 0.39 shoots/explant respectively (Table 21).

4.1.2.3.2 Effect of cytokinins on multiple shoot regeneration Shoot tip explants excised from 21 days old seedlings cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of three cytokinins (BA, Kn and 2iP) to evaluate the regeneration potential of the explants. Induction of shoot buds started after 16 days of culture in BA supplemented media and all the explants showed multiple shoot regeneration, while the number of shoots increased linearly with an increase in the concentration of BA up to 5.0 µM. The medium containing 1.0 µM BA produced 8.63 ± 0.23 shoots/explant with an average shoot length of 3.40 ± 0.23 cm exhibiting 27.66 ± 2.40% regeneration response after 6 weeks of culture (Figure 18 A). The number of shoots further increased at 5.0 µM BA to a maximum of 12.56 ± 0.34 shoots/explant with shoot length of 3.40 ± 0.23 cm in 60.00 ± 3.46% cultures after 6 weeks of incubation (Figure 18 B, C & D). Higher concentrations (7.5 and 10.0 µM) of BA proved to be inhibitory for shoot regeneration and the number of shoots reduced to 7.56 ±

Table 21. Effect of age of shoot tips on direct shoot regeneration in C. angustifolia cultured on MS medium containing different concentrations of BA.

BA (µM) 14 days old explant 21 days old explant 28 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00e 0.00  0.00d 00.00  0.00e 00.00  0.00e 00.00  0.00d 0.00  0.00e 1.0 25.66  1.45d 3.00  0.11c 27.66  2.40d 8.63  0.23c 24.33  2.60bc 2.63  0.23d 2.5 29.66  1.45c 5.50  0.28b 34.66  2.40c 10.13  0.23b 27.66  1.45b 4.90  0.20b 5.0 49.66  1.45a 8.60  0.20a 60.00  3.46a 12.56  0.34a 46.33  2.33a 5.96  0.39a 7.5 38.33  2.02b 5.23  0.14b 42.33  1.45b 9.86  0.20b 29.00  2.08b 4.00  0.17c 10.0 24.00  2.08d 2.93  0.23c 32.66  1.76c 7.56  0.23d 19.33  2.96c 2.23  0.14d

Explanation of Figure 18 In vitro morphogenic responses of shoot tip explants of C. angustifolia. A. Induction of multiple shoots on MS medium supplemented with 2.5 µM BA - 2 weeks old culture. (Bar = 0.65 cm)

B. Induction of multiple shoots on MS medium supplemented with 5.0 µM BA - 2 weeks old culture. (Bar = 0.80 cm)

C. Multiplication of shoots on 5.0 µM BA - 3 weeks old culture. (Bar = 0.73 cm)

D. Multiplication and elongation of shoots on medium comprised of BA (5.0 µM) - 5 weeks old culture. (Bar = 0.75 cm)

E. Multiplication of shoots on medium comprised of Kn (7.5 µM) - 5 weeks old culture. (Bar = 0.89 cm)

F. Multiplication of shoots on medium comprised of 2iP (7.5 µM) - 5 weeks old culture. (Bar = 0.86 cm)

Figure 18

B C

D E F

0.23 shoots/explant at 10.0 µM BA with 32.66 ± 1.76% response having shoot length of 2.50 ± 0.17 cm after 6 weeks of culture (Table 22).

Shoot tip explants displayed moderate response on Kn and 2iP supplemented media and lesser number of shoots/explant was produced as compared to BA supplemented media. Lower concentration (1.0 µM) of both Kn and 2iP found to be ineffective for shoot regeneration, however, beyond 1.0 µM both cytokinins influenced the production of multiple shoots. The medium comprised of MS + 7.5 µM Kn proved to be optimal and exhibited 34.00 ± 2.30% response with maximum 6.93 ± 0.23 shoots/explant attaining highest shoot length of 3.23 ± 0.14 cm after 6 weeks of culture (Figure 18 E). While, 2iP at the same concentration provided only 3.66 ± 0.17 shoots/explant with 2.80 ± 0.30 cm shoot length in 18.00 ± 1.15% cultures (Figure 18 F, Table 22).

4.1.2.3.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration The optimal concentrations of BA (5.0 µM), Kn and 2iP (7.5 µM) also tested with various concentrations of auxins (IAA, IBA and NAA) to evaluate the combined effect of cytokinins and auxins on multiple shoot regeneration. The induction of shoot buds found to be early and good than single hormone treatments. Medium comprised of MS + BA (5.0 µM) + NAA (0.4 µM) produced 18.96 ± 0.14 shoots/explant in 65.33 ± 2.40% cultures (Figure 19 A). The best response was obtained in the medium containing BA (5.0 µM) + NAA (0.6 µM) wherein the shoot buds were differentiated within 14 days of incubation producing the maximum 22.46 ± 0.29 shoots/explant in 69.00 ± 2.64% cultures having maximum shoot length of 4.00 ± 0.11 cm after 6 weeks of culture (Figure 19 B). The higher concentration of NAA (0.8 µM NAA) beyond optimal level proved to be inhibitory for shoot multiplication and produced an average of 15.46 ± 0.29 shoots/explant having shoot length of 3.20 ± 0.11 cm in 62.00 ± 1.15% cultures. The combination treatments of BA + IBA and BA + IAA at various concentrations provided moderate response. The medium comprised of MS + BA (5.0 µM) + IBA (0.6 µM) produced a maximum of 18.26 ± 0.17 shoots/explant with shoot length of 3.76 ± 0.20 cm in 64.66 ± 2.90% cultures after 6 weeks of culture. While the medium containing 0.6 µM IAA along with 5.0 µM BA produced an

Table 22. Effect of different cytokinins on direct shoot regeneration from shoot tips of C. angustifolia on MS medium.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 27.66  2.40d 8.63  0.23c 2.43  0.23cd 2.5 - - 34.66  2.40c 10.13  0.23b 2.70  0.20bc 5.0 - - 60.00  3.46a 12.56  0.34a 3.40  0.23a 7.5 - - 42.33  1.45b 9.86  0.20b 2.80  0.11bc 10.0 - - 32.66  1.76c 7.56  0.23d 2.50  0.17c - 1.0 - 00.00  0.00g 0.00  0.00h 0.00  0.00e - 2.5 - 16.00  2.30f 3.60  0.26f 2.43  0.26cd - 5.0 - 24.66  1.76d 5.20  0.20e 2.66  0.17bc - 7.5 - 34.00  2.30c 6.93  0.23d 3.23  0.14ab - 10.0 - 22.33  1.45de 3.90  0.20f 2.36  0.20cd - - 1.0 00.00  0.00g 0.00  0.00h 0.00  0.00e - - 2.5 12.66  1.76f 2.16  0.33g 1.86  0.13d - - 5.0 16.00  1.15f 2.36  0.20g 2.46  0.24cd - - 7.5 18.00  1.15ef 3.66  0.17f 2.80  0.30bc - - 10.0 14.66  1.76f 2.06  0.23g 2.26  0.14cd

Explanation of Figure 19 In vitro morphogenic responses of shoot tip explants of C. angustifolia. A. Multiple shoots production on MS medium supplemented with BA (5.0 µM) and NAA (0.4 µM) - 5 weeks old culture. (Bar = 0.75 cm)

B. Multiplication and proliferation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.70 cm)

C. Multiplication of shoots on medium comprised of MS + Kn (7.5 µM) + NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.85 cm)

D. Multiplication of shoots on medium comprised of MS + 2iP (7.5 µM) + NAA (0.6 µM) - 5 weeks old culture. (Bar = 0.76 cm)

Figure 19

A B

C D

average of 14.83 ± 0.32 shoots/explant with 3.46 ± 0.24 cm shoot length in 62.33 ± 1.45% cultures. Higher concentration of both IBA and IAA reduced the regeneration potential of the explant due to heavy callusing (Table 23).

The optimal concentration (7.5 µM) of Kn and 2iP tested with three different auxins at various concentrations exhibited better response than the single cytokinin containing media. Among three auxins tested, Kn provided best response (47.33 ± 5.45%) with 0.6 µM of NAA, producing a maximum of 12.13 ± 0.20 shoots/explant having an average shoot length of 4.23 ± 0.14 cm after 6 weeks of culture (Figure 19 C). Further increase in the concentration (0.8 µM) of NAA reduced the regeneration potential (38.00 ± 2.30%) of the explant and therefore the number of shoots/explant (8.30 ± 0.20) was also reduced. IBA proved to be better than IAA in combination with Kn and produced an average of 11.90 ± 0.20 shoots/explant with 4.00 ± 0.11 cm shoot length at MS + Kn (7.5 µM) + IBA (0.6 µM) while IAA at the same concentration produced 9.26 ± 0.14 shoots/explant having shoot length of 3.76 ± 0.14 cm after 6 weeks of culture (Table 24).

Similar trend of response was obtained with the cultures on 7.5 µM 2iP with different auxins. The maximum 6.26 ± 0.17 shoots/explant attaining an average shoot length of 3.96 ± 0.14 cm were produced in the medium containing 2iP (7.5 µM) + NAA (0.6 µM) with 34.66 ± 1.45% response after 6 weeks of culture (Figure 19 D). Among various combinations treatments of 2iP with IBA and IAA, the optimal growth of cultures was obtained at 0.6 µM concentration of each of the two auxins. The medium comprised of MS + 2iP (7.5 µM) + IBA (0.6 µM) produced 5.90 ± 0.20 shoots/explant while IAA at the same concentration produced an average of 5.00 ± 0.11 shoots/explant after 6 weeks of culture (Table 25).

4.1.2.3.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration The regeneration potential of ST explants also tested in MS medium augmented with different concentrations of TDZ (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM). The explants showed enlargement and swelling at the base and subsequently differentiated multiple shoot buds within 10 days of incubation on 2.5 µM TDZ (Figure 20 A). A maximum of 15.06 ± 0.17 shoots/explant with shoot

Table 23. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from shoot tips of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.4 - - 65.33  2.40ab 18.96  0.14b 3.66  0.17ab 5.0 0.6 - - 69.00  2.64a 22.46  0.29a 4.00  0.11a 5.0 0.8 - - 62.00  1.15b 15.46  0.29c 3.20  0.11bcd 5.0 - 0.4 - 62.00  3.05b 15.46  0.20c 3.46  0.24abc 5.0 - 0.6 - 64.66  2.90ab 18.26  0.17b 3.76  0.20a 5.0 - 0.8 - 60.00  1.15b 11.90  0.20d 3.00  0.11cd 5.0 - - 0.4 60.66  1.76b 11.93  0.34d 3.20  0.11bcd 5.0 - - 0.6 62.33  1.45ab 14.83  0.32c 3.46  0.24abc 5.0 - - 0.8 60.66  1.15b 11.40  0.30d 2.80  0.11d

Table 24. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from shoot tips of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.4 - - 42.66  3.52ab 10.60  0.20b 3.83  0.14abc 7.5 0.6 - - 47.33  5.45a 12.13  0.20a 4.23  0.14a 7.5 0.8 - - 38.00  2.30ab 8.30  0.20de 3.46  0.14bcd 7.5 - 0.4 - 37.33  1.76ab 8.43  0.23d 3.56  0.23bcd 7.5 - 0.6 - 40.00  4.61ab 11.90  0.20a 4.00  0.11ab 7.5 - 0.8 - 36.00  3.46b 7.86  0.23def 3.43  0.26cd 7.5 - - 0.4 35.33  1.76b 7.76  0.14ef 3.30  0.11cd 7.5 - - 0.6 39.33  1.76ab 9.26  0.14c 3.76  0.14abcd 7.5 - - 0.8 34.00  1.15b 7.26  0.17f 3.23  0.14d

Table 25. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from shoot tips of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.4 - - 30.33  1.45a 4.23  0.14c 3.66  0.17ab 7.5 0.6 - - 34.66  1.45a 6.26  0.17a 3.96  0.14a 7.5 0.8 - - 25.33  1.45b 3.80  0.20cd 3.23  0.14bcd 7.5 - 0.4 - 24.00  2.08b 3.80  0.11cd 3.53  0.20abc 7.5 - 0.6 - 32.33  1.45a 5.90  0.20a 3.63  0.14ab 7.5 - 0.8 - 22.00  1.15bc 3.56  0.23de 3.06  0.14cd 7.5 - - 0.4 17.66  1.45d 3.23  0.14ef 3.00  0.11d 7.5 - - 0.6 25.00  1.15b 5.00  0.11b 3.23  0.14bcd 7.5 - - 0.8 20.66  2.40bc 2.73  0.14f 2.73  0.14d

length of 3.36 ± 0.14 cm was obtained on MS + TDZ (2.5 µM) with a regeneration potential of 72.33 ± 1.45% after 6 weeks of culture (Figure 20 B). Further increase in the concentration of TDZ from optimal level resulted in basal callus production which adversely affected the regeneration capacity of the explant and hampered the growth and development of new shoots. The higher concentrations of TDZ were thus, proved to be inhibitory and produced only 3.60 ± 0.26 shoots/explant with regeneration potential of 42.33 ± 1.45% and attained only 1.23 ± 0.14 cm shoot length after 6 weeks of culture at 10.0 µM of TDZ (Table 26).

4.1.2.3.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Continuous transferring of the regenerative tissue on the same concentration of TDZ resulted in distortion and fasciation of regenerated shoots. Therefore, the regenerated cultures were transferred to TDZ free MS basal medium as well as MS medium containing lower concentrations (0.5. 1.0, 2.5 and 5.0 µM) of BA after 6 weeks of incubation on TDZ. The MS medium devoid of BA was found to be ineffective for further shoot multiplication. Whereas, the media supplemented with BA (0.5 µM) enhanced the number of shoots (16.96 ± 0.26) with an average shoot length of 3.70 ± 0.17 cm after 6 weeks of transfer. The maximum 25.90 ± 0.20 shoots/explant was obtained on 2.5 µM BA having shoot length of 4.56 ± 0.23 cm after 6 weeks of culture (Figure 20 C). Further increase in the concentration of BA (5.0 µM) suppressed the growth and development of shoots and their number was reduced to 17.56 ± 0.23 having shoot length of 3.90 ± 0.20 cm (Table 27).

4.1.2.3.5 Effect of different media

ST explants were cultured on four different media (B5, L2, MS and WPM) containing optimized concentrations of PGRs i.e. BA (5.0 µM) and NAA (0.6 µM) to evaluate the effect of different culture media. ST provided best results with MS medium, producing a maximum of 22.46 ± 0.29 shoots/explant having an average shoot length of 4.00 ± 0.11 cm after 6 weeks of culture. WPM showed slow growth as compared to MS medium and lesser shoots (16.23 ± 0.31) were obtained with reduced shoot length (3.73 ± 0.25 cm). L2 medium delivered

Explanation of Figure 20 In vitro morphogenic responses of shoot tip explants of C. angustifolia. A. Differentiation of multiple shoots on TDZ (2.5 µM) supplemented medium - 2 weeks old culture. (Bar = 0.43 cm)

B. Production of deformed and stunted shoots on prolonged exposure to TDZ (2.5 µM) supplemented medium - 5 weeks old culture. (Bar = 0.65 cm)

C. Multiplication and elongation of TDZ (2.5 µM) induced cultures on BA (2.5 µM) supplemented medium - after 4 weeks of transfer. (Bar = 0.70 cm)

Figure 20

Table 26. Effect of various concentrations of TDZ on direct shoot regeneration from shoot tips of C. angustifolia.

TDZ (M) Regeneration Mean number of Mean shoot (%) shoots/explants length (cm)

0.1 45.00  1.73e 4.56  0.17e 1.73  0.14d 0.25 55.00  1.73cd 6.90  0.20d 2.00  0.11cd 0.5 60.00  1.15bc 7.96  0.29c 2.26  0.14bc 1.0 65.00  2.88b 10.56  0.23b 2.53  0.14b 2.5 72.33  1.45a 15.06  0.17a 3.36  0.14a 5.0 62.33  1.45b 8.50  0.17c 2.56  0.23b 7.5 52.00  1.15d 5.00  0.11e 2.03  0.14cd 10.0 42.33  1.45e 3.60  0.26f 1.23  0.14e

Table 27. Effect of various concentrations of BA on TDZ (2.5 M) induced cultures from shoot tips of C. angustifolia for further multiplication and proliferation.

PGR (M) Mean number of Mean shoot shoots/explant length (cm)

MS 15.06  0.17d 3.36  0.14c MS + BA (0.5) 16.96  0.26c 3.70  0.17bc MS + BA (1.0) 20.83  0.37b 4.00  0.11ab MS + BA (2.5) 25.90  0.20a 4.56  0.23a MS + BA (5.0) 17.56  0.23c 3.90  0.20bc

moderate response with shoot tip explants and 10.93 ± 0.34 shoots/explant having 3.53 ± 0.25 cm shoot length were produced after the same time period.

B5 medium proved to be the least effective and produced only 7.16 ± 0.20 shoots/explant with an average shoot length of 3.23 ± 0.25 cm after 6 weeks of culture (Figure 21).

4.1.2.3.6 Effect of pH The effect of different pH values (5.0, 5.4. 5.8, 6.2 and 6.6) was tested with ST explants in the MS medium containing optimized concentrations of BA (5.0 µM) and NAA (0.6 µM). The lower pH values (5.0 and 5.4) proved to be inhibitory for shoot multiplication and produced 8.56 ± 0.34 and 15.83 ± 0.27 shoots/explant at pH 5.0 and 5.4 respectively after 6 weeks of culture. The optimum pH value found to be 5.8, where a maximum of 22.46 ± 0.29 shoots/explant with 4.00 ± 0.11 cm shoot length were harvested after the same time period. High pH values also retarded the rate of shoot multiplication because at higher pH values the medium became hard in texture and hampered the development of shoots. The number of shoots was reduced to 13.23 ± 0.49 and 7.26 ± 0.17 shoots/explant at pH 6.2 and 6.6 respectively (Figure 22).

4.1.2.3.7 Effect of sucrose concentrations Different concentrations (1, 2, 3, 4 and 5%) of sucrose were also tested with ST explants on the optimal medium i.e. MS + BA (5.0 µM) + NAA (0.6 µM) to investigate the effect of sucrose concentrations on shoot morphogenesis. The best response was obtained with the optimal 3% sucrose, producing a maximum of 22.46 ± 0.29 shoots/explant having maximum shoot length of 4.00 ± 0.11 cm after 6 weeks of culture. However, the lower concentrations (1 and 2%) of sucrose retarded the rate of shoot multiplication and produced 9.56 ± 0.34 and 15.80 ± 0.26 shoots/explant at 1% and 2% sucrose respectively. Further increase in the concentration of sucrose from optimal 3% value also reduced the number of shoots to 17.90 ± 0.20 and 11.83 ± 0.27 shoots/explant at 4% and 5% of sucrose respectively after same incubation period (Figure 23).

Mean number of shoots/explant Mean shoot length (cm) 25 5 a

a 20 a 4 ab b b

15 3

c 10 2 d

Mean shoot length (cm) Mean shoot length 5 1

Mean number of shoots/explant Mean number of

0 0 MS WPM L2 B5 Different media

Figure 21. Effect of different culture media supplemented with optimal concentration of BA (5.0 µM) and NAA (0.6 µM) on shoot regeneration from shoot tip explants of C. angustifolia after 6 weeks of culture.

mean number of shoots/explant Mean shoot length (cm) 25 5 a a 20 a 4 ab bc b 15 c 3 c

10 d 2 e

length (cm)Mean shoot 5 1 shoots/explant Mean number of

0 0 5.0 5.4 5.8 6.2 6.6 Different pH of medium

Figure 22. Effect of different pH values on shoot regeneration from shoot tip explants of C. angustifolia on MS medium containing optimal concentration of BA (5.0 µM) and NAA (0.6 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 25 6 a

20 b 5 c 15 a ab d 4 ab 10 e bc

c 3 Mean shootlength (cm) 5 Mean number of shoots/explant

0 2 1 2 3 4 5 Concentration of sucrose (%)

Figure 23. Effect of different concentrations of sucrose on shoot regeneration from shoot tip explants of C. angustifolia on MS medium containing optimal concentration of BA (5.0 µM) and NAA (0.6 µM) after 6 weeks of culture.

4.1.2.4 Effect of subculture passages on shoot multiplication and proliferation Amongst all the three explants (CN, NS and ST) tested for direct shoot regeneration, maximum number of shoots was obtained through CN explants by adopting a two-step culture procedure. This was done by culturing the explants on a primary medium containing TDZ and then transferring the shoot cultures to a secondary medium augmented with BA for further multiplication and elongation. Maximum 45.5 ± 0.51 shoots/CN explant were obtained by culturing on 2.5 µM of TDZ for 6 weeks and then transferring to BA (2.5 µM), elongated shoots were harvested after 6 weeks and transferred to rooting medium for the induction of roots, while the mother explant was further cultured onto the fresh medium of same composition (MS + 2.5 µM BA). The mother explant exhibited the development of new shoots up to two subculture passages only and retained the same number of shoots (45.50 ± 0.51). Thereafter, a decline in the regeneration potential as well as number of shoots was observed.

Thus, to maintain the cultures for long duration with high regeneration potential and vigorous growth, cytokinin-auxin treated cultures (39.16 ± 0.14 shoots/CN explant) were regularly subcultured on to the fresh medium of optimized phytohormones [BA (5.0 µM) + NAA (0.6 µM)]. Observations were recorded up to six subculture passages at an interval of 6 weeks each. The elongated and healthy shoots with 3-4 internodes were harvested at the end of each passage and transferred to rooting medium, while mother explant (CN) was regularly subcultured on to the fresh medium of same composition. The mother tissue retained the regeneration potential up to the fourth subculture passage and thereafter, a decline was observed. During the first subculture passage new shoot buds were differentiated from the mother tissue and a maximum of 42.90 ± 0.20 shoots were obtained with 5.96 ± 0.14 cm shoot length after 6 weeks, but it was observed that some of the regenerated shoots showed the sign of premature leaf fall, yellowing of leaves and shoot tip necrosis (STN) (Figure 24 A & B). To overcome these anomalies, the regeneration medium was amended with different concentrations (10, 20, 30, 40 and 50 µM) of adenine sulphate (AdS) along with optimized PGRs. Incorporation of 30.0 µM of AdS proved to be highly effective for the control of STN, yellowing and abscission of leaves, it also

Explanation of Figure 24 Shoot tip necrosis, yellowing and abscission of leaves in C. angustifolia. A. Yellowing and abscission of leaves observed during first subculture passage on regeneration medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) - after 2 weeks of transfer. (Bar = 0.36 cm)

B. Shoot tip necrosis observed during first subculture passage on regeneration medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) - after 3 weeks of transfer. (Bar = 0.44 cm)

Figure 24

A B

enhanced the rate of shoot multiplication and the new shoots developed were quite healthy and having normal dark green leaves. Therefore, the medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) + AdS (30.0 µM) found to be the most suitable and optimized combination of PGRs for shoot multiplication and proliferation. Thus, from second passage onwards, cultures were transferred to this optimized medium and by the end of 2nd subculture a maximum of 51.53 ± 0.64 shoots were harvested (Figure 25). The new shoots continued to develop up to 4th subculture passage where the highest 64.90 ± 0.32 shoots with maximum shoot length of 6.56 ± 0.23 cm were obtained (Figure 26). From 5th subculture passage onwards a decline in the regeneration potential was noticed and by the end of sixth subculture passage, number of shoots was reduced to 41.06 ± 0.49 shoots/explant.

The cultures obtained through NS and ST explants were also maintained and transferred to fresh medium of same composition after every 6 weeks up to six subculture passages but they exhibited poor response as compared to the cultures obtained from CN explants (Data not shown).

4.1.3 Indirect Organogenesis For the induction of organogenic calli, green cotyledonary leaves and roots isolated from aseptic seedlings of C. angustifolia were cultured on different concentrations of auxins and cytokinins.

4.1.3.1 Cotyledonary leaf (CL) explant 4.1.3.1.1 Effect of explant age on callus production To select the most responsive age of the explant, CL explants of different age group (7, 14 and 21 days old) excised from axenic seedlings were cultured on MS medium containing 1.0, 2.5, 5.0, 7.5 and 10.0 µM of 2,4-D. MS medium without 2,4-D was served as control treatment on which explants remained green and did not show any sign of callus induction. However, addition of 2,4-D stimulated callus induction and the frequency of callus growth varied with the age of the explants as well as concentration of 2,4-D. Callus cells started to differentiate from cut ends and gradually extended to cover the entire surface of the explant. The MS medium amended with 1.0 µM 2,4-D showed poor response, while increasing the concentration to 5.0 µM, resulted in enhanced

Mean number of shoots/explant Mean shoot length (cm) 70 7.0 a b a 6.8 60 c c 6.6 50 d ab ab e 6.4 40 6.2 bc bc 30 6.0

20 c 5.8 (cm) length shoot Mean

shoots/explant of number Mean 10 5.6

0 5.4 I II III IV V VI Subculture passages

Figure 25. Effect of subculture passages on multiplication and proliferation of shoots through cotyledonary nodes of C. angustifolia on optimized regeneration medium*. [*Regeneration medium: I subculture passage - MS + BA (5.0 µM) + NAA (0.6 µM), II-VI subculture passage - MS + BA (5.0 µM) + NAA (0.6 µM) + AdS (30.0 µM)].

Explanation of Figure 26 Maintenance of cultures in C. angustifolia Maintained cultures obtained through regenerative tissue of cotyledonary nodes after 4th subculture passage on optimal medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) + AdS (30.0 µM). (Bar = 1.33 cm)

Figure 26

callus growth with the production of green, compact and nodular callus in 84.66 ± 2.60% cultures through 14 days old explants. Higher concentrations of 2,4-D decreased the rate of callus growth to 37.66 ± 3.20% at 10.0 µM 2,4-D (Table 28).

Explants excised from 7 and 21 days old seedlings exhibited poor response compared to the explants of 14 days old seedling. The MS medium containing 5.0 µM 2,4-D produced loose and friable callus through 7 days old CL explants in 36.00 ± 2.30% cultures, while 21 days old explants produced hard and dry callus in 39.66 ± 3.23% cultures at the same concentration. Higher concentrations of 2,4-D further reduced the callus growth. Hence the optimal growth of callus obtained from the explants of 14 days old seedlings on 5.0 µM of 2,4-D was used for shoot organogenesis (Table 28).

4.1.3.2 Root (R) explant 4.1.3.2.1 Effect of explant age on callus production For the induction of organogenic calli and shoot regeneration, roots of aseptic seedlings were also used as an explant and cultured on various PGRs. To evaluate the effect of explant age, root segments were excised from 20, 30 and 40 days old axenic seedlings and cultured on different concentrations of 2,4-D. MS medium devoid of 2,4-D was served as control, explants failed to respond on MS basal medium and remained as such even after 4 weeks of incubation. However, addition of 2,4-D facilitated the induction of callus from cut ends of the explant which covered the entire surface of the explant. The root explants collected from 30 days old seedlings produced green, compact and nodular calli on 5.0 µM 2,4-D in 57.66 ± 1.45% cultures, although, higher concentrations (7.5 and 10.0 µM) resulted in the formation of loose and friable calli. Explants collected from 20 and 40 days old seedlings at the same concentration produced calli in 45.00 ± 2.88% and 39.00 ± 2.08% cultures respectively (Table 29).

4.1.3.3 Effect of different auxins on callus induction from cotyledonary leaf (CL) and root (R) explants Cotyledonary leaf explants (14 days old) and root explants (30 days old) were cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of 2,4-D and 2,4,5-T for the production of callus. Induction of callus started from cut ends of

Table 28. Effect of age of cotyledonary leaf explant on callus induction in C. angustifolia cultured on MS medium containing different concentrations of 2,4-D.

PGR (µM) 7 days old explant 14 days old explant 21 days old explant

2,4-D % Rate of % Rate of % Rate of Response callus growth Response callus growth Response callus growth

- 00.00  0.00i NR 00.00  0.00e NR 00.00  0.00g NR 1.0 27.33  1.80ab - 33.00  2.91d + 24.66  2.91bc - 2.5 30.00  2.31ab + 40.30  3.21c + + 28.70  3.50abc - 5.0 36.00  2.31a + + 84.66  2.60a + + + + 39.66  3.23a + 7.5 31.00  2.01ab + 59.00  3.76b + + 34.71  2.62ab + + 10.0 25.33  2.40ab + 37.66  3.23d + 29.33  1.76abc +

Table 29. Effect of age of root explant on callus induction in C. angustifolia cultured on MS medium containing different concentrations of 2,4-D.

PGR (µM) 20 days old explant 30 days old explant 40 days old explant

2,4-D % Rate of % Rate of % Rate of Response callus growth Response callus growth Response callus growth

- 00.00  0.00d NR 00.00  0.00d NR 00.00  0.00e NR 1.0 30.00  1.73b - 32.66  1.76c + 15.66  2.33d - 2.5 32.33  1.45b + 45.66  2.33b + 22.33  1.45c - 5.0 45.00  2.88a + 57.66  1.45a + + 39.00  2.08a + 7.5 39.33  1.76a + 49.00  2.08b + 29.00  2.08b + 10.0 29.00  2.08b + 35.66  2.33c + 22.33  1.45c +

the explants and then spread all over the surface of the explants. Between two auxins tested, 2,4-D provided better response than 2,4,5-T at various concentrations, the lower concentration (1.0 µM) of 2,4-D was not much effective as only 33.00 ± 2.91% CL explants and 32.66 ± 1.76% root explants showed induction of callus after 6 weeks of culture. The MS medium containing 5.0 µM 2,4-D influenced callus induction within 12 days of inoculation and the optimal growth of green, compact and nodular callus was obtained in 84.66 ± 2.60% cultures from CL explants after 6 weeks. While root explants were less responsive at the same concentration and exhibited 57.66 ± 1.45% response with the production of loose and friable callus (Table 30). The callus produced by CL explants on 5.0 µM 2,4-D was initially green in colour but later on turned dark brown (Figure 27 A & B). On further enhancement in the concentration of 2,4-D from 5.0 µM, the frequency of callus growth reduced with the production of loose and friable callus from both type of explants. The calli induced on various concentrations of 2,4,5-T were yellow, friable and loose. At 1.0 µM of 2,4,5-T, root explants failed to induce any response but 35.66 ± 3.52% CL explants produced callus on the same concentration. The rate of callus growth increased with an enhancement in the concentration of 2,4,5-T and an optimal growth of callus was obtained at 2.5 µM of 2,4,5-T in 60.00 ± 5.10% cultures from CL explants (Figure 27 C). Root explants exhibited poor response with 2,4,5-T and only 39.33 ± 2.33% cultures produced calli on 5.0 µM of 2,4,5-T. On further increasing the concentration of 2,4,5-T, reduction in the percent response as well as rate of callus growth was observed (Table 30).

4.1.3.4 Effect of different cytokinins on callus induction from cotyledonary leaf (CL) and root (R) explants The cotyledonary leaf and root explants were also cultured on various concentrations (0.1, 0.5, 1.0, 2.5 and 5.0 µM) of three different cytokinins (BA, Kn and TDZ) to induced organogenic calli. The lower concentrations (0.1 and 0.5 µM) of BA and Kn found to be ineffective for callus induction from both CL and root explants, however, an increase in the concentration of BA and Kn stimulated the explants to induce callus. The MS medium supplemented with 2.5 µM BA induced callus from cut ends of the explants which was light green and friable in nature and exhibited 35.66 ± 2.33% and 50.66 ± 1.45% response through CL

100

Table 30. Effect of auxins on callus induction through cotyledonary leaf and root explants of C. angustifolia.

PGR (µM) Cotyledon explant Root explant

2,4-D 2,4,5-T % Rate of % Rate of Response callus growth Response callus growth

1.0 - 33.00  2.91cd + 32.66  1.76c + 2.5 - 40.30  3.21c + + 45.66  2.33b + 5.0 - 84.66  2.60a + + + + 57.66  1.45a + + 7.5 - 59.00  3.76b + + 49.00  2.08b + 10.0 - 37.66  3.23cd + 35.66  2.33c + - 1.0 35.66  3.52cd + 00.00  0.00g NR - 2.5 60.00  5.10b + + + 27.66  1.45e - - 5.0 27.66  4.34cd + 39.33  2.33c + - 7.5 22.00  4.21d + 29.66  1.45e + - 10.0 21.33  2.02d + 22.33  1.45f -

Explanation of Figure 27 Production of callus through cotyledonary leaf (CL) and root (R) explants in C. angustifolia. A. Production of green, compact and nodular callus from cut ends of the CL explant on MS medium containing 5.0 µM 2,4-D - 4 weeks old culture. (Bar = 0.69 cm)

B. Callus turned dark brown in colour on the same medium - 6 weeks old culture. (Bar = 0.54 cm)

C. Production of yellow, friable and loose callus through CL explant on medium comprised of MS + 2,4,5-T (2.5 µM) - 6 weeks old culture. (Bar = 0.58 cm)

D. Production of green, compact and nodular callus through root explant on MS medium containing 1.0 µM TDZ - 2 weeks old culture. (Bar = 0.73 cm)

E. -do- 3 weeks old culture. (Bar = 0.52 cm)

F. The green callus turned brown on the same medium - 6 weeks old culture. (Bar = 0.50 cm)

Figure 27

A B C

D E F

and root explants respectively. The CL and root explants cultured on 2.5 µM Kn produced soft, watery and yellowish calli in 22.33 ± 1.45% and 30.33 ± 1.15% cultures respectively (Table 31).

Amongst all the three cytokinins tested, TDZ proved to be more effective for the induction of regenerative calli, however, CL explants were comparatively less responsive than root explants on TDZ supplemented medium. The lower concentration (0.1 µM) of TDZ produced yellowish and loose calli with 27.33 ± 1.45% and 40.33 ± 1.15% response from CL and root explants respectively. Optimal growth of green, compact and nodular callus was obtained at 1.0 µM TDZ with root explant in 90.66 ± 1.45% cultures. Initially the callus was green in colour which later on converted to dark brown (Figure 27 D, E & F). The CL explants produced loose and friable callus at 1.0 µM of TDZ in 49.00 ± 2.08% cultures after 6 weeks of incubation. Higher concentrations (2.5 and 5.0 µM) of TDZ proved to be inhibitory for both CL and root explants and produced yellowish and loose calli. At 5.0 µM TDZ the rate of callus induction was reduced to 39.66 ± 2.02% and 40.00 ± 1.73% from CL and root explants respectively (Table 31).

4.1.3.5 Shoot differentiation from cotyledonary leaf derived callus 4.1.3.5.1 Effect of cytokinins on multiple shoot differentiation The dark green or brown, compact and nodular calli raised on optimized medium (MS + 5.0 μM of 2,4-D) from 14 days old CL explants were selected for organogenesis and transferred to shoot regeneration medium comprised of different cytokinins (BA, Kn and TDZ) at various concentrations (Table 32). Shoot differentiation from the calli was determined by the type of growth regulator, its concentration and combination. Amongst three cytokinins tested, BA was found to be more effective than Kn and TDZ. On BA (5.0 µM) supplemented medium the dark brown and compact callus turned green within 6 days of transfer with the differentiation of small nodular structures and after 10- 12 days started to develop shoot bud primordial in most of the cultures (Figure 28 A & B). Differentiated shoot buds emerged out leading to healthy shoot development. The MS medium supplemented with 1.0 µM of BA exhibited 70.66 ± 2.31% response and produced 3.10 ± 0.60 shoots/explant, the rate of shoot

101

Table 31. Effect of different cytokinins on callus induction through cotyledonary leaf and root explants of C. angustifolia.

PGR (µM) Cotyledon explant Root explant

BA Kn TDZ % Rate of % Rate of Response callus growth Response callus growth

0.1 - - 00.00  0.00g NR 00.00  0.00h NR 0.5 - - 00.00  0.00g NR 00.00  0.00h NR 1.0 - - 22.33  1.45e - 40.00  1.15d + + 2.5 - - 35.66  2.33c + 50.66  1.45b + 5.0 - - 27.66  1.45d + 30.33  1.73f + - 0.1 - 00.00  0.00g NR 00.00  0.00h NR - 0.5 - 00.00  0.00g NR 00.00  0.00h NR - 1.0 - 20.33  1.45e + 20.33  1.45g + - 2.5 - 22.33  1.45e + 30.33  1.15f + - 5.0 - 12.33  1.45f - 25.00  1.73g + - - 0.1 27.33  1.45d - 40.33  1.15e + + - - 0.5 36.00  2.30c + 60.66  1.45c + + + - - 1.0 49.00  2.08a + + 90.66  1.45a + + + + - - 2.5 44.33  2.96ab + 70.33  2.08b + + + - - 5.0 39.66  2.02bc - 40.00  1.73e + +

Table 32. Effect of different cytokinins on shoot differentiation from cotyledonary leaf derived callus of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn TDZ

- - - 00.00  0.00k 00.00  0.00e 0.00  0.00g 1.0 - - 70.66  2.31d 3.10  0.60cd 1.43  0.26def 2.5 - - 77.33  2.60c 5.50  0.79b 2.10  0.26bcd 5.0 - - 91.00  2.60a 10.73  0.95a 3.96  0.20a 10.0 - - 84.66  2.85b 4.93  0.58b 2.56  0.34b - 1.0 - 55.33  2.55f 2.66  0.23d 1.03  0.14ef - 2.5 - 62.00  2.31e 3.16  0.43cd 1.73  0.27de - 5.0 - 66.66  3.52d 4.30  0.62bc 2.60  0.26b - 10.0 - 61.66  3.52e 2.20  0.17d 2.20  0.26bc - - 1.0 42.66  1.47h 2.53  0.29d 1.23  0.14ef - - 2.5 50.33  1.64g 2.90  0.20cd 2.10  0.20bcd - - 5.0 37.66  2.60i 2.43  0.23d 1.10  0.20ef - - 10.0 32.66  2.31j 2.23  0.14d 0.90  0.20f

differentiation further increased and at 5.0 µM BA, a maximum of 10.73 ± 0.95 shoots/explant were produced in 91.00 ± 2.60% cultures after 6 weeks of transfer (Figure 28 C). However, higher concentration (10.0 µM) of BA had inhibitory effects reducing the regeneration potential to 84.66 ± 2.85% and the number of shoots also reduced to 4.93 ± 0.58 shoots/explant after 6 weeks of transfer (Table 32).

Between Kn and TDZ, Kn exhibited better response and at an optimal concentration of 5.0 µM, 66.66 ± 3.52% cultures differentiated multiple shoot buds, producing an average of 4.3 ± 0.62 shoots/explant with 2.60 ± 0.26 cm of shoot length after 6 weeks of culture. TDZ supplemented media did not support for shoot bud differentiation but favored intensive growth of callus masses. At 2.5 µM of TDZ only 2.90 ± 0.20 shoots/explant were produced with shoot length of 2.10 ± 0.20 cm in 50.33 ± 1.64% cultures after 6 weeks of transfer. Further increase of TDZ concentration adversely affected the regeneration potential with heavy callusing and retarded the growth and development of new shoots (Table 32).

4.1.3.5.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation The dark, compact and nodular calli obtained from 14 days old CL explants on 5.0 µM of 2,4-D were transferred to cytokinin-auxin combinations also. Incorporation of NAA at various concentrations (0.2, 0.4 and 0.6 µM) with the optimal concentration (5.0 µM) of BA enhanced the rate of shoot multiplication and shoot buds were differentiated within 10 days of incubation. The medium comprised of MS + BA (5.0 µM) + NAA (0.4 µM) produced maximum of 23.16 ± 1.44 shoots/explant with 96.33 ± 1.45% response after 6 weeks of transfer (Figure 28 D & E, Table 33). However, addition of 0.2 μM and 0.6 μM of NAA to the medium did not show any pronounced increase in the number of shoots per culture and percent response. The MS medium supplemented with BA (5.0 μM) + NAA (0.2 μM) influenced the production of an average of 11.43 ± 1.43 shoots/explant in 90.50 ± 2.31% cultures compared to MS + BA (5.0 μM) wherein 10.73 ± 0.95 shoots/explant were produced in 91.00 ± 2.60% cultures. While higher concentration of NAA (0.6 μM) resulted in heavy growth of callus

102

Explanation of Figure 28 Differentiation of shoots from cotyledonary leaf (CL) derived callus in C. angustifolia. A. The dark brown callus induced at 5.0 µM 2,4-D turned green and nodular along with the differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (5.0 µM) - after 1 week of transfer. (Bar = 0.50 cm)

B. Differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (5.0 µM) - 2 weeks old culture. (Bar = 0.50 cm)

C. Multiplication and proliferation of shoots on MS medium containing BA (5.0 µM) - 4 weeks old culture. (Bar = 0.50 cm)

D. Differentiation and multiplication of shoots on MS medium supplemented with BA (5.0 µM) and NAA (0.4 µM) - 4 weeks old culture. (Bar = 0.48 cm)

E. Proliferation and elongation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (0.4 µM) - 6 weeks old culture. (Bar = 0.75 cm)

Figure 28

C E

Table 33. Effect of optimal concentration of BA with different auxins on shoot differentiation from cotyledonary leaf derived callus of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IAA IBA

5.0 0.2 - - 90.50  2.31a 11.43  1.43bc 4.13  0.20b 5.0 0.4 - - 96.33  1.45a 23.16  1.44a 5.00  0.26a 5.0 0.6 - - 79.33  1.81b 9.20  1.90b 2.66  0.23cde 5.0 - 0.2 - 58.86  3.13d 9.03  0.26cd 3.03  0.31cd 5.0 - 0.4 - 67.56  1.47c 12.33  1.12b 3.50  0.28bc 5.0 - 0.6 - 49.53  2.23e 8.36  0.61cd 2.70  0.32cde 5.0 - - 0.2 35.30  1.41f 6.23  0.43de 2.56  0.31de 5.0 - - 0.4 45.56  2.71e 8.46  1.07cd 2.90  0.20cde 5.0 - - 0.6 32.70  1.61f 5.13  0.58e 2.00  0.28e

which adversely affected the regeneration response (79.33 ± 1.81%) and also retarded the growth and development of new shoots (9.20 ± 1.90 shoots/explant).

The other two auxins (IAA and IBA) failed to enhance the rate of shoot differentiation and multiplication. Addition of IAA and IBA at various concentrations provided moderate results; heavy callusing reduced the percent response as well as number of shoots/explant. A maximum of 12.33 ± 1.12 and 8.46 ± 1.07 shoots/explant with 67.56 ± 1.46% and 45.56 ± 2.71% response were obtained in the medium containing BA (5.0 µM) + IAA (0.4 µM) and BA (5.0 µM) + IBA (0.4 µM) respectively. Further increase in the concentration of IAA and IBA reduced the percent response as well as number of shoots/culture (Table 33).

4.1.3.6 Shoot differentiation from root derived callus 4.1.3.6.1 Effect of cytokinins on multiple shoot differentiation The regenerative callus obtained from 30 days old root explants at 1.0 µM of TDZ was used for the differentiation of shoots and transferred to shoot regeneration medium. The fast growing nodular calli produced on 1.0 µM of TDZ started to develop green pigmentation and nodulation within one week of transfer to the regeneration medium containing cytokinins (BA, Kn and TDZ) at various concentrations (1.0, 2.5 and 5.0 µM). The appearance of first shoot bud was noticed in the form of dark green organoid or the green nodular structure, which further transformed into shoot (Figure 29 A & B). Among three cytokinins tested, BA at 2.5 µM proved to be the best for shoot differentiation and development where a maximum of 24.56 ± 1.97 shoots/explant were obtained attaining a shoot length of 4.70 ± 0.26 cm after 6 weeks of transfer. Kn and TDZ supplemented media provided lesser number of shoots as compared to BA supplemented media. The MS medium containing an optimal concentration of 2.5 µM of Kn differentiated 18.36 ± 1.18 shoots/culture while the TDZ at the same concentration produced a maximum of only 12.16 ± 0.86 shoots/explant after 6 weeks of culture. Further, increase in the concentration of hormones (Kn and TDZ) beyond their optimal level retarded the growth and development of shoots (Table 34).

103

Explanation of Figure 29 Differentiation of shoots from root derived callus in C. angustifolia. A. The dark brown compact callus started to turn greenish along with the differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (2.5 µM) - after 1 week of transfer. (Bar = 0.48 cm)

B. -do- after 2 weeks of transfer. (Bar = 0.33 cm)

C. Differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM) - 2 weeks old culture. (Bar = 0.32 cm)

D. -do- 3 weeks old culture. (Bar = 0.41 cm)

E. Development of multiple shoots on MS medium containing BA (2.5 µM) and NAA (0.6 µM) - 4 weeks old cultures. (Bar = 0.48 cm)

F. Proliferation and elongation of shoots on same medium - 6 weeks old culture. (Bar = 1.0 cm)

Figure 29

A B

C D

E F

Table 34. Effect of different cytokinins on shoot differentiation from root explant derived callus of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn TDZ

- - - 00.00  0.00g 00.00  0.00f 0.00  0.00d 1.0 - - 40.00  1.15d 19.20  1.43b 3.63  0.14b 2.5 - - 75.33  1.45a 24.56  1.97a 4.70  0.26a 5.0 - - 60.66  1.76b 22.76  1.36a 3.56  0.31b - 1.0 - 35.66  1.76d 13.10  1.47cd 3.23  0.14b - 2.5 - 50.66  1.76c 18.36  1.18b 3.56  0.26b - 5.0 - 30.33  1.45e 15.46  0.70b 2.43  0.12c - - 1.0 40.33  1.45d 7.13  0.98e 2.43  0.12c - - 2.5 30.66  1.76e 12.16  0.86cd 3.23  0.14b - - 5.0 25.66  1.76f 10.03  1.24de 2.26  0.17c

4.1.3.6.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation The optimal concentration of BA (2.5 µM) was also tested with 3 different auxins (NAA, IAA and IBA) for the differentiation of shoots from root derived organogenic calli produced on MS + TDZ (1.0 µM). Augmentation of NAA enhanced the rate of shoot multiplication as well as the number of shoots/culture as compared to the cultures containing BA alone (Figure 29 C & D). The regeneration potential was increased to 90.33 ± 1.45% producing a maximum of 35.63 ± 0.75 shoots/explant with highest 5.43 ± 0.20 cm shoot length on MS + BA (2.5 µM) + NAA (0.6 µM) after 6 weeks of transfer (Figure 29 E & F). However, the higher concentration (0.8 µM) of NAA retarded the regeneration potential (80.00 ± 1.76%) as well as number of shoots/explant (25.33 ± 1.9). Addition of IAA and IBA provided moderate response and a maximum of 22.56 ± 1.01 and 20.80 ± 1.30 shoots/explant were obtained in the medium containing 2.5 µM of BA along with the optimal concentration (0.6 µM) of IAA and IBA respectively. Thus, for the continuous growth and development of shoots, the regenerative tissues were regularly subcultured onto the MS medium containing 2.5 µM BA and 0.6 µM NAA at an internal of 6 weeks (Table 35).

4.1.3.7 Effect of subculturing and maintenance of cultures For long-term maintenance of cultures and further multiplication of shoots, the organogenic calli obtained on TDZ (1.0 µM) through root explants were repeatedly subcultured on the optimized medium containing MS + BA (2.5 µM) + NAA (0.6 µM). However, the calli induced through cotyledon explants at 2,4-D (5.0 µM) showed a sudden decrease in the differentiation of shoot buds just after first subculture passage, thus for multiplication and maintenance of cultures only root derived callus was preferred. Elongated shoots were regularly harvested at the end of each passage and transferred to rooting medium; observations were recorded up to 6 subculture passages at an interval of 6 weeks each (Figure 30). An increase in the rate of shoot multiplication and development of new shoots was recorded up to 4th subculture passage which became constant at fifth subculture passage, where the maximum 42.66 ± 1.47 shoots/explant were obtained from the root derived calli (Figure 31). Beyond 5th subculture passage,

104

Table 35. Effect of optimal concentration of BA with different auxins on shoot differentiation from root derived callus of C. angustifolia.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IAA IBA

2.5 0.4 - - 75.66  1.45b 27.76  1.25b 3.60  0.23bcd 2.5 0.6 - - 90.33  1.45a 35.63  0.75a 5.43  0.20a 2.5 0.8 - - 80.00  1.76b 25.33  1.90b 4.26  0.14bc 2.5 - 0.4 - 70.00  1.15c 17.13  1.83ef 3.63  0.14bcd 2.5 - 0.6 - 75.33  1.45b 22.56  1.01cd 4.33  0.24b 2.5 - 0.8 - 60.66  1.45d 20.80  1.30de 4.06  0.17bc 2.5 - - 0.4 65.00  2.88cd 15.20  1.02f 3.16  0.37d 2.5 - - 0.6 71.00  2.08c 20.80  1.30de 4.26  0.14b 2.5 - - 0.8 60.00  3.05d 17.60  1.38ef 3.36  0.20cd

Mean number of shoots/explant Mean shoot length (cm) 50 6.8

ab a a 6.6 bc a bc 40 a c a a 6.4

ab 30 6.2

b 6.0 20

5.8

Mean shoot length (cm) length shoot Mean 10

Mean number of shoots/explant number of Mean 5.6

0 5.4 I II III IV V VI Subculture passages

Figure 30. Effect of subculture passages on shoot differentiation efficiency of root derived callus of C. angustifolia on optimal regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM).

Explanation of Figure 31 Subculturing and maintenance of root derived cultures in C. angustifolia. Maintenance of cultures obtained from root derived callus on optimal medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM) - after 4th subculture passage. (Bar = 2.0 cm)

Figure 31

regeneration potential of the tissue reduced and decline in mean shoot number was recorded.

4.1.4 Somatic embryogenesis Immature cotyledons (IC) excised from green seeds of semi mature pods (Figure 32 A & B) were cultured on MS medium containing different concentrations of auxins (2,4-D, 2,4,5-T, IAA, IBA and NAA) singly or in combination with cytokinins (BA, Kn and 2iP) to induce embryogenic calli (Table 36). MS medium without any PGR served as control medium on which explants failed to induce embryogenic callus even after 3 weeks of incubation. Augmentation of auxins stimulated swelling of explants within 1 week of culture and yellowish to light green calli started to differentiate from cut ends of the explant (Figure 33 A & B). Induction of globular embryoids was observed after 3 weeks of culture and later on heart shaped and torpedo embryoids were also observed under the stereozoom microscope (Figure 33 C & D). Cotyledonary staged embryos were most frequently seen and clearly visible with naked eye. Among various concentrations of different auxins tested, the lower concentrations of 2,4-D were not much effective and at 5.0 µM about 65.20 ± 1.53% cultures exhibited the induction of embryogenic callus with the differentiation of cotyledonay staged embryos after 6 weeks (Figure 33 E & F). 2,4-D at 10.0 µM found to be the most effective for inducing embryogic calli in 83.90 ± 1.70% cultures (Figure 34 A & B), wherein a maximum of 9.23 ± 0.67 embryoids/explant were produced and a few started germination on the same medium (Figure 34 C & D). 2,4,5-T and all other 3 auxins produced loose and watery callus which was light brown in colour and showed moderate embryogenic response, while lower concentrations (1.0 or 2.5 µM) failed to induce somatic embryogenesis. IAA and IBA were found to be least effective and exhibited only 8.63 ± 2.07% and 9.63 ± 1.50% response at 5.0 µM IAA and IBA respectively. While, NAA at the same concentration exhibited 42.96 ± 1.43% response producing an average of 6.26 ± 0.40 embryoids/explant. Some of the embryoids started germination on the same medium containing 5.0 µM of NAA and showed development of root and shoot apices (Figure 35 A & B) which ultimately resulted in the formation of complete plantlets (Table 36).

105

Explanation of Figure 32 Source of explants for somatic embryogenesis in C. angustifolia. A. Green immature pods. (Bar = 1.0 cm)

B. Immature seeds. (Bar = 0.53 cm)

Figure 32

Table 36. Effect of different auxins on induction of somatic embryogenesis from immature green cotyledons of C. angustifolia.

PGR (M) Response Mean number of (%) embyoids/explant 2,4-D 2,4,5-T IAA IBA NAA

- - - - - 00.00  0.00j 0.00  0.00e 1.0 - - - - 26.43  1.86e 3.90  0.37c 2.5 - - - - 38.30  1.90d 6.23  0.53b 5.0 - - - - 65.20  1.53b 8.30  0.62a 10.0 - - - - 83.90  1.70a 9.23  0.67a - 1.0 - - - 00.00  0.00j 0.00  0.00e - 2.5 - - - 12.76  1.29g 1.83  0.32d - 5.0 - - - 19.73  2.21f 3.90  0.20c - 10.0 - - - 10.26  0.95gh 1.53  0.31d - - 1.0 - - 00.00  0.00j 0.00  0.00e - - 2.5 - - 00.00  0.00j 0.00  0.00e - - 5.0 - - 8.63  2.07hi 2.23  0.53d - - 10.0 - - 5.86  1.41i 1.50  0.28d - - - 1.0 - 00.00  0.00j 0.00  0.00e - - - 2.5 - 00.00  0.00j 0.00  0.00e - - - 5.0 - 9.63  1.50ghi 3.63  0.23c - - - 10.0 - 00.00  0.00j 0.00  0.00e - - - - 1.0 00.00  0.00j 0.00  0.00e - - - - 2.5 29.46  2.11e 3.83  0.32c - - - - 5.0 42.96  1.43c 6.26  0.40b - - - - 10.0 30.30  1.02e 3.60  0.20c

Explanation of Figure 33 Somatic embryogenesis in C. angustifolia. A. Production of embryogenic callus through immature cotyledon explant on MS medium supplemented with 5.0 µM 2,4-D, arrows represent globular stage embryoids - 3 weeks old culture. (Bar = 0.38 cm)

B. -do- 4 weeks old culture. (Bar = 0.36 cm)

C. Embryogenic callus under stereozoom microscope. (Bar = 100.0 µm)

D. An enlarged view of globular embryo under stereozoom microscope. (Bar = 100.0 µm)

E. Differentiation of mature somatic embryos on MS medium containing 5.0 µM 2,4-D, arrows represent cotyledonary stage embryoids - 6 weeks old culture. (Bar = 0.35 cm)

F. -do- (Bar = 0.35 cm)

Figure 33

A B

C D

E F

Explanation of Figure 34 Somatic embryogenesis in C. angustifolia. A. Production of embryogenic callus through immature cotyledon explant on MS medium supplemented with 10.0 µM 2,4-D, arrow represents cotyledonary stage embryoids - 3 weeks old culture. (Bar = 0.31 cm)

B. -do- 4 weeks old culture. (Bar = 0.38 cm)

C. Maturation and germination of cotyledonay stage embryoids on MS medium containing 10.0 µM 2,4-D, arrows represent germinating embryoid with proper shoot primordia - 6 weeks old culture. (Bar = 0.37 cm)

D. -do- arrows represent root primordia of germinating embryoids - 6 weeks old culture. (Bar = 0.39 cm)

Figure 34

A B

C D

Explanation of Figure 35 Somatic embryogenesis in C. angustifolia. A. Development of somatic embryoids on MS medium containing 5.0 µM NAA along with germination of mature embryoids on the same medium, arrows represent germinating embryoid with proper shoot and root primordia - 5 weeks old cultures. (Bar = 0.21 cm)

B. -do- (Bar = 0.20 cm)

Figure 35

The optimal concentration (10.0 µM) of 2,4-D was also tested with three different cytokinins at various concentrations to produce embryogenic calli. Addition of cytokinins along with 2,4-D improved the rate of embryogenesis and also facilitated the germination of embryoids on the same medium. Best embryogenic callus was obtained on MS medium amended with 10.0 µM 2,4-D and 1.0 µM BA producing a maximum of 22.80 ± 1.59 embryoids/explant in 90.56 ± 1.88% cultures (Figure 36 A & B). A maximum of 35.33 ± 2.90% germination rate was recorded on the same medium, the cotyledons of the germinating embryoids started to expand along with the emergence of root primordia (Figure 36 C & D). Further increase in the concentration of BA resulted in the formation of non embryogenic calli and the rate of embryogenesis reduced to 83.43 ± 0.34% on MS + 2,4-D (10.0 µM) + BA (5.0 µM) (Figure 37 A). Kn provided moderate response in 78.03 ± 1.59% cultures on medium containing MS + 2,4-D (10.0 µM) + Kn (2.5 µM) (Figure 37 B), while, 2iP proved to be least effective and exhibited 64.50 ± 2.32% response on medium comprised of MS + 2,4-D (10.0 µM) + 2iP (2.5 µM) (Table 37).

The germinating embryoids (8 days old) measuring 0.5-0.6 cm, when transferred to the hormone free half strength MS and MS basal medium, started to expand the cotyledonary leaves along with the elongation of radicle after 4 days of transfer. Expansion in cotyledonary leaves and growth in root system was more pronounced in half strength MS medium (30%) than the MS basal medium (20%). Apical shoot growth with the appearance of leaf primordial was observed after 10 days of transfer on both the medium (Figure 37 C & D). However, growth and differentiation of shoot system was better on half strength MS medium and took about 14 more days to attain a length of 2.20 cm followed by 1.07 cm in MS basal medium.

4.1.5 Rooting in microshoots For the production of complete plantlets and their establishment in the external environment, proper root system has to be developed in the regenerated microshoots. Roots can be induced either in vitro by transferring the microshoots onto the rooting media or ex vitro by short pulse treatment with an auxin.

106

Explanation of Figure 36 Somatic embryogenesis in C. angustifolia. A. Development of somatic embryos on MS medium supplemented with 10.0 µM 2,4-D and 1.0 µM BA, arrows represent globular stage embryoids - 2 weeks old culture. (Bar = 0.40 cm)

B. Development of cotyledonary stage somatic embryos on medium comprised of MS + 2,4-D (10.0 µM) + BA (1.0 µM) - 4 weeks old culture. (Bar = 0.38 cm)

C. Maturation of somatic embryos on the same medium along with expansion of cotylenoray leaves; arrows represent expanded cotyledonary leaves and root primordia - 6 weeks old culture. (Bar = 0.40 cm)

D. -do- (Bar = 0.41 cm)

Figure 36

A B

C D

Table 37. Effect of optimal concentration of 2,4-D with different cytokinins on somatic embryogenesis from immature green cotyledons of C. angustifolia.

PGR (M) Response Mean number of Germination (%) embryoids/explant (%) 2,4-D BA Kn 2iP

10.0 1.0 - - 90.56  1.88a 22.80  1.59a 35.33  2.90a 10.0 2.5 - - 86.06  0.58ab 18.20  1.20b 30.00  1.15ab 10.0 5.0 - - 83.43  0.34b 17.06  0.74b 26.00  2.30b 10.0 - 1.0 - 68.23  0.95d 13.23  1.43cd 12.33  1.45cd 10.0 - 2.5 - 78.03  1.59c 14.33  0.76c 15.66  2.33b 10.0 - 5.0 - 66.56  1.82d 11.40  0.45d 10.00  1.15cd 10.0 - - 1.0 52.73  1.75e 11.73  0.62cd 8.66  2.02d 10.0 - - 2.5 64.50  2.32d 13.10  0.20cd 11.33  1.76cd 10.0 - - 5.0 50.46  1.35e 11.43  0.31d 7.66  1.45d

Explanation of Figure 37 Somatic embryogenesis in C. angustifolia. A. Development of somatic embryos on MS medium supplemented with 10.0 µM 2,4-D and 5.0 µM BA, arrow represents a germinating embryoid - 3 weeks old culture. (Bar = 0.40 cm)

B. Development of somatic embryos at different stages on MS medium supplemented with 10.0 µM 2,4-D and 2.5 µM Kn - 3 weeks old culture. (Bar = 0.38 cm)

C. Development of complete plantlets from germinating embryoids on transferring to MS basal medium, arrows represent developing root - after 3 weeks of transfer. (Bar = 0.43 cm)

D. Development of complete plantlets from germinating embryoids on transferring to half strength MS medium, arrows represent developing root - after 3 weeks of transfer. (Bar = 0.55 cm)

Figure 37

C D

4.1.5.1 In vitro rooting The regenerated shoots of appropriate length (3-4 cm) were cut and transferred to rooting media for the induction of roots to produce complete plantlets. The rooting media comprised of full and half strength MS medium without any PGR was served as control. Microshoots failed to induce rooting on full strength MS medium while half strength MS medium was found to induce rooting after 14 days in 11.00 ± 2.08% cultures with 1.33 ± 0.20 roots/shoot. Augmentation of three different auxins (IAA, IBA and NAA) at various concentrations (0.5, 1.0 and 2.0 µM) enhanced the rooting percentage and helped in the development of healthy root system. Induction of roots started after 9-10 days; initially roots were thin and milky white in colour which later on developed secondary branches, became thick and dark brown to black. The half strength MS medium containing 1.0 µM IBA produced the maximum 3.23 ± 0.52 roots/shoot having 2.66 ± 0.35 cm root length in 59.00 ± 7.81% microshoots after 4 weeks of culture (Figure 38 A). Further increase in the concentration of IBA resulted in the formation of callus at the base of microshoots and thus hampered the growth of roots. The rooting percentage was reduced to 34.33 ± 3.48% on half strength MS + IBA (2.0 µM) and the number of roots/shoot was also reduced to 2.00 ± 0.26 (Figure 38 B). Among three auxins tested, IBA was found to be the best for rooting while NAA and IAA provided moderate response. An average of 2.50 ± 0.28 and 1.60 ± 0.20 roots/shoot were produced in the medium containing half strength MS + 1.0 µM NAA (Figure 38 C) and half strength MS + 1.0 µM IAA respectively (Table 38).

Addition of phloroglucinol (PG) with the optimal concentration of IBA enhanced the rooting percentage as well as the mean number of roots/shoot. PG used at different concentrations (2.5, 5.0 and 10.0 µM) facilitated the root induction within 7-8 days and the best response was obtained on half strength MS + IBA (1.0 µM) + PG (5.0 µM) where 4.80 ± 0.17 roots/shoot were obtained in 82.00 ± 6.42% microshoots having root length of 4.36 ± 0.14 cm after 4 weeks of culture (Figure 38 D & E, Table 38).

The liquid rooting medium (without agar) containing three different auxins at the same concentrations was also used for in vitro root induction. MS basal medium without any auxin (control) failed to induce rooting, while half strength MS

107

Explanation of Figure 38 In vitro rooting on agar gelled medium in C. angustifolia. A. Development of thin long root on half strength MS medium supplemented with 1.0 µM IBA - 4 weeks old culture. (Bar = 0.75 cm)

B. Development of thick short roots along with basal callusing on half strength MS medium supplemented with 2.0 µM IBA - 4 weeks old culture. (Bar = 0.67 cm)

C. Development of single, thin root along with basal callusing on half strength MS medium supplemented with 1.0 µM NAA - 4 weeks old culture. (Bar = 0.92 cm)

D. Development of long healthy and branched roots on half strength MS medium supplemented with 1.0 µM IBA and 5.0 µM PG - 4 weeks old culture. (Bar = 0.83 cm)

E. -do- (Bar = 0.89 cm)

Figure 38

A B C

D E

Table 38. Effect of different auxins and phloroglucinol on in vitro root induction in microshoots of C. angustifolia in agar gelled medium.

Treatment (M) Response Mean number of Mean root (%) roots/shoot length (cm)

MS 00.00  0.00g 0.00  0.00g 0.00  0.00g ½ MS 11.00  2.08ef 1.33  0.20ef 1.20  0.17ef ½ MS + IBA (0.5) 26.00  2.64cd 2.13  0.23cd 1.56  0.38e ½ MS + IBA (1.0) 59.00  7.81b 3.23  0.52b 2.66  0.35cd ½ MS + IBA (2.0) 34.33  3.48c 2.00  0.26cde 1.10  0.20ef ½ MS + NAA (0.5) 17.33  1.45de 1.90  0.20cdef 1.40  0.30ef ½ MS + NAA (1.0) 28.66  2.02cd 2.50  0.28c 2.43  0.23d ½ MS + NAA (2.0) 22.33  1.45cde 1.76  0.14cdef 0.96  0.14ef ½ MS + IAA (0.5) 00.00  0.00g 0.00  0.00g 0.00  0.00g ½ MS + IAA (1.0) 14.66  1.45de 1.60  0.20def 2.30  0.17d ½ MS + IAA (2.0) 10.00  1.15ef 1.16  0.12f 0.76  0.14f ½ MS + IBA (1.0) + PG (2.5) 62.66  10.10b 3.66  0.17b 3.16  0.14bc ½ MS + IBA (1.0) + PG (5.0) 82.00  6.42a 4.80  0.17a 4.36  0.14a ½ MS + IBA (1.0) + PG (10.0) 62.33  7.76b 3.80  0.11b 3.53  0.20b

induced rooting after 10 days and showed 20.33 ± 2.02% rooting response with the production of 1.60 ± 0.20 roots/shoot attaining an average root length of 1.50 ± 0.17 cm after 4 weeks. Addition of different auxins at various concentrations in liquid medium found to be significantly more responsive for root induction compared to semisolid rooting medium. Addition of IBA (1.0 µM) in the medium triggered early response as the root induction was noticed after 6-7 days of transfer. On this medium a maximum of 3.93 ± 0.29 roots/shoot, attaining an average root length of 3.00 ± 0.28 cm, were obtained after 4 weeks in 67.33 ± 2.60% microshoots (Figure 39 A). The higher concentration (2.0 µM) of IBA resulted in basal callusing which hampered the root development (Figure 39 B). The rooting efficiency of the microshoots was further enhanced when the optimal medium fortified with phloroglucinol at various concentrations (Figure 39 C). roots were induced within 4-5 days and a maximum of 5.56 ± 0.23 roots/shoot having root length of 6.23 ± 0.14 cm was obtained after 4 weeks in 87.33 ± 2.33% cultures on medium comprised of half strength MS + 1.0 µM IBA + 5.0 µM PG (Figure 39 D, Table 39). The rooting response was further tested on phytagel gelled rooting medium containing the same hormonal treatments, but the results were quite unsatisfactory as very short and stunted root clumps were obtained along with heavy basal callusing (Figure 39 E) (Data not shown).

4.1.5.2 Ex vitro rooting During in vitro rooting, yellowing and premature leaf fall was observed additionally the development of callus at the basal cut ends also hampered the induction of roots. Rooting in the external environment is an alternative method which helped to overcome these problems of in vitro rooting. Microshoots measuring 3-4 cm in length were used for ex vitro rooting through pulse treatment in different concentrations (50, 100, 150, 200, 250 and 300 µM) of IBA for about 30 min. Treated shoots were transferred to thermocol cups containing sterile soilrite and covered with polythene bags to ensure high relative humidity. Shoots were irrigated with quarter strength MS salt solution for 2 weeks followed by tap water. Polythene bags were removed gradually upon emergence of new leaves in order to acclimatize the plants. After 6 weeks of treatment, shoots were taken out from the soilrite and healthy roots with secondary branches were observed from the cut ends of the shoots (Figure 40). Through ex vitro rooting

108

Explanation of Figure 39 In vitro rooting in liquid and phytagel gelled medium in C. angustifolia. A. Development of thin delicate roots on half strength MS medium supplemented with 1.0 µM IBA - 4 weeks old culture. (Bar = 1.0 cm)

B. Development of thick roots along with basal callusing on half strength MS medium supplemented with 2.0 µM IBA - 4 weeks old culture. (Bar = 1.04 cm)

C. Development of short thick roots on half strength MS medium supplemented with 1.0 µM IBA and 2.5 µM PG - 4 weeks old culture. (Bar = 1.13 cm)

D. Development of long healthy roots with secondary branches on half strength MS medium supplemented with 1.0 µM IBA and 5.0 µM PG - 4 weeks old culture. (Bar = 0.83 cm)

E. Development of short stunted roots along with basal callusing on half strength MS medium gelled with 0.25% phytagel and supplemented with 1.0 µM IBA and 5.0 µM PG - 4 weeks old culture. (Bar = 0.86 cm)

Figure 39

A B C

D E

Table 39. Effect of different auxins and phloroglucinol on in vitro root induction in microshoots of C. angustifolia in liquid medium.

Treatment (M) Response Mean number of Mean root (%) roots/shoot length (cm)

MS 00.00  0.00g 0.00  0.00h 0.00  0.00d ½ MS 20.33  2.02fg 1.60  0.20efg 1.50  0.17c ½ MS + IBA (0.5) 40.33  2.60e 2.73  0.14cd 2.33  0.44b ½ MS + IBA (1.0) 67.33  2.60c 3.93  0.29b 3.00  0.28b ½ MS + IBA (2.0) 51.66  2.02d 2.13  0.23de 1.30  0.20c ½ MS + NAA (0.5) 25.66  2.33fg 2.16  0.24de 1.60  0.26c ½ MS + NAA (1.0) 31.66  2.02f 2.63  0.24cd 2.63  0.23b ½ MS + NAA (2.0) 21.33  2.96gh 1.90  0.20e 1.26  0.14c ½ MS + IAA (0.5) 12.33  1.45i 1.23  0.14fg 1.23  0.14c ½ MS + IAA (1.0) 17.66  1.45gh 1.76  0.14ef 2.53  0.20b ½ MS + IAA (2.0) 14.00  3.05gh 1.06  0.66g 1.03  0.14c ½ MS + IBA (1.0) + PG (2.5) 76.00  2.30b 5.26  0.14a 5.90  0.20a ½ MS + IBA (1.0) + PG (5.0) 87.33  2.33a 5.56  0.23a 6.23  0.14a ½ MS + IBA (1.0) + PG (10.0) 65.00  2.64c 4.40  0.30b 5.56  0.23a

Response (%) 100 a A

b b b 60

c 40

Response (%) 20

d 0

0 50 100 150 200 250 300 350

IBA µM

Mean number of roots/shoot Mean root length (cm) 7 7 B a 6 6 a 5 5

b 4 b b 4 b c 3 c 3 c c 2 2

1 1

0 0 50 100 150 200 250 300

IBA (µM)

Figure 40. Effect of pulse treatment of different concentrations of IBA for half an hour to induce ex vitro rooting in microshoots of C. angustifolia. (A) Line represents percentage response (%) for root induction. (B) Bars represent average number of roots/shoot and average root length (cm).

technique, leaf abscission and basal callusing has been minimized. The best response (90.33 ± 2.60%) with 4.83 ± 0.24 roots/shoot having an average root length of 5.80 ± 0.23 cm was obtained with the microshoots dipped in 200 µM of IBA for about half an hour (Figure 41 A- E).

4.1.6 Hardening and acclimatization Hardening and acclimatization of regenerated plantlets is the most crucial and important phase of plant tissue culture. Regenerated plantlets with well- developed root system were successfully hardened off under controlled conditions in three different planting substrates (garden soil, soilrite and vermiculite). Hardening of plantlets was done by the procedure described in the materials and methods. Out of three planting substrates used, maximum survival percentage (89.33 ± 2.33%) was observed in soilrite. However, plantlets transferred to vermiculite provided moderate response (64.66 ± 2.60%), while, garden soil showed minimum survival rate (54.66 ± 2.90%) (Figure 42 & 43 A-D).

About 90% plantlets survived after transferring to the field conditions and the regenerated plantlets grew well with no detectable morphological variations when compared with the in vivo grown plants (Figure 44).

4.1.7 Synthetic seeds The synseeds or artificial seeds of C. angustifolia were prepared by encapsulation of nodal segments excised from 1 month old microshoots to assess the regeneration potential of the explant under various culture conditions.

4.1.7.1 Effect of different concentrations of sodium alginate on beads formation For the production of uniform and viable synseeds, various concentrations of sodium alginate (Na2-alginate) (1, 2, 3, 4 and 5%) were tested with 100 mM calcium chloride (CaCl2·2H2O) in different combinations. The texture of beads is highly influenced by different concentrations of gelling matrix and complexing agent. Table 40 depicted the texture and rate of conversion of beads into shoots at different concentrations of Na2-alginate with 100 mM of CaCl2·2H2O. Very soft and friable beads were produced at lower concentrations (1 and 2%) of gelling matrix which were difficult to handle and no bead was converted into microshoot.

109

Explanation of Figure 41 Ex vitro rooting in C. angustifolia. A. Development of single thin root through pulse treatment of 30 min with 100 µM IBA - after 4 weeks of treatment. (Bar = 1.0 cm)

B. Development of thin short roots through pulse treatment of 30 min with 150 µM IBA - after 4 weeks of treatment. (Bar = 1.0 cm)

C. Development of long healthy roots through pulse treatment of 30 min with 200 µM IBA - after 4 weeks of treatment. (Bar = 1.2 cm)

D. Development of thin highly branched roots through pulse treatment of 30 min with 250 µM IBA - after 4 weeks of treatment. (Bar = 1.0 cm)

E. Development of thick, highly branched long roots through pulse treatment of 30 min with 200 µM IBA - after 6 weeks of treatment. (Bar = 0.90 cm)

Figure 41

A B C

D E

Garden soil Soilrite Vermiculite 100 a

60 c

(%) percentage Survival 20

0 Garden soil Soilrite Vermiculite

Planting substrate

Figure 42. Effect of different planting substrates on survival percentage (%) of regenerated plantlets of C. angustifolia during ex vitro acclimatization.

Explanation of Figure 43 Hardening and acclimatization of regenerated plantlets in C. angustifolia. A. Successfully acclimatized in vitro rooted plantlets in sterile soilrite - after 3 weeks of transfer. (Bar = 1.33 cm)

B. Ex vitro rooted plantlets with simultaneous acclimatization in sterile soilrite - after 6 weeks of pulse treatment. (Bar = 2.02 cm)

C. In vitro regenerated plantlet in soil - after 1 week of transfer from soilrite. (Bar = 2.50 cm)

D. Regenerated plantlet with normal inflorescence development – after 1 month of transfer from soilrite. (Bar = 6.42 cm)

Figure 43

C D

Explanation of Figure 44 In vitro raised 2 months old plants of C. angustifolia in field conditions with normal flowering and fruit development.

Figure 44

The optimal combination for the production of uniform, easy to handle, firm and clear beads was found to be 3% Na2-alginate with 100 mM CaCl2·2H2O showing 74.06 ± 1.56% conversion response. The higher concentrations (4 and 5%) resulted in the formation of hard beads which showed poor conversion response i.e. 57.40 ± 1.59% and 32.90 ± 1.57% at 4% and 5% Na2-alginate respectively (Table 40).

4.1.7.2 Effect of different concentrations of calcium chloride on beads formation Different concentrations (25, 50, 75, 100 and 200 mM) of complexing agent

(CaCl2·2H2O) were also tested with 3% Na2-alginate to produce synseeds and cultured on MS medium. 25 mM and 50 mM of CaCl2·2H2O produced very soft and fragile beads which were difficult to handle and showed zero conversion response. 75 mM CaCl2·2H2O with 3% Na2-alginate exhibited 60.50 ± 1.21% conversion response, however, the beads were soft. The uniform and ideal synseeds with maximum conversion response of 74.06 ± 1.56% were produced with 100 mM CaCl2·2H2O and 3% gelling matrix. Increasing the concentration of complexing agent to 200 mM resulted in the production of hard beads which showed 47.33 ± 1.64% conversion response (Table 41).

4.1.7.3 Effect of PGRs on conversion of synthetic seeds into plantlets

The ideal beads produced by encapsulating NS in 3% Na2-alginate and 100 mM

CaCl2·2H2O were cultured on MS basal medium without any PGR or with various concentrations (1.0, 2.5, 5.0 and 10.0 µM) of BA either singly or in combination of NAA (0.2, 0.4 and 0.6 µM). The MS basal medium showed 74.06 ± 1.56% conversion response and the regeneration of shoots occurred after 3 weeks of culture (Figure 45 A & B). Addition of BA enhanced the regeneration potential of the beads and the shoots emerged out within 2 weeks of transfer to the regeneration medium. 4-5 shoots/beads were produced in the medium containing 2.5 µM of BA with 82.63 ± 1.22% conversion response after 6 weeks of culture (Figure 45 C). The regenerated microshoots were failed to develop into complete plantlets on the same medium. Addition of NAA with optimal concentration (2.5 µM) of BA did not help in the formation of roots from the microshoots (Figure 46 A). However, the medium containing MS + BA (2.5 µM) +

110

Table 40. Effect of different concentrations of sodium alginate with 100 mM calcium chloride on encapsulated nodal segments of C. angustifolia.

Sodium alginate Conversion response Texture of beads (% w/v) (%)

1.0 00.00  0.00d very soft and fragile beads 2.0 00.00  0.00d very soft and fragile beads 3.0 74.06  1.56a soft and uniform beads 4.0 57.40  1.59b hard beads 5.0 32.90  1.57c hard beads

Table 41. Effect of different concentrations of calcium chloride with 3% sodium alginate on encapsulated nodal segments of C. angustifolia.

CaCl2·2H2O Conversion response Texture of beads (mM) (%)

25 00.00  0.00d very soft and fragile beads 50 00.00  0.00d very soft and fragile beads 75 60.50  1.21b soft and fragile beads 100 74.06  1.56a soft and uniform beads 200 47.33  1.64c hard beads

Explanation of Figure 45 Synthetic seed production in C. angustifolia. A. Uniform synseeds prepared through encapsulation of aseptic nodal segments with 3% sodium alginate and 100 mM calcium chloride on MS basal medium - 1 day old culture. (Bar = 1.03 cm)

B. Germination of synseeds on MS basal medium - 3 weeks old culture. (Bar = 0.85 cm)

C. Germination of synseeds on MS medium supplemented with 2.5 µM BA - 3 weeks old culture. (Bar = 1.57 cm)

Figure 45

A B

Explanation of Figure 46 Synthetic seed production in C. angustifolia. A. Germination of synseeds along with development of multiple shoots on MS medium supplemented with 2.5 µM BA and 0.2 µM NAA - 2 weeks old culture. (Bar = 0.34 cm)

B. Germination of synseeds along with development of multiple shoots on MS medium supplemented with 2.5 µM BA and 0.4 µM NAA - 2 weeks old culture. (Bar = 0.36 cm)

C. -do- 4 weeks old culture. (Bar = 0.43 cm)

Figure 46

B C

NAA (0.4 µM) showed maximum conversion (94.06 ± 1.56%) of beads into microshoots (Figure 46 B & C, Table 42). The regenerated shoots after attaining suitable length were isolated and transferred to optimized rooting medium comprised of half strength MS + IBA (1.0 µM) + PG (5.0 µM) for in vitro root induction and the development of complete plantlets. Ex vitro root induction by pulse treatment with IBA (200 µM) also helped in the development of roots.

4.1.7.4 Low temperature storage To evaluate the reproducibility of encapsulated NS, the beads were kept at 4ºC for different time periods (0, 1, 2, 4, 6 and 8 weeks). The non-encapsulated NS were also stored at the same temperature and time period. The encapsulated and non-encapsulated NS showed maximum conversion into microshoots at 0 days of storage. As long as the time period was increased the conversion response of the explants was reduced. Encapsulated NS explants showed a linear decrease in the conversion response from 94.06 ± 1.56% to 72.30 ± 1.21% at 0 days and 4 weeks of storage at 4ºC respectively. The non- encapsulated NS showed drastic decrease in the regeneration potential and after 4 weeks of storage only 33.33 ± 1.35% explants exhibited shoot regeneration on MS medium supplemented with BA (2.5 µM) + NAA (0.4 µM). However, further increase in the storage duration decreased the regeneration potential of the beads and after 8 weeks of storage 43.90 ± 1.49% beads showed conversion into microshoots (Table 43).

4.1.7.5 Ex vitro germination of synthetic seeds Encapsulated NS were sown into the sterilized soilrite for ex vitro conversion of beads into the plantlets and soilrite was moistened with quarter strength MS salt solutions. Sowing of synthetic seed directly into the soilrite facilitated the development of roots and production of complete plantlets with 20% conversion rate after 6 weeks. The regenerated plantlets showed 2-3 roots/shoot.

4.1.8 Physiological studies Physiological studies were carried out in in vitro regenerated plantlets to estimate the content of photosynthetic pigments (Chlorophyll a, b, total chlorophyll and carotenoids) as well as the rate of photosynthesis (PN ratio) during different acclimatization periods.

111 Table 42. Effect of different plant growth regulators on encapsulated nodal segments of C. angustifolia.

PGRs Conversion response (µM) (%)

MS 74.06  1.56cd MS + BA (1.0) 77.60  1.28c MS + BA (2.5) 82.63  1.22b MS + BA (5.0) 72.26  1.12d MS + BA (10.0) 57.56  1.40f MS + BA (2.5) + NAA (0.2) 86.50  1.53b MS + BA (2.5) + NAA (0.4) 94.06  1.56a MS + BA (2.5) + NAA (0.6) 67.90  1.53e

Table 43. Effect of storage at 4ºC for different time periods on conversion of encapsulated and non-encapsulated nodal segments of C. angustifolia on MS medium containing BA (2.5 µM) + NAA (0.4 µM).

Storage period Conversion response Conversion response (weeks) of encapsulated NS of non-encapsulated NS (%) (%)

0 94.06  1.56a 96.13  1.38a 1.0 89.40  1.53a 60.60  1.34b 2.0 82.66  1.44b 43.56  1.61c 4.0 72.30  1.21c 33.33  1.75d 6.0 60.70  1.65d 22.53  1.35e 8.0 43.90  1.79e 12.80  1.32f

4.1.8.1 Chlorophyll a, b and total chlorophyll content During different periods (0, 1, 2, 3 and 4 weeks) of acclimatization, it was observed that the contents of chlorophyll a, b as well as total chlorophyll initially decreased and later on increased linearly with an increase in acclimatization period. During first week of transfer from in vitro to ex vitro conditions, a decrease in chlorophyll a (0.44 to 0.29 mg/g), chlorophyll b (0.16 to 0.15 mg/g) and the total chlorophyll (0.60 to 0.45 mg/g) was noticed. After second week of acclimatization a linear increase in pigments content was observed (Figure 47 & 48). At the end of fourth week the content of chlorophyll a (1.04 mg/g), chlorophyll b (0.35 mg/g) and the total chlorophyll (1.31 mg/g) got stabilized with a very gradual increase which suggested that plants are now fully adapted to the external environment.

4.1.8.2 Carotenoids content Similar to the chlorophyll pigments, carotenoids also showed the same trend during different acclimatization periods. Initially the carotenoids content was reduced to 0.10 mg/g from 0.17 mg/g in the first week of transfer to the ex vitro conditions. Later on the content was increased to 0.29 mg/g after second week and by the end of fourth week the carotenoids content was recorded 0.52 mg/g (Figure 49).

4.1.8.3 Net photosynthetic rate (PN ratio)

Net photosynthetic rate is the estimation of amount of CO2 absorbed by per unit area of the plant per second. It was estimated during different periods of acclimatization of regenerated plantlets. Initially, when plantlets were transferred to the external environment, a decrease in the amount of CO2 absorbed per unit -2 -1 area per second (2.50 to 1.20 µmol CO2 m s ) was recorded during first week. As soon as the plantlets get adapted to the external environment, the net -2 -1 photosynthetic rate increased linearly and 8.16 µmol CO2 m s and 9.23 µmol -2 -1 CO2 m s was recorded by the end of third and fourth week of acclimatization respectively (Figure 50).

112

Chlorophyll a Chlorophyll b 1.2 a 1.0 b 0.8

c 0.6 d 0.4 a e a c

Chlorophylls content (mg/g) Chlorophylls content b 0.2 b

0.0 0 1 2 3 4 Acclimatization period (weeks)

Figure 47. Change in chlorophyll a and b contents (mg/l) of in vitro raised C. angustifolia plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

Total chlorophyll (mg/g) 1.6

a 1.4

a 1.2

1.0 b

0.8 c

0.6

Chlorophyll (mg/g) content c

0.4

0.2 0 1 2 3 4 Acclimatization period (weeks)

Figure 48. Change in total chlorophyll content (mg/l) of in vitro raised C. angustifolia plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

Carotenoids 0.6 a

0.5

0.4 b

b 0.3

0.2 c

Carotenoids (mg/g) content c 0.1

0.0 0 1 2 3 4 Acclimatization period (weeks)

Figure 49. Change in carotenoid content (mg/l) of in vitro raised C. angustifolia plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

Net photostnthetic rate 10 a

-1 ) s 8

-2

c 4

2 e

Net photosynthetic rate (µmolCO 0 0 1 2 3 4 Acclimatization period (weeks) Figure 50. Change in Net Photosynthetic rate (μmol CO2 m-2 s-1) of in vitro raised C. angustifolia plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

4.1.9 Histological studies The mode of shoot organogenesis via direct regeneration through cotyledonary node (CN) or callus formation through cotyledonary leaf (CL) and root (R) explants of C. angustifolia was also studied through histological sections and confirmed the origin of shoot morphogenesis.

The regenerating tissue obtained through CN explant cultured on MS + BA (5.0 µM) was analysed at various stages of development. The longitudinal section of 2 weeks old CN explant cultured on BA (5.0 µM), as shown in Figure 51 A & B which is the left (A) and right (R) portion of histological section of the same explant, revealed the presence of large number of organized meristematic zones which actually took part in the differentiation of crown of shoot buds around the node junction of the explant, proving the direct origin of the shoot buds. These meristematic regions after several divisions of cells transformed into shoot buds having prominent apical dome flanked by leaf primordia (Figure 51 C) and later on developed into complete shoots. The CN explants cultured on various concentrations of TDZ were also fixed and analysed under histological sections. The regenerating tissue fixed after 4 weeks of culture on TDZ (5.0 µM) supplemented MS medium, showed deformed and abnormal shoot buds development. Such abnormal shoot buds were having deformed apical dome with suppressed growth of leaf primordial and most of the shoot buds possessed single leaf primordium which was deformed in shape (Figure 52 A & B). The higher concentration of TDZ (10.0 µM) also produced abnormal shoot buds showing swollen apical dome with multiple, deformed leaf primordia (Figure 52 C).

The histological analysis of CL derived callus tissue, produced on 5.0 µM 2,4-D, at various developmental stages revealed the organization of several meristematic zones (meristemoids) within the callus tissue. These meristemoids were constituted of densely cytoplasmic, isodiametric cells with prominent nuclei. Majority of meristemoids developed at the peripheral region of the calli (Figure 53 A). However, several deep seated meristemoids were also observed which were embedded in callus tissue and showed distinct demarcation from parenchymatous cells of callus (Figure 53 B). The peripheral as well as

113

Explanation of Figure 51 Histological sections showing differentiation pattern in cotyledonary node (CN) culture of C. angustifolia.

A. LS of CN explant showing differentiation of multiple meristematic zones (arrows) in the left (L) swollen portion of explant on MS + BA (5.0 µM). (Bar = 100 µm)

B. LS of the right (R) portion of the same explant showing differentiation of meristematic zone (arrows). (Bar = 100 µm)

C. A developed shoot bud with well differentiated leaf primordia (lp) and apical dome (ap). (Bar = 120 µm)

Figure 51

lp lp ap

Explanation of Figure 52 Histological sections showing differentiation pattern in CN explants of C. angustifolia cultured on TDZ supplemented medium.

A. Differentiation of a deformed shoot bud having abnormal apical dome (ap) and single leaf primordium (lp) on MS medium supplemented with 5.0 µM TDZ. (Bar = 120 µm) B. -do- (Bar = 120 µm) C. An abnormal shoot bud showing deformed and swollen structure of apical dome (ap) and leaf primordial on MS medium supplemented with 10.0 µM TDZ. (Bar = 60 µm)

Figure 52

lp lp ap

A B

lp ap

Explanation of Figure 53 Histological sections showing differentiation pattern in cotyledonary leaf (CL) derived callus of C. angustifolia.

A. Organization of meristematic zones (meristemoids) in the callus tissue (arrow head) which later transformed into shoot buds. (Bar = 140 μm) B. Well organised meristemoids (arrow head) embedded in callus tissue. (Bar = 50 μm) C. A developing shoot bud at the peripheral surface showing apical dome (ap). (Bar = 50 μm) D. Development of a deep seated shoot bud in callus tissue showing apical dome (ap) surrounded by well-organized leaf primordia (lp). (Bar = 120 μm)

Figure 53

A B

lp ap ap lp

C D

embedded meristemoids developed into shoot buds having prominent apical dome surrounded by leaf primordia (Figure 53 C & D) on transferring to the regeneration medium comprised of MS + BA (5.0 µM) + NAA (0.4 µM) which later transformed into healthy shoots. The histological sections of root derived callus tissue under the microphotograph revealed a very clear and distinctive feature of morphogenesis. It was well demonstrated that the meristematic zone enlarged in size because of fast meristematic activity leading to the formation of nodular structures (Figure 54 A-C). After 2 weeks of nodule formation, the meristematic sphere started to differentiate into organoids, initially it appears as dome like structure at several places around the sphere, thereafter in the subsequent weeks the organoid regions flanked with small primordia leading to the differentiation of shoot buds (Figure 54 D). Further it was observed that the meristematic zone first enlarged in size, became determinant at several places for polarity and then started to differentiate into multiple shoot buds (Figure 54 E & F).

The embryogenic callus produced through immature cotyledon explant on various hormonal supplements was fixed at different stages of embryoids differentiation and revealed the mode of differentiation under histological sections. The embryogenic callus produced on MS + 2,4-D (5.0 µM) showed the differentiation of globular embryoids with well-marked boundaries and distinction from the callus tissue (Figure 55 A). The globular embryoids grew in size and became pear shaped (Figure 55 B). The heart shaped embryoids were also clearly observed under microphotographs having well differentiated boundary with apical notch (Figure 55 C & D). The mature and cotyledonary staged embryoids were distinctly present in the embryogenic callus obtained on MS + 2,4-D (10.0 µM) and showed well differentiated cotyledonary leaves under histological sections (Figure 55 E & F).

114

Explanation of Figure 54 Histological sections showing differentiation pattern in root derived callus of C. angustifolia.

A. Organization of meristemoids (m) in callus tissue and differentiation of shoot buds (db). (Bar = 70 µm) B. Enlarged view of developing buds (db) with well-marked boundaries. (Bar = 50 µm) C. Large number of meristemoids (m) and developing buds (db) differentiated in the subperipheral region of the callus. (Bar = 110 µm) D. Deep-seated shoot bud (sb) differentiation flanked with leaf primordia (l). (Bar = 70 µm) E. Differentiation of shoot buds (sb) from peripheral callus tissue. (Bar = 100 µm) F. Enlarged view of differentiation of shoot buds (sb) at the peripheral surface. (Bar = 120 µm)

Figure 54

m m db db

m db m m

C A B C

sb sb sb sb lp

F db D E F

Explanation of Figure 55 Histological sections showing differentiation of somatic embryos of C. angustifolia through immature cotyledon at different developmental stages.

A. Formation of globular embryos at the peripheral surface of callus tissue on MS + 2,4-D (5.0 µM). (Bar = 50 µm) B. Conversion of globular embryos to pear shaped at later satges of development on the same medium. (Bar = 30 µm) C. Heart shaped embryos showing cotyledonay initials (arrows) and differentiating apical meristem region (arrow head) with suspensor like structure (s) on MS + 2,4-D (5.0 µM). (Bar = 40 µm) D. Heart shaped embryo at later stage of development. (Bar = 100 µm) E. Formation of mature, cotyledonary satged somatic embryos on MS + 2,4-D (10.0 µM). (Bar = 400 µm) F. -do- (Bar = 400 µm)

Figure 55

A B

C D

E F

4.2 C. sophera Linn. 4.2.1 In vitro seed germination The sterilized seeds of C. sophera (Figure 56 A & B) were inoculated on full and half strength MS media with or without GA3 under controlled conditions. Full and half strength MS medium without GA3 served as control treatment. Half strength MS medium provided better response than full strength MS medium (Table 44). A maximum of 90.33 ± 1.45% seeds germinated on half strength MS medium without GA3 and required 6.56 ± 0.23 days for germination while full strength MS medium showed 62.33 ± 1.45% seed germination which took 10.90 ± 0.20 days for initiation of germination. Addition of GA3 at various concentrations (0.5, 1.0 and 2.5 µM) further enhanced the rate of seed germination and reduced the minimum number of days required for the start of germination of seeds.

Application of 1.0 µM GA3 in half strength MS showed germination within 4.46 ± 0.14 days of incubation with the maximum 99.33 ± 0.66% seed germination after 4 weeks of inoculation (Figure 57). While full strength MS medium having the same concentration of GA3 exhibited 72.33 ± 1.45% seed germination in 7.13 ± 0.23 days for the start of germination. Further increase in the concentration (2.0

µM) of GA3 reduced the rate of germination (91.66 ± 1.45%) and increased the number of days (6.53 ± 0.24) for germination on half strength MS medium (Table

44). Therefore half strength MS + GA3 (1.0 µM) proved to be the optimal medium for seed germination in C. sophera and was used throughout the study for germination of seeds to collect different explants.

4.2.2 Direct shoot regeneration 4.2.2.1 Cotyledonary node (CN) explant 4.2.2.1.1 Effect of explant age on multiple shoot regeneration To evaluate the efficiency of shoot regeneration, cotyledonary node explants excised from seedlings of different age group (14, 21 and 28 days old) were cultured on MS medium containing various concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA. MS medium without BA served as control treatment and all the three different aged explants failed to respond on hormone free MS medium and died within 2 weeks of inoculation. The CN explants excised from 21 days old seedlings produced a maximum of 12.43 ± 0.29 shoots/explant on MS + BA (5.0 µM) medium after 6 weeks of culture in 78.66 ± 2.33% cultures. On this medium

115

Explanation of Figure 56 Source of explant for shoot organogenesis in C. sophera. A. Mature and dried pods. (Bar = 0.88 cm)

B. Mature seeds. (Bar = 0.85 cm)

Figure 56

Explanation of Figure 57 In vitro seed germination in C. sophera. Aseptic seedlings (30 days old) on half strength MS medium supplemented with 1.0 µM GA3. (Bar = 0.72 cm)

Figure 57

Table 44. Effect of GA3 on seed germination of C. sophera in MS basal and half strength MS media.

Germination Mean number of Germination Medium days to germination rate (%)

MS 10.90  0.20a 62.33  1.45ef c e MS + GA3 (0.5 µM) 8.90  0.20 66.00  1.15 d d MS + GA3 (1.0 µM) 7.13  0.23 72.33  1.45 b f MS + GA3 (2.5 µM) 9.56  0.23 59.33  1.76

½ MS 6.56  0.23d 90.33  1.45c e ab ½ MS + GA3 (0.5 µM) 5.36  2.90 96.00  1.15 f a ½ MS + GA3 (1.0 µM) 4.46  0.14 99.33  0.66 d c ½ MS + GA3 (2.5 µM) 6.53  0.24 91.66  1.45

induction of shoot buds started after 9-10 days of culture. On the same composition of medium explants isolated from 14 days old seedlings exhibited 62.33 ± 1.45% response producing 9.06 ± 0.23 shoots/explant while, explants obtained from 28 days old seedlings produced only 6.56 ± 0.23 shoots/explant in 56.33 ± 2.33% cultures after 6 weeks of inoculation (Table 45).

4.2.2.1.2 Effect of cytokinins on multiple shoot regeneration The best responsive CN explants excised from 21 days old seedlings were cultured on MS medium containing three different cytokinins (BA, Kn and 2iP) for multiple shoot regeneration through direct organogenesis. MS medium without cytokinins (control) did not stimulate any morphogenic response and the explants died within two weeks of culture. However, supplementation of various cytokinins at different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) facilitated induction of multiple shoots from CN explants. Among all the three cytokinins tested, BA was proved to be the best and the number of shoots increased with an increase in the concentration up to 5.0 µM, beyond that a decline in percent response as well as number of shoots was recorded (Table 46). Enlargement and swelling of the explants was observed within 4-5 days of incubation followed by emergence of green protuberances from the swelled region after 9-10 days (Figure 58 A). At 5.0 µM BA, a maximum of 12.43 ± 0.29 shoots/explant attaining an average shoot length of 4.23 ± 0.26 cm was obtained in maximum 78.66 ± 2.33% cultures after 6 weeks of inoculation (Figure 58 B). Higher concentrations of BA (7.5 and 10.0 µM) found to be inhibitory for shoot regeneration because of huge basal callusing which hampered the growth and development of new shoots. At 10.0 µM of BA, a decrease in the percent response (64.00 ± 3.05%) was noticed and the number of shoots was reduced to 5.90 ± 0.20 shoots/explant after 6 weeks of culture.

In contrast to BA, Kn and 2iP were effective at higher concentration i.e. 7.5 µM where induction of shoot buds began after 14 days of inoculation in both Kn and 2iP supplemented media. Shoots were not as healthy as that of BA supplemented media and were having small leaves with thin stem. The lower concentration (1.0 µM) of both Kn and 2iP failed to provide any response However as the concentration of cytokinins was further increased, a linear increase in the number of shoots was observed up to an optimal level

116

Table 45. Effect of age of cotyledonary nodes on direct shoot regeneration in C. sophera cultured on MS medium containing different concentrations of BA.

BA (µM) 14 days old explant 21 days old explant 28 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00e 0.00  0.00e 00.00  0.00e 00.00  0.00d 00.00  0.00e 0.00  0.00e 1.0 45.66  3.48d 3.30  0.20c 55.66  2.33d 4.66  0.20c 35.00  2.88d 3.20  0.17d 2.5 54.33  2.33b 5.23  0.14b 67.33  2.33c 8.83  0.32b 48.33  2.90bc 3.96  0.14bc 5.0 62.33  1.45a 9.06  0.23a 78.66  2.33a 12.43  0.29a 56.33  2.33a 6.56  0.23a 7.5 52.66  1.76bc 5.80  0.20b 72.66  1.45b 9.76  0.43b 50.00  2.86ab 4.56  0.23b 10.0 47.33  2.02cd 3.93  0.23d 64.00  3.05c 5.90  0.20c 42.33  1.45c 3.66  0.26cd

Table 46. Effect of different cytokinins on direct shoot regeneration from cotyledonary nodes of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 55.66  2.33ef 4.66  0.20f 3.00  0.11cde 2.5 - - 67.33  2.33bc 8.83  1.32c 3.46  0.14bc 5.0 - - 78.66  2.33a 12.43  0.29a 4.23  0.26a 7.5 - - 72.66  1.45b 9.76  0.43b 3.90  0.20ab 10.0 - - 64.00  3.05cd 5.90  0.20de 3.26  0.14cd - 1.0 - 00.00  0.00l 0.00  0.00j 0.00  0.00g - 2.5 - 40.66  2.33ij 3.63  0.23g 2.60  0.20ef - 5.0 - 44.33  2.96hi 5.63  0.23e 3.00  0.11cde - 7.5 - 60.33  1.45de 6.56  0.34d 3.56  0.29bc - 10.0 - 52.33  1.45fg 4.43  0.23f 3.23  0.14cd - - 1.0 00.00  0.00l 0.00  0.00j 0.00  0.00f - - 2.5 33.00  2.08k 2.06  0.23i 2.43  0.23f - - 5.0 36.33  2.02jk 2.90  0.20h 2.76  0.14def - - 7.5 47.66  1.45gh 4.23  0.14fg 3.23  0.14cd - - 10.0 37.66  1.45k 2.53  0.20hi 2.73  0.14def

(7.5 µM) (Table 46). The MS medium containing 7.5 µM of Kn produced an average of 6.56 ± 0.34 shoots/explant with a regeneration potential of 60.33 ± 1.45% (Figure 58 C). But, 2iP at the same concentration yielded 4.23 ± 0.14 shoots/explant in 47.66 ± 1.45% cultures after 6 weeks of inoculation (Figure 58 D). Beyond the optimal concentration (7.5 µM) a decrease in the response as well as number of shoots was recorded due to basal callusing (Table 46). To check the growth of basal callus, the cultures were frequently transferred onto the fresh medium of same composition after removing the callus mass after every 2 weeks.

4.2.2.1.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration The optimal concentrations i.e. 5.0 µM of BA and 7.5 µM of Kn and 2iP were also tested with three auxins to evaluate the synergistic effect of cytokinin–auxin combinations. Among various combinations, BA + NAA combination treatments provided best response (Figure 59 A). Induction of shoot buds took place within 8 days of incubation with slight swelling of the explant on MS + BA (5.0 µM) + NAA (1.0 µM) and yielded a maximum 19.50 ± 0.51 shoots/explant in 86.00 ± 2.08% cultures with 5.23 ± 0.14 cm shoot length after 6 weeks of inoculation (Figure 59 B). However, the addition of higher concentration (2.0 µM) of NAA resulted in callus formation and thereby decline in mean number of shoots/explant (13.93 ± 0.23) as well as in percent response (80.33 ± 1.45%) was recorded. In other combination treatments, BA + IBA combination was better than BA + IAA. The medium comprised of MS with optimal concentration of BA (5.0 µM) and IBA (1.0 µM) exhibited 83.00 ± 1.15% response producing an average of 14.83 ± 0.37 shoots/explant, while IAA at the same concentration provided 13.56 ± 0.23 shoots/explant with 77.33 ± 1.45% response after 6 weeks of culture (Table 47).

The combinations of Kn + NAA at different concentrations provided better response than 2iP + NAA at similar concentrations. The explants cultured on MS medium augmented with Kn (7.5 µM) + NAA (0.5 µM) regenerated an average of 8.20 ± 0.17 shoots/explant with a percent response of 64.33 ± 2.33% after 6 weeks of culture. The regeneration potential of the explant further enhanced

117

Explanation of Figure 58 In vitro morphogenic responses through cotyledonary nodes of C. sophera. A. Induction of multiple shoots on MS medium containing 2.5 µM BA - 3 weeks old culture. (Bar = 0.64 cm) B. Induction of multiple shoots on MS medium supplemented with 5.0 µM BA - 3 weeks old culture. (Bar = 0.60 cm) C. Induction of multiple shoots on MS medium comprised of 7.5 µM Kn - 3 weeks old culture. (Bar = 0.55 cm) D. Induction of multiple shoots on MS medium supplemented with 7.5 µM 2iP - 3 weeks old culture. (Bar = 0.64 cm)

Figure 58

A B

C D

Explanation of Figure 59 In vitro morphogenic responses through cotyledonary nodes of C. sophera. A. Multiplication and proliferation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (0.5 µM) - 6 weeks old culture. (Bar = 0.83 cm)

B. Multiplication and proliferation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.78 cm)

C. Multiple shoots production on MS medium containing 7.5 µM Kn and 1.0 µM NAA - 6 weeks old culture. (Bar = 0.82 cm)

D. Multiple shoots production on MS medium containing 7.5 µM 2iP and 1.0 µM NAA - 6 weeks old culture. (Bar = 0.81 cm)

Figure 59

A B

C D

Table 47. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from cotyledonary nodes of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.5 - - 82.33  1.45abc 15.26  0.17b 4.76  0.14ab 5.0 1.0 - - 86.00  2.08a 19.50  0.51a 5.23  0.14a 5.0 2.0 - - 80.33  1.45bcd 13.93  0.23c 4.26  0.14c 5.0 - 0.5 - 79.33  1.76bcd 12.90  0.20de 4.23  0.14c 5.0 - 1.0 - 83.00  1.15ab 14.83  0.37b 4.96  0.14ab 5.0 - 2.0 - 77.66  1.45cd 12.53  0.20e 4.10  0.20c 5.0 - - 0.5 75.66  1.76d 12.23  0.14ef 4.00  0.11c 5.0 - - 1.0 77.33  1.45cd 13.56  0.23cd 4.50  0.17bc 5.0 - - 2.0 75.00  1.73d 11.46  0.31f 4.03  0.14c

(72.33 ± 1.45%) at Kn (7.5 µM) + NAA (1.0 µM) where induction of multiple shoots took place after 11 days and a maximum of 10.90 ± 0.20 shoots/explant having an average shoot length of 4.76 ± 0.14 cm were harvested after same incubation period (Figure 59 C). On further increasing the concentration of NAA a decrease in response as well as in number of shoots was observed (Table 48).

Similarly, 2iP also provided good response with NAA where a maximum of 7.56 ± 0.23 shoots/explant having average shoot length of 4.23 ± 0.14 cm with 54.90 ± 0.20% response was recorded at MS + 2iP (7.5 µM) + NAA (1.0 µM) (Figure 59 D). On this medium differentiation of shoot buds occurred after 14 days of culture (Table 49).

4.2.2.1.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration The CN explants were cultured on MS medium containing different concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) of TDZ to investigate the effect of TDZ on shoot multiplication. Initial response was noticed by swelling and enlargement of the explant tissue in TDZ supplemented media within 4-5 days of inoculation. Differentiation of multiple shoot buds occurred after 10-12 days, which grew in size and later on developed into healthy shoots (Figure 60 A). Multiple shoots were produced in all the concentrations of TDZ and their number increased with an increase in the concentration from 0.1-2.5 μM. However beyond 2.5 μM of TDZ a sudden decline in explant response was noticed which resulted in decreased the number of shoots per explant as well as shoot length. The MS medium supplemented with TDZ (2.5 μM) induced a maximum of 6.73 ± 0.17 shoots/explant in 93.00 ± 1.52% of cultures with an average shoot length of 2.26 ± 0.14 cm (Figure 60 B). It was further observed that the higher concentrations of TDZ (5.0-10.0 µM) were inhibitory for shoot development and resulted in reduced shoot length because of heavy basal callusing. Explants cultured on 10.0 µM TDZ produced an average of only 2.20 ± 0.15 shoots/explant in 45.66 ± 3.48% cultures, which were highly stunted attaining merely 0.80 ± 0.15 cm of shoot length after 6 weeks of incubation (Table 50).

118

Table 48. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from cotyledonary nodes of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.5 - - 64.33  2.33a 8.20  0.17c 4.56  0.23ab 7.5 1.0 - - 72.33  1.45a 10.90  0.20a 4.76  0.14a 7.5 2.0 - - 62.66  1.43bc 7.20  0.17d 4.23  0.14bc 7.5 - 0.5 - 62.33  1.45bc 7.90  0.20c 3.76  0.14cd 7.5 - 1.0 - 65.00  1.73b 9.23  0.14b 4.16  0.20bc 7.5 - 2.0 - 60.33  1.45bc 6.60  0.20e 3.70  0.11cd 7.5 - - 0.5 57.66  1.45cd 5.30  0.20f 3.40  0.20d 7.5 - - 1.0 63.33  1.45bc 6.96  0.14de 3.96  0.14c 7.5 - - 2.0 54.00  2.08d 5.60  0.20f 3.23  0.14d

Table 49. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from cotyledonary nodes of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.5 - - 52.50  1.38bc 5.90  0.20b 3.76  0.14ab 7.5 1.0 - - 54.90  0.20a 7.56  0.23a 4.23  0.14a 7.5 2.0 - - 50.40  0.83d 5.40  0.11bcd 3.73  0.14ab 7.5 - 0.5 - 47.66  0.44e 5.56  0.23bc 3.03  0.08c 7.5 - 1.0 - 53.40  0.45ab 5.90  0.20b 3.73  0.14ab 7.5 - 2.0 - 47.16  0.44e 4.56  0.23e 3.23  0.14bc 7.5 - - 0.5 47.40  0.30e 4.90  0.20de 2.86  0.23c 7.5 - - 1.0 51.30  0.55cd 5.23  0.14cd 3.13  0.23c 7.5 - - 2.0 45.90  0.37e 3.96  0.14f 2.90  0.20c

Explanation of Figure 60 In vitro morphogenic responses through cotyledonary nodes of C. sophera. A. Basal swelling and callusing along with differentiation of multiple shoots on MS medium supplemented with 2.5 µM TDZ - 2 weeks old culture. (Bar = 0.47 cm) B. Multiplication of shoots on medium comprised of MS + TDZ (2.5 µM) - 5 weeks old culture. (Bar = 0.82 cm) C. Multiplication and elongation of TDZ (2.5 µM) induced cultures on BA (1.0 µM) supplemented medium - 6 weeks old culture. (Bar = 0.71 cm)

Figure 60

B C

Table 50. Effect of different concentrations of TDZ on multiple shoot induction from cotyledonary nodes of C. sophera.

TDZ % Response Mean number of Mean shoot (μM) shoots/explant length (cm)

0.1 40.00 ± 1.15g 3.23 ± 0.14d 1.26 ± 0.14cde 0.25 49.00 ± 2.08ef 4.00 ± 0.26c 1.50 ± 0.17cd 0.5 61.00 ± 2.08d 4.63 ± 0.29c 1.60 ± 0.20bc 1.0 79.66 ± 2.60b 5.33 ± 0.24b 2.06 ± 0.17ab 2.5 93.00 ± 1.52a 6.73 ± 0.17a 2.26 ± 0.14a 5.0 69.66 ± 3.17c 2.83 ± 0.20de 1.26 ± 0.14cde 7.5 55.66 ± 2.33de 2.46 ± 0.24e 1.00 ± 0.17e 10.0 45.66 ± 3.48fg 2.20 ± 0.15e 0.80 ± 0.15e

-Data recorded after 6 weeks of culture. -Value represents Mean ± SE of three repeated experiments with 10 replicates each. Means followed by the same letter within columns are significantly not different (P = 0.05) using Duncan’s test.

4.2.2.1.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Multiple shoots induced on TDZ supplemented media exhibited stunted growth and also the prolonged exposure to TDZ resulted in distortion and fasciation of regenerated shoots. In order to avoid these deleterious effects of TDZ, the clumps of regenerated shoots were separated from the parent tissue and transferred to TDZ free MS basal medium as well as MS medium supplemented with different concentrations (0.5, 1.0 and 2.5 μM) of BA for further growth and elongation of shoots. When TDZ induced cultures were transferred to MS medium without BA, no growth and development of shoots occurred, shoots failed to elongate and ultimately shed off all their leaves after 3 weeks. However, addition of different concentrations of BA facilitated better response and at 1.0 μM BA the number of shoots increased to 14.90 ± 1.35 shoots/explant from initial 6.73 ± 0.17 shoots, attaining an enhanced shoot length of 5.76 ± 0.23 cm after 6 weeks of transfer (Figure 60 C). The higher concentration (2.5 µM) of BA was not very much effective and an average of 9.50 ± 1.30 shoots/explant were obtained having shoot length of 4.16 ± 0.17 cm after 6 weeks (Table 51).

4.2.2.1.5 Effect of different media

Four different media (B5, L2, MS and WPM) were tried for CN explants having optimized concentration of PGRs i.e. BA (5.0 µM) and NAA (1.0 µM) to evaluate the effect of different nutrients composition on shoot morphogenesis. Amongst all the four media tested, MS medium was found to be the most suitable medium for multiple shoots regeneration and yielded maximum 19.50 ± 0.51 shoots/explant with highest shoot length of 5.23 ± 0.14 cm after 6 weeks of culture. On MS medium shoot buds were induced within 8 days of culture. WPM exhibited less response as compared to MS medium and a maximum of 11.23 ± 0.53 shoots/explant with shoot length of 4.56 ± 0.23 cm were obtained from CN explants after 6 weeks of inoculation. Explants cultured on L2 medium delivered moderate response with the production of an average 6.63 ± 0.23 shoots/explant, while, B5 medium provided least response with merely 3.63 ± 0.23 shoots/explant after 6 weeks (Figure 61).

119

Table 51. Effect of different concentrations of BA on TDZ (2.5 μM) induced cultures of C. sophera for further multiplication and elongation.

PGR (μM) Mean number of Mean shoot shoots/explant length (cm)

MS 6.73 ± 0.17c 2.26 ± 0.14d MS + BA (0.5) 10.23 ± 1.64ab 5.03 ± 0.14b MS + BA (1.0) 14.90 ± 1.35a 5.76 ± 0.23a MS + BA (2.5) 9.50 ± 1.30b 4.16 ± 0.17c

Mean number of shoots/explant Mean shoot length (cm) 25 6

a ab 5 20 bc c 4 15

3 b 10 2 c (cm) length shoot Mean 5 shoots/explant of number Mean d 1

0 0 MS WPM L2 B5 Different media

Figure 61. Effect of different culture media supplemented with BA (5.0 µM) and NAA (1.0 µM) on shoot regeneration from cotyledonary nodes of C. sophera after 6 weeks of culture.

4.2.2.1.6 Effect of pH The effect of different pH (5.0, 5.4. 5.8, 6.2 and 6.6) was tested in MS medium comprised of optimized concentrations of BA (5.0 µM) and NAA (1.0 µM) on shoot regeneration from CN explants. The ideal value of pH for the maximum regeneration proved to be 5.8 where a maximum of 19.50 ± 0.51 shoots/CN explant was obtained after 6 weeks of culture. pH values below the optimal resulted in loose or watery medium which was slight acidic in nature and declined the number of shoots to 10.63 ± 0.29 and 6.83 ± 0.27 shoots/explant at pH 5.4 and 5.0 respectively. High pH values produced alkaline medium which affected the regeneration potential of the explants and thus, reduced the number of shoots to 8.43 ± 0.29 and 5.50 ± 0.17 shoots/explant at pH 6.2 and 6.6 respectively (Figure 62).

4.2.2.1.7 Effect of sucrose concentrations Different concentrations (1, 2, 3, 4 and 5%) of sucrose were tested with CN explants on the optimized medium i.e. MS + BA (5.0 µM) + NAA (1.0 µM). The best response was exhibited by the explants on medium comprised of 3% sucrose, producing a maximum of 19.50 ± 0.51 shoots/explant attaining the highest shoot length of 5.23 ± 0.14 cm after 6 weeks of culture. While, below this optimal concentration of sugar the explants showed less response and consequently the number of shoots declined to 9.63 ± 0.34 and 5.66 ± 0.29 shoots/explant at 2% and 1% sucrose respectively. Higher concentrations of sucrose also reduced the regeneration efficacy of the explant and number of shoots was reduced to 11.86 ± 0.23 and 7.63 ± 0.29 shoots/explant at 4% and 5% of sucrose respectively (Figure 63).

4.2.2.2 Nodal segment (NS) explant 4.2.2.2.1 Effect of explant age on multiple shoot regeneration NS explants excised from seedlings of three different age group (14, 21 and 28 days old) were cultured on MS medium containing different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA to select the best responsive age of the explant for maximum shoot regeneration. The MS medium without BA served as control on which the explants failed to respond. NS excised from 21 days old seedlings provided best response in 90.33 ± 3.17% cultures with multiple shoot induction

120

Mean number of shoots/explant Mean shoot length (cm)

25 8

a 20 6 a

15 b b b 4 b c 10 c

d e 2 Mean shootlength (cm) 5 Mean numbershoots/explant of

0 0 5.0 5.4 5.8 6.2 6.6 Different pH of medium

Figure 62. Effect of different pH values on shoot regeneration from cotyledonary nodes of C. sophera on MS medium ccontaining optimal concentration of BA (5.0 µM) and NAA (1.0 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 25 6 a

a 5 20 b b

c b c 3 10 c d 2

e Mean shootlength (cm) 5 Mean number of shoots/explant 1

0 0 1 2 3 4 5

Cncentration of sucrose (%)

Figure 63. Effect of different concentrations of sucrose on shoot regeneration from cotyledonary nodes of C. sophera on optimal medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) after 6 weeks of culture.

(16.46 ± 1.21 shoots/explant) after six weeks of incubation on MS + BA (5.0 µM). Explants isolated from 14 and 28 days old seedlings exhibited less regeneration and induced lesser number of shoots at the same treatment i.e. 10.20 ± 0.46 and 8.20 ± 0.51 shoots/explant respectively (Table 52).

4.2.2.2.2 Effect of cytokinins on multiple shoot regeneration The best responsive NS (21 days old) of C. sophera were cultured on three different cytokinins (BA, Kn and 2iP) at various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 µM) to evaluate the morphogenic response of the explant. Different cytokinins showed differential response and accordingly the number of shoots/explant varied with the type and concentration of PGRs. The number of shoots increased linearly with an increase in the concentration of BA from 1.0 µM up to an optimal concentration of 5.0 µM. The MS medium containing 1.0 µM BA produced 7.53 ± 1.05 shoots/explant having shoot length of 3.00 ± 0.23 cm with 64.66 ± 2.90% regeneration response (Figure 64 A). BA at 5.0 µM proved to be critical wherein shoot buds were induced within 8 days of culture and a maximum of 16.46 ± 1.21 shoots/explant were harvested in 90.33 ± 3.17% cultures with shoot length of 4.80 ± 0.37 cm after 6 weeks of incubation (Figure 64 B & C). While higher concentrations (7.5 µM and 10.0 µM) of BA proved inhibitory for both shoot regeneration as well as multiplication and the number of shoots reduced to 6.16 ± 0.63 shoots/explant at 10.0 µM BA with 66.33 ± 2.33% regeneration response (Table 53).

The nodal segments delivered moderate response on Kn and 2iP supplemented media and the number of shoots/explant was comparatively low to BA supplemented media. The explants on Kn supplemented medium began induction of shoot buds after 10 days of incubation. Kn at 7.5 µM provided optimal response (62.33 ± 1.45%) wherein an average of 7.60 ± 0.34 shoots/explant with shoot length of 3.66 ± 0.20 cm were produced after 6 weeks of culture (Figure 64 D). Explants expressed least response on medium containing 2iP; the regenerated shoots were not as healthy as in BA and Kn supplemented media and possessed reduced leaf lamina. The MS medium comprised of 7.5 µM 2iP produced only 5.16 ± 0.49 shoots/explant with shoot

121

Table 52. Effect of age of nodal segments on direct shoot regeneration in C. sophera cultured on MS medium containing different concentrations of BA.

BA (µM) 14 days old explant 21 days old explant 28 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00e 00.00  0.00e 00.00  0.00e 00.00  0.00d 00.00  0.00e 0.00  0.00e 1.0 42.33  1.45c 3.93  0.23d 64.66  2.90d 7.53  1.05c 32.33  1.45cd 2.93  0.17d 2.5 49.00  2.08bc 5.83  0.32c 75.66  2.33c 10.63  1.25b 37.33  1.45c 4.96  0.35b 5.0 63.00  2.86a 10.20  0.46a 90.33  3.17a 16.46  1.21a 54.33  2.92a 8.20  0.51a 7.5 50.66  2.33b 7.90  0.37b 82.33  1.45b 10.83  0.76b 42.33  1.45b 5.83  0.32b 10.0 35.33  2.90d 4.80  0.32d 66.33  2.33d 6.16  0.63c 29.33  1.76d 3.93  0.23c

Explanation of Figure 64 In vitro morphogenic responses through nodal segments of C. sophera. A. Basal swelling and enlargement of explant along with Induction of multiple shoots on medium comprised of MS + BA (1.0 µM) - 2 weeks old culture. (Bar = 0.47 cm)

B. Basal swelling and enlargement of explant along with Induction of multiple shoots on medium comprised of MS + BA (5.0 µM) - 2 weeks old culture. (Bar = 0.59 cm)

C. Multiplication of shoots on MS medium containing 5.0 µM BA - 3 weeks old culture. (Bar = 0.86 cm)

D. Multiple shoots development on MS medium containing 7.5 µM Kn - 3 weeks old culture. (Bar = 0.45 cm)

E. Production of multiple shoots on MS medium containing 7.5 µM 2iP - 4 weeks old culture. (Bar = 0.46 cm)

Figure 64

A B C

D E

Table 53. Effect of different cytokinins on direct shoot regeneration from nodal segments of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 64.66  2.90d 7.53  1.05c 3.00  0.23bcde 2.5 - - 75.66  2.33c 10.63  1.25b 3.53  0.26b 5.0 - - 90.33  3.17a 16.46  1.21a 4.80  0.37a 7.5 - - 82.33  1.45b 10.83  0.76b 4.50  0.28a 10.0 - - 66.33  2.33d 6.16  0.63cde 3.33  0.24b - 1.0 - 34.33  2.33ij 2.93  0.23fg 2.53  0.26cde - 2.5 - 39.00  1.73hi 4.23  0.14ef 2.93  0.17bcde - 5.0 - 49.66  1.45ef 6.56  0.23cd 3.23  0.14bc - 7.5 - 62.33  1.45d 7.60  0.34c 3.66  0.20ab - 10.0 - 45.66  2.96g 5.83  0.27cde 3.23  0.14bc - - 1.0 00.00  0.00k 0.00  0.00h 0.00  0.00f - - 2.5 29.00  2.08j 1.86  0.35jg 2.33  0.17e - - 5.0 45.00  2.08gh 3.20  0.17fg 2.53  0.20cde - - 7.5 53.33  2.02e 5.16  0.49de 3.10  0.20bcd - - 10.0 35.66  2.96i 4.36  0.32ef 2.46  0.29de

ength of 3.10 ± 0.20 cm in 53.33 ± 2.02% cultures after 6 weeks of culture (Figure 64 E, Table 53).

4.2.2.2.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration The nodal segments were also cultured in cytokinin-auxin combinations to observe the combined effect of PGRs on direct shoot regeneration. The optimal concentrations of each cytokinin (BA, Kn and 2iP) were tested with different concentrations (0.5, 1.0 and 2.0 µM) of three auxins (IAA, IBA and NAA). The synergistic effect of optimal concentration (5.0 µM) of BA with auxins is depicted in Table 54. It was observed that addition of NAA enhanced the rate of shoot multiplication; facilitated better growth and proliferation of shoots and shoot buds were induced within 5-6 days of inoculation. The MS medium augmented with BA (5.0 µM) + NAA (0.5 µM) yielded 22.20 ± 0.56 shoots/explant with 92.33 ± 1.45% regeneration attaining an average shoot length of 5.50 ± 0.17 cm after 6 weeks of culture (Figure 65 A). When the concentration of NAA was further increased to 1.0 µM, the regeneration response was enhanced (97.33 ± 1.45%) and the maximum 25.36 ± 0.34 shoots/explant having shoot length of 6.23 ± 0.14 cm were harvested during the same time period (Figure 65 B). On increasing the concentration of NAA up to 2.0 µM, there was a gradual decrease in regeneration potential (91.00 ± 1.52%) and number of shoots/explant (20.63 ± 0.44). The elevated concentration of NAA (2.0 µM) caused basal callusing which hampered the growth and multiplication of shoots. Among the various combinations of BA and IBA used, the MS medium containing BA (5.0 µM) and IBA (1.0 µM) produced the maximum 18.63 ± 0.86 shoots/explant with highest shoot length of 5.26 ± 0.14 cm in 92.33 ± 1.45% cultures after 6 weeks. Among various BA-IAA combinations, the maximum response (87.66 ± 1.45%) of shoot bud formation and highest number of shoots/explant (16.43 ± 0.29) was obtained on MS medium containing 5.0 µM BA along with 1.0 µM IAA. At higher concentration (2.0 µM) of the auxins (IBA and IAA), regeneration potential as well as number of shoots get reduced (Table 54).

When the NS were cultured on optimal concentration (7.5 µM) of Kn along with three different auxins at varying concentrations, induction of multiple shoots took

122

Table 54. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from nodal segments of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.5 - - 92.33  1.45ab 22.20  0.56b 5.50  0.17b 5.0 1.0 - - 97.33  1.45a 25.36  0.34a 6.23  0.14a 5.0 2.0 - - 91.00  1.52b 20.63  0.44c 4.76  0.14cd 5.0 - 0.5 - 87.66  1.45bc 16.43  0.29e 4.40  0.20de 5.0 - 1.0 - 92.33  1.45ab 18.63  0.86d 5.26  0.14bc 5.0 - 2.0 - 84.66  1.45cd 15.80  0.20e 4.23  0.14e 5.0 - - 0.5 82.00  1.15d 15.63  0.20e 4.10  0.20e 5.0 - - 1.0 87.66  1.45bc 16.43  0.29e 5.00  0.11bc 5.0 - - 2.0 74.00  3.05e 15.00  0.40e 3.96  0.14e

Explanation of Figure 65 In vitro morphogenic responses through nodal segments of C. sophera. A. Multiplication and elongation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (0.5 µM) - 6 weeks old culture. (Bar = 0.83 cm)

B. Multiplication and proliferation of shoots on MS medium containing BA (5.0 µM) and NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.84 cm)

C. Multiple shoots production on MS medium supplemented with Kn (7.5 µM) and NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.92 cm)

D. Multiplication of shoots on medium comprised of MS + 2iP (7.5 µM) + NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.81 cm)

Figure 65

A B

C D

place with lesser frequency as compared to BA. Shoot buds were induced after 8 days of incubation on MS medium containing optimal concentration of Kn with different concentration of NAA. The medium comprised of Kn (7.5 µM) and NAA (1.0 µM) produced a maximum of 12.30 ± 0.20 shoots/explant in 74.66 ± 1.45% cultures having shoot length of 4.20 ± 0.15 cm after 6 weeks of incubation (Figure 65 C). IBA and IAA proved to be less efficient than NAA with reduction in regeneration potential as well as number of shoots/explant (Table 55).

Among various combinations of 2iP and auxins, the MS medium containing 2iP (7.5 µM) and NAA (1.0 µM) provided best regeneration response (62.33 ± 1.45%) producing an average of 9.56 ± 0.23 shoots/explant, attaining shoot length of 4.00 ± 0.11 cm after 6 weeks of incubation (Figure 65 D). Further increase in the concentration of NAA up to 2.0 µM, reduced the percent response (55.00 ± 2.88%) as well as number of shoots/explant (6.70 ± 0.20). IBA and IAA were found to be less effective than NAA in combination with optimal concentration of 2iP (Table 56).

4.2.2.2.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration In another experiment, NS of C. sophera were cultured at various concentrations (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) of TDZ to evaluate the regeneration efficacy of the nodal segments. Augmentation of TDZ influenced the induction of multiple shoot buds within 7 days of culture and the number of shoots/explant increased with an increase in the concentration from 0.1 µM up to 2.5 µM TDZ. The explants swelled and slight callusing was also observed at the base of the explants. The medium comprised of MS + TDZ (0.1 µM) yielded 3.90 ± 0.20 shoots/explant having an average shoot length of 1.50 ± 0.17 cm in 45.00 ± 2.51% cultures after 6 weeks of inoculation. While at 2.5 µM of TDZ, a maximum of 13.76 ± 0.38 shoots/explant were produced with a regeneration potential of 95.00 ± 1.15% having an average shoot length of 3.26 ± 0.14 cm after 6 weeks of incubation (Figure 66 A & B). Higher concentrations of TDZ were found to restrict the growth of new shoots and produced heavy basal callusing. Consequently, beyond the optimal level a decline in percent response (65.66 ± 2.96%) as well as number of shoots (2.90 ± 0.20) was recorded at 10.0 µM TDZ (Table 57).

123

Table 55. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from nodal segments of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.5 - - 67.66  1.45b 9.93  0.38b 3.76  0.14abc 7.5 1.0 - - 74.66  1.45a 12.30  0.20a 4.20  0.15a 7.5 2.0 - - 65.00  1.15b 9.33  0.32bc 3.56  0.14bc 7.5 - 0.5 - 59.66  1.45c 6.96  0.14d 3.63  0.20bc 7.5 - 1.0 - 67.33  1.45b 8.86  0.24c 4.00  0.11ab 7.5 - 2.0 - 57.66  1.45c 6.43  0.23de 3.33  0.17c 7.5 - - 0.5 57.66  1.45c 6.30  0.37de 3.26  0.14c 7.5 - - 1.0 63.33  1.45b 6.90  0.20d 3.70  0.11bc 7.5 - - 2.0 56.33  2.33c 5.93  0.23e 3.46  0.14c

Table 56. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from nodal segments of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.5 - - 57.66  1.45ab 6.90  0.20b 3.53  0.26abc 7.5 1.0 - - 62.33  1.45a 9.56  0.23a 4.00  0.11a 7.5 2.0 - - 55.00  2.88bc 6.70  0.20b 3.66  0.08ab 7.5 - 0.5 - 54.66  1.45bc 6.26  0.14bc 2.96  0.14c 7.5 - 1.0 - 59.66  1.45ab 6.90  0.20b 3.50  0.23abc 7.5 - 2.0 - 50.33  1.45bc 5.76  0.14cd 3.23  0.14bc 7.5 - - 0.5 47.33  1.45c 5.43  0.29d 3.03  0.14c 7.5 - - 1.0 54.66  1.45bc 5.90  0.20cd 3.40  0.23abc 7.5 - - 2.0 45.66  2.33c 5.36  0.23d 2.93  0.23c

Explanation of Figure 66 In vitro morphogenic responses through nodal segments of C. sophera. A. Induction of multiple shoots on medium comprised of 2.5 µM TDZ - 2 weeks old culture. (Bar = 0.56 cm)

B. Multiplication of shoots on TDZ (2.5 µM) containing medium - 5 weeks old culture. (Bar = 1.13 cm)

C. Proliferation and elongation of TDZ (2.5 µM) induced cultures on BA (1.0 µM) supplemented medium, after 5 weeks of transfer. (Bar = 0.40 cm)

Figure 66

B C

Table 57. Effect of various concentrations of TDZ on direct shoot regeneration from nodal segments of C. sophera.

TDZ (M) Regeneration Mean number of Mean shoot (%) shoots/explants length (cm)

0.1 45.00  2.51f 3.90  0.20f 1.50  0.17d 0.25 52.33  1.45e 5.23  0.14e 1.73  0.14cd 0.5 65.66  2.33d 6.56  0.34d 2.10  0.20cd 1.0 85.00  2.08b 9.53  0.31b 2.73  0.14b 2.5 95.00  1.15a 13.76  0.38a 3.26  0.14a 5.0 76.66  1.76c 8.26  0.17c 2.70  0.17b 7.5 66.66  2.72d 3.90  0.20f 2.23  0.14bc 10.0 65.66  2.96d 2.90  0.20g 1.23  0.14d

4.2.2.2.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Cultures on TDZ containing media showed distortion and stunting of microshoots after six weeks of incubation, thus, to nullify these deleterious effects cultures were transferred to MS medium devoid of TDZ. The MS medium without any PGR could not improve the growth and development of shoots, therefore, medium was supplemented with different concentrations (0.5, 1.0 and 2.5 µM) of BA for further enhancement in response. The medium comprised of MS + BA (0.5 µM) showed a considerable enhancement in shoot number with 16.23 ± 0.14 shoots/explant compared to 13.76 ± 0.38 shoots/explant on TDZ (2.5 µM) and with an increase in shoot length (5.50 ± 0.17 cm) after 6 weeks of culture. On further increasing the concentration of BA (1.0 µM) the number of shoots was increased to 19.83 ± 0.02 shoots/explant with highest shoot length of 6.00 ± 0.11 cm after 6 weeks of culture (Figure 66 C). However, beyond 1.0 µM of BA, a decline in the number of shoots (14.63 ± 0.23) and shoot length (5.23 ± 0.14 cm) was observed (Table 58).

4.2.2.2.5 Effect of different media

The effect of nutrients composition of four different media (B5, L2, MS and WPM) was tested with the NS explants on optimized concentration of PGRs i.e. BA (5.0 µM) and NAA (1.0 µM). Among four media tested, MS medium was proved to be the most appropriate medium for multiplication and proliferation of shoots and provided maximum of 25.36 ± 0.34 shoots/explant with maximum shoot length of 6.23 ± 0.14 cm. On MS medium induction of shoot buds took place within 5-6 days of culture. WPM was found to deliver moderate results, while, L2 and B5 were less effective as compared to MS medium. A maximum of 18.96 ± 0.64 shoots/explant were obtained from NS cultured on WPM after 6 weeks of inoculation and induction of shoots started after 10 days of incubation. The number of shoots was further reduced to 14.90 ± 0.20 shoots/explant on L2 medium while on B5 medium least number of shoots were produced (9.53 ± 0.37 shoots/explant) (Figure 67).

124

Table 58. Effect of various concentrations of BA on TDZ (2.5 M) induced cultures from nodal segments of C. sophera for further multiplication and proliferation.

PGR (M) Mean number of Mean shoot shoots/explant length (cm)

MS 14.23  0.14c 3.33  0.08c MS + BA (0.5) 16.23  0.14b 5.50  0.17b MS + BA (1.0) 19.83  0.02a 6.00  0.11b MS + BA (2.5) 14.63  0.23c 5.23  0.14b

4.2.2.2.6 Effect of pH The pH of the medium had a significant role in the induction and multiplication of shoots, therefore, the effect of different pH (5.0, 5.4. 5.8, 6.2 and 6.6) was tested with MS medium comprised of optimized concentration of BA (5.0 µM) and NAA (1.0 µM)]. The optimum value of pH for the induction of maximum 25.36 ± 0.34 shoots/NS was found to be 5.8. The low pH of the medium resulted in acidic nature and formed loose or watery medium which declined the number of shoots to 16.16 ± 0.26 and 10.53 ± 0.26 shoots/explant at pH 5.4 and 5.0 respectively. High pH value formed comparatively firm medium which eventually affected the growth and multiplication of shoots and thus, reduced the number of shoots to 19.96 ± 0.31 and 13.86 ± 0.30 shoots/explant at pH 6.2 and 6.6 respectively (Figure 68).

4.2.2.2.7 Effect of sucrose concentrations Effect of different concentrations (1, 2, 3, 4 and 5%) of sucrose was also tested with NS explants on the optimized medium i.e. MS + BA (5.0 µM) + NAA (1.0 µM). The NS explants exhibited best response on the medium containing 3% sucrose where a maximum of 25.36 ± 0.34 shoots/explant were produced having a maximum shoot length of 6.23 ± 0.14 cm. However, the number of shoots reduced to 10.56 ± 0.23 and 7.56 ± 0.23 shoots/explant at 2% and 1% sucrose respectively. Further increase in the concentration of sucrose from optimal 3% also reduced the number of shoots to 16.56 ± 0.34 and 11.36 ± 0.44 shoots/explant at 4% and 5% of sucrose respectively (Figure 69).

4.2.2.3 Shoot tip (ST) explant 4.2.2.3.1 Effect of explant age on multiple shoot regeneration The shoot tip explants excised from aseptic seedlings of three different ages (14, 21 and 28 days) were cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of BA to select the best responsive age of the explant. Explants cultured on hormone free MS medium failed to induce any morphogenic response and died within 2 weeks of culture. Shoot tips excised from 21 days old seedlings delivered maximum response of 60.00 ± 1.15% on MS + BA (5.0 µM) producing a maximum of 6.10 ± 0.26 shoots/explant after 6 weeks of culture. On this medium induction of multiple shoot buds started after 12-13 days of culture.

125

Mean number of shoots/explant Mean shoot length (cm)

30 7.0

a 6.5 25 a 6.0 20 b

5.5 b c 15 5.0

d 10 c 4.5 (cm) length shoot Mean d Mean number os shoots/explant number Mean 5 4.0

0 3.5 MS WPM L2 B5 Different Media

Figure 67. Effect of different culture media supplemented with optimal concentration of BA (5.0 µM) and NAA (1.0 µM) on shoot regeneration from nodal segments of C. sophera after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm)

30 7.0

a 6.5 25 a 6.0 b 20 5.5 c 15 b d 5.0

e bc 4.5 10 cd

(cm) Mean shoot length d 4.0

shoots/explant Mean number of 5 3.5

0 3.0 5.0 5.4 5.8 6.2 6.6 Different pH Figure 68. Effect of different pH of the medium on shoot regeneration from nodal explants of C. sophera on optimized medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm)

30 7 a 25 a 6

20 b 5 b b 15

c 4 c 10 d

(cm) length shoot Mean c c 3 Mean number of shoots/explant of number Mean 5

0 2 1 2 3 4 5 Concentration of sucrose (%)

Figure 69. Effect of different concentrations of sucrose on shoot regeneration from nodal segments of C. sophera on MS medium containing optimal concentration of BA (5.0 µM) and NAA (1.0 µM) after 6 weeks of culture.

The explants collected from 14 and 28 days old seedlings were less responsive as well as exhibited delayed shoot buds differentiated i.e. after 15 days of incubation in both the explants. On MS + BA (5.0 µM) an average of 3.93 ± 0.17 and 3.23 ± 0.14 shoots/explant were produced by the explants from 14 and 28 days old seedlings respectively (Table 59).

4.2.2.3.2 Effect of cytokinins on multiple shoot regeneration The ST explants of 21 days old seedlings were chosen as the best responsive explant and cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of three cytokinins (BA, Kn and 2iP) to assess the morphogenic potential of the explants. Induction of shoot buds started after 12-13 days of culture in BA supplemented media. ST explants revealed least response in comparison to NS and CN explants. The MS medium augmented with 1.0 µM BA produced only 2.06 ± 0.23 shoots/explant with shoot length of 2.73 ± 0.14 cm in 38.33 ± 3.75% cultures after 6 weeks. The regeneration potential increased linearly with an increase in the concentration of BA up to 5.0 µM, where maximum 6.10 ± 0.26 shoots/explant, attaining an average shoot length of 3.96 ± 0.20 cm were harvested in 60.00 ± 1.15% cultures during the same incubation period (Figure 70 A). Higher concentrations of BA were found to be inhibitory for shoot regeneration because of basal callusing and the number of shoots was reduced to 2.66 ± 0.27 shoots/explant at 10.0 µM BA in 39.00 ± 2.08% cultures (Table 60).

Explants cultured on Kn supplemented media revealed moderate response, and lesser number of shoots/explant were obtained as compared to cultures on BA supplemented media. Lower concentration (1.0 µM) of Kn was inhibitory and no response was induced. However, as the concentration of cytokinin was increased, there was an increase in the percent response as well as number of shoots/explant. Explants cultured on 7.5 µM Kn showed induction of shoot buds after 15 days and revealed maximum response (52.33 ± 1.45%) with 3.66 ± 0.24 shoots/explant after 6 weeks of culture (Figure 70 B). 2iP delivered least response with lower concentrations (1.0 and 2.5 µM) being unable to stimulate any morphogenic change, while the higher concentration facilitated the induction of multiple shoots within 17-18 days of incubation. The medium comprised of 7.5

126

Table 59. Effect of age of shoot tips on direct shoot regeneration in C. sophera cultured on MS medium containing different concentrations of BA.

BA (µM) 14 days old explant 21 days old explant 28 days old explants

Regeneration Mean number of Regeneration Mean number of Regeneration Mean number of % shoots/explant % shoots/explant % shoots/explant

- 00.00  0.00e 0.00  0.00e 00.00  0.00e 0.00  0.00e 00.00  0.00d 0.00  0.00d 1.0 29.33  2.33d 1.63  0.23d 38.33  3.75d 2.06  0.23d 24.66  1.45c 1.40  0.26c 2.5 35.66  2.33c 2.36  0.17bc 44.33  2.96c 2.90  0.20c 32.66  1.45b 1.76  0.14c 5.0 54.00  2.64a 3.93  0.17a 60.00  1.15a 6.10  0.26a 46.33  2.33a 3.23  0.14a 7.5 42.33  1.45b 2.86  0.24b 49.33  2.33b 4.76  0.14b 36.66  2.02b 2.46  0.17b 10.0 32.33  1.45cd 2.23  0.14c 39.00  2.08d 2.66  0.27cd 25.66  2.33c 1.70  0.20c

Explanation of Figure 70 In vitro morphogenic responses through shoot tip explants of C. sophera. A. Induction of multiple shoots on MS medium supplemented with 5.0 µM BA - 3 weeks old culture. (Bar = 0.65 cm)

B. Induction of multiple shoots on MS medium supplemented with 7.5 µM Kn - 3 weeks old culture. (Bar = 0.71 cm)

C. Induction of multiple shoots on MS medium supplemented with 7.5 µM 2iP - 3 weeks old culture. (Bar = 0.58 cm)

Figure 70

A B C

Table 60. Effect of different cytokinins on direct shoot regeneration from shoot tips of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn 2iP

1.0 - - 38.33  3.75de 2.06  0.23efg 2.73  0.14bcde 2.5 - - 44.33  2.96cd 2.90  0.20d 3.10  0.20bc 5.0 - - 60.00  1.15a 6.10  0.26a 3.96  0.20a 7.5 - - 49.33  2.33bc 4.76  0.14b 2.90  0.20bc 10.0 - - 39.00  2.08de 2.66  0.27de 2.63  0.23cde - 1.0 - 00.00  0.00h 0.00  0.00h 0.00  0.00g - 2.5 - 24.33  3.48g 2.23  0.14ef 2.73  0.17bcde - 5.0 - 35.66  2.33ef 2.43  0.20de 3.23  0.14b - 7.5 - 52.33  1.45b 3.66  0.24c 2.23  0.14ef - 10.0 - 33.66  2.02ef 1.73  0.14fg 1.96  0.14f - - 1.0 00.00  0.00h 0.00  0.00h 0.00  0.00g - - 2.5 00.00  0.00h 0.00  0.00h 0.00  0.00g - - 5.0 30.66  1.76fg 1.50  0.28g 2.33  0.20def - - 7.5 35.66  2.33ef 2.70  0.20de 2.76  0.14bcd - - 10.0 29.66  1.45fg 1.43  0.29g 1.90  0.20f

µM 2iP yielded an average of 2.70 ± 0.20 shoots/explant in 35.66 ± 2.33% cultures after 6 weeks of inoculation (Figure 70 C, Table 60).

4.2.2.3.3 Effect of cytokinin-auxin combinations on multiple shoot regeneration The shoot tip explants were also cultured on cytokinin-auxin combinations to evaluate the combined effect of PGRs on multiple shoot regeneration. The optimal concentration of BA (5.0 µM) was tested with various concentrations of auxins (IAA, IBA and NAA) to improve the regeneration efficacy of the explants. Among various combination treatments, BA (5.0 µM) + NAA (1.0 µM) was proved to be the best where a maximum of 10.56 ± 0.23 shoots/explant, attaining an average shoot length of 4.80 ± 0.11 cm were produced in 69.66 ± 1.45% cultures after 6 weeks of culture (Figure 71 A & B). On this medium shoot buds started to differentiate within 10-11 days of incubation. The concentrations of NAA beyond optimal level (1.0 µM) were less effective and yielded an average of 8.26 ± 0.14 and 7.53 ± 0.2 shoots/explant at BA (5.0 µM) + NAA (0.5 µM) and BA (5.0 µM) + NAA (2.0 µM) respectively. Higher concentrations of auxins resulted in basal callusing and thus hampered the growth and development of shoots. Similar to CN and NS explants, ST explants also provided moderate response with other two auxins i.e. IBA and IAA. The medium comprised of MS + BA (5.0 µM) + IBA (1.0 µM) produced an average of 9.00 ± 0.25 shoots/explant with 4.50 ± 0.17 cm shoot length in 65.00 ± 1.73% cultures after 6 weeks, while, the medium comprised of IAA at the same concentration provided 6.73 ± 0.14 shoots/explant with shoot length of 4.23 ± 0.24 cm in 60.33 ± 1.45% cultures during the same time period (Table 61).

The optimal concentration (7.5 µM) of Kn and 2iP were also tested with three different auxins. Augmentation of auxins at various concentrations enhanced the frequency of shoot regeneration compared to the medium containing single Kn or 2iP. Amongst various combinations tested, ST explants provided optimal response at Kn (7.5 µM) + NAA (1.0 µM) on which induction of shoot buds started after 14 days of incubation and an average of 5.83 ± 0.2 shoots/explant, having shoot length of 4.50 ± 0.11 cm were obtained in 62.66 ± 1.45% cultures after 6 weeks (Figure 71 C). Further increase in the concentration (2.0 µM) of

127

Explanation of Figure 71 In vitro morphogenic responses through shoot tip explants of C. sophera. A. Multiplication of shoots on MS medium supplemented with BA (5.0 µM) and NAA (1.0 µM) - 4 weeks old culture. (Bar = 0.83 cm)

B. Proliferation and elongation of shoots on medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.75 cm)

C. Multiplication of shoots on medium comprised of MS + Kn (7.5 µM) + NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.76 cm)

D. Multiplication of shoots on medium comprised of MS + 2iP (7.5 µM) + NAA (1.0 µM) - 6 weeks old culture. (Bar = 0.80 cm)

Figure 71

A B

C D

Table 61. Effect of optimal concentration of BA with different auxins on direct shoot regeneration from shoot tips of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IBA IAA

5.0 0.5 - - 65.00  1.73ab 8.26  0.14bc 4.53  0.20ab 5.0 1.0 - - 69.66  1.45a 10.56  0.23a 4.80  0.11a 5.0 2.0 - - 62.00  2.08bc 7.53  0.20c 4.10  0.20bc 5.0 - 0.5 - 62.33  1.45bc 6.30  0.45de 3.73  0.14cd 5.0 - 1.0 - 65.00  1.73ab 9.00  0.25b 4.50  0.17ab 5.0 - 2.0 - 59.33  1.76bcd 5.60  0.23ef 3.53  0.20de 5.0 - - 0.5 57.66  1.45cd 5.13  0.23fg 3.50  0.17de 5.0 - - 1.0 60.33  1.45bcd 6.73  0.14d 4.23  0.14abc 5.0 - - 2.0 55.33  2.40d 4.56  0.23g 3.10  0.20e

NAA produced basal callusing thereby reduced the regeneration potential (54.66 ± 1.45%) as well as the number of shoots/explant (3.96 ± 0.14). IBA proved to be better than IAA in combination with Kn and produced 5.43 ± 0.29 shoots/explant with shoot length of 4.23 ± 0.14 cm on MS + Kn (7.5 µM) + IBA (1.0 µM) while IAA at the same concentration produced 4.73 ± 0.26 shoots/explant with 3.90 ± 0.20 cm of shoot length after 6 weeks of culture (Table 62).

Similar trend of response was achieved with the cultures on MS medium containing optimal concentration (7.5 µM) of 2iP and auxins. Induction of shoot buds was somewhat delayed, started after 16-17 days of inoculation and the maximum 4.60 ± 0.20 shoots/explant having shoot length of 4.20 ± 0.17 cm with 47.33 ± 1.45% response were produced on medium containing 2iP (7.5 µM) and NAA (1.0 µM) (Figure 71 D). Among various combinations of IBA and IAA with 2iP, the optimal growth of shoots was recorded at 1.0 µM of each IBA and IAA. The medium comprised of MS + 2iP (7.5 µM) + IBA (1.0 µM) produced 4.23 ± 0.14 shoots/explant having shoot length of 4.00 ± 0.17 cm, while, IAA at the same concentration produced an average of 3.73 ± 0.14 shoots/explant after 6 weeks of culture (Table 63).

4.2.2.3.4 Effect of thidiazuron (TDZ) on multiple shoot regeneration The ST explants cultured on MS medium containing different concentrations of TDZ (0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 µM) exhibited induction of multiple shoot buds in all the treatments except 0.1 µM after 12 days of culture. ST explants provided less number of shoots than CN and NS on TDZ supplemented media (Table 64). The number of shoots/explant increased linearly with an increase in the concentration of TDZ up to 2.5 µM. A maximum of 4.70 ± 0.20 shoots/explant with shoot length of 2.26 ± 0.14 cm was obtained on MS + TDZ (2.5 µM) with a regeneration potential of 74.66 ± 1.45% after 6 weeks of culture (Figure 72 A & B). Beyond the optimal concentration (2.5 µM) a decrease in the percent response (36.66 ± 2.02%) as well as number of shoots/explant (1.56 ± 0.34) was observed at 10.0 µM of TDZ.

128

Table 62. Effect of optimal concentration of Kn with different auxins on direct shoot regeneration from shoot tips of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) Kn NAA IBA IAA

7.5 0.5 - - 60.33  1.45b 4.63  0.23bc 4.23  0.14ab 7.5 1.0 - - 62.66  1.45a 5.83  0.20a 4.50  0.11a 7.5 2.0 - - 54.66  1.45bc 3.96  0.14de 4.00  0.17ab 7.5 - 0.5 - 53.33  2.40bcd 4.06  0.12cd 3.76  0.14bc 7.5 - 1.0 - 57.33  1.45ab 5.43  0.29a 4.23  0.14ab 7.5 - 2.0 - 52.66  1.45bcd 3.73  0.17def 3.40  0.20cd 7.5 - - 0.5 49.66  1.45cd 3.40  0.17ef 3.23  0.14d 7.5 - - 1.0 53.33  2.02bcd 4.73  0.26b 3.90  0.20bc 7.5 - - 2.0 48.33  2.02d 3.26  0.14f 3.03  0.14d

Table 63. Effect of optimal concentration of 2iP with different auxins on direct shoot regeneration from shoot tips of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) 2iP NAA IBA IAA

7.5 0.5 - - 42.33  1.45b 3.10  0.20c 3.90  0.20ab 7.5 1.0 - - 47.33  1.45a 4.60  0.20a 4.20  0.17a 7.5 2.0 - - 37.66  1.45bc 2.76  0.14cd 3.66  0.20abc 7.5 - 0.5 - 34.00  2.08cde 2.90  0.20cd 3.76  0.14abc 7.5 - 1.0 - 41.66  2.02b 4.23  0.14ab 4.00  0.17ab 7.5 - 2.0 - 30.00  1.15e 2.56  0.23cd 3.46  0.26bc 7.5 - - 0.5 32.33  1.45de 2.76  0.14cd 3.46  0.14bc 7.5 - - 1.0 37.33  1.45bcd 3.73  0.14b 3.73  0.14abc 7.5 - - 2.0 29.00  2.08e 2.46  0.14d 3.23  0.14c

Table 64. Effect of various concentrations of TDZ on direct shoot regeneration from shoot tips of C. sophera.

TDZ (M) Regeneration Mean number of Mean shoot (%) shoots/explants length (cm)

0.1 00.00  0.00g 0.00  0.00e 0.00  0.00e 0.25 33.00  2.08f 2.00  0.40cd 1.23  0.14d 0.5 42.66  1.76de 2.23  0.14cd 1.46  0.14cd 1.0 63.66  2.72b 3.43  0.20b 1.86  0.24abc 2.5 74.66  1.45a 4.70  0.20a 2.26  0.14a 5.0 54.66  2.60c 3.56  0.23b 1.96  0.14ab 7.5 46.33  2.33d 2.56  0.23c 1.66  0.16bcd 10.0 36.66  2.02ef 1.56  0.34d 1.26  0.14d

Explanation of Figure 72 In vitro morphogenic responses through shoot tip explants of C. sophera. A. Induction of multiple shoots on MS medium supplemented with 2.5 µM TDZ - 2 weeks old culture. (Bar = 0.52 cm)

B. Production of stunted shoots on prolonged exposure to TDZ (2.5 µM) supplemented medium - 6 weeks old culture. (Bar = 0.82 cm)

C. Multiplication and elongation of TDZ (2.5 µM) induced cultures on BA (1.0 µM) supplemented medium - after 6 weeks of transfer. (Bar = 0.78 cm)

Figure 72

B C

4.2.2.3.4.1 Effect of BA on TDZ (2.5 µM) induced cultures for further shoot multiplication and proliferation Prolonged exposure of the regenerative tissue on same concentration of TDZ resulted in the distortion and stunting of regenerated shoots. Therefore, the regenerative cultures were transferred to TDZ free MS medium as well as MS medium augmented with different concentrations (0.5. 1.0, 2.5 and 5.0 µM) of BA after 6 weeks of incubation on TDZ. The MS medium devoid of BA showed only a slight increase in the number of shoots/explant (5.00 ± 0.11) and shoot length (2.50 ± 0.11 cm). However, the medium supplemented with BA (1.0 µM) enhanced the number of shoots to 8.56 ± 0.23 shoots/explant as well as the growth in the regenerated microshoots (5.20 ± 0.11 cm) was also recorded (Figure 72 C). The higher concentration (2.5 µM) of BA suppressed the growth and development of shoots and their number was reduced to 6.90 ± 0.20 shoots/explant having shoot length of 4.63 ± 0.23 cm (Table 65).

4.2.2.3.5 Effect of different media

Four different media (B5, L2, MS and WPM) were evaluated to assess the regeneration efficacy of ST explants on optimized concentration of BA (5.0 µM) and NAA (1.0 µM). Like CN and NS explants, MS medium was proved to be the best regeneration medium for ST explants producing a maximum of 10.56 ± 0.23 shoots/explant attaining highest shoot length of 4.80 ± 0.11 cm after 6 weeks of culture. WPM was found to be less effective than MS medium and produce a maximum of 6.90 ± 0.20 shoots/explant having 3.90 ± 0.20 cm shoot length from

ST explants after 6 weeks of culture. L2 and B5 medium further reduced the regeneration potential of the explants and an average of 4.56 ± 0.23 shoots/explant were produced on L2 medium whereas least number of shoots

(2.63 ± 0.29 shoots/explant) were produced on B5 medium (Figure 73).

4.2.2.3.6 Effect of pH The effect of different pH (5.0, 5.4. 5.8, 6.2 and 6.6) was also tested with ST explants on MS medium supplemented with optimal BA (5.0 µM) + NAA (1.0 µM)]. The best morphogenic response was obtained on pH 5.8 where a maximum of 10.56 ± 0.23 shoots/ST explant with 4.80 ± 0.11 cm shoot length was produced after 6 weeks of culture. Below this optimal value of pH a

129

Table 65. Effect of various concentrations of BA on TDZ (2.5 M) induced cultures from shoot tips of C. sophera for further multiplication and proliferation.

PGR (M) Mean number of Mean shoot shoots/explant length (cm)

MS 5.00  0.11d 2.50  0.11c MS + BA (0.5) 5.93  0.23c 4.23  0.14b MS + BA (1.0) 8.56  0.23a 5.20  0.11a MS + BA (2.5) 6.90  0.20b 4.63  0.23b

Mean number of shoots/explant Mean shoot length (cm) 12 6

10 a 5

b 8 bc 4

b c

6 3

c 4 2 d (cm) Mean shoot length shoots/explant Mean number of 2 1

0 0 MS WPM L2 B5 Different media

Figure 73. Effect of different culture media supplemented with optimal concentration of BA (5.0 µM) and NAA (1.0 µM) on shoot regeneration from shoot tip explants of C. sophera after 6 weeks of culture.

decrease in regeneration potential was noticed which ultimately affected the number of shoots/explant and reduced to 6.26 ± 0.17 and 4.33 ± 0.20 shoot/explant at pH 5.4 and 5.0 respectively. Higher pH values also exhibited inhibitory effects on the explants and thus, reduced the number of shoots to 4.90 ± 0.20 and 3.56 ± 0.23 shoots/explant at pH 6.2 and 6.6 respectively (Figure 74).

4.2.2.3.7 Effect of sucrose concentrations Sucrose, the only source of carbon and energy was also tested at different concentrations (1, 2, 3, 4 and 5%) with ST explants on the optimized medium i.e. MS + BA (5.0 µM) + NAA (1.0 µM). The medium comprised of 3% sucrose provided best response producing a maximum of 10.56 ± 0.23 shoots/explant attaining an average shoot length of 4.30 ± 0.11 cm after 6 weeks of culture. Lower concentrations of sugar delivered less response and subsequently reduced the number of shoots to 5.63 ± 0.29 and 3.56 ± 0.23 shoots/explant at 2% and 1% sucrose respectively. Higher concentrations also proved to be inhibitory for shoot regeneration and thus reduced the number of shoots to 7.70 ± 0.28 and 4.03 ± 0.14 at 4% and 5% of sucrose respectively (Figure 75).

4.2.2.4 Effect of subculture passages Among three different explants (CN, NS and ST) tested for morphogenic potential and regeneration of multiple shoots in C. sophera, NS explants elicited best response in terms of maximum regeneration potential as well as provided highest number of shoots/explant on the optimized medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM). On this medium a maximum of 25.36 ± 0.34 shoots/explant, attaining an average shoot length of 6.23 ± 0.14 cm were harvested after 6 weeks of culture. The elongated shoots were excised and transferred to rooting medium for root induction, while the remaining regenerative tissue was again cultured onto the fresh medium of same composition for further multiplication and proliferation. New shoot buds differentiated from the mother tissue and after first subculture passage 28.56 ± 0.23 shoots were obtained. However, the regenerated shoots showed premature leaf fall or yellowing of leaves (Figure 76 A & B), thus to overcome these abnormalities, proliferation medium was supplemented with different concentrations (10, 20, 30, 40 and 50 µM) of AdS. Among various

130

Mean number of shoots/explant Mean shoot length (cm)

12 6

a 10 5 b 8 d 4 d b 6 e 3 c c 4 d 2

Mean shoot length (cm)

Mean number of shoots/explant 2 1

0 0 5.0 5.4 5.8 6.2 6.6 Different pH of medium Figure 74. Effect of different pH values on shoot regeneration from shoot tip explants of C. sophera on MS medium containing optimal concentration of BA (5.0 µM) and NAA (1.0 µM) after 6 weeks of culture.

Mean number of shoots/explant Mean shoot length (cm) 12 6

a 10 a 5 b 8 bc ab cd 6 c d 4

d 4 d

3 (cm) Mean shoot length

shoots/explant Mean number of 2

0 2 1 2 3 4 5

Concentrataion of sucrose

Figure 75. Effect of different concentrations of sucrose on shoot regeneration from shoot tip explants of C. sophera on MS medium containing optimal concentration of BA (5.0 µM) and NAA (1.0 µM) after 6 weeks of culture.

Explanation of Figure 76 Shoot tip necrosis, yellowing and premature leaf fall in C. sophera. A. Shoot tip necrosis observed during first subculture passage on regeneration medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) - after 3 weeks of transfer. (Bar = 0.36 cm)

B. Premature leaf fall along with shoot tip necrosis observed during first subculture passage on regeneration medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) - after 3 weeks of transfer. (Bar = 0.54 cm)

Figure 76

A B

concentrations of AdS, 20 µM proved to be the ideal for prevention of yellowing and premature leaf fall. Hence after first subculture passage the mother tissue was subcultured onto the best hormonal composition of MS + BA (5.0 µM) + NAA (1.0 µM) + AdS (20 µM) (Figure 77). The number of shoots continued to enhance up to 4th subculture passage where a maximum of 42.33 ± 0.32 shoots/explant were obtained (Figure 78). Observations were recorded till 6th subculture passages at an interval of six weeks each. Beyond 4th subculture a decline in regeneration potential was noticed and consequently number of shoots reduced to 34.88 ± 0.24 and 29.16 ± 0.52 shoots/explant at the end of 5th and 6th subculture passages respectively. The cultures obtained through CN and ST explants of C. sophera also subcultured on to the fresh medium of optimal hormonal concentrations after every 6 weeks and maintained up to six subculture passages but the highest regeneration potential was exhibited by NS explants at various subculture passages followed by CN and ST explants (Data not shown).

4.2.3 Indirect organogenesis For indirect organogenesis in C. sophera various explants viz.: cotyledonary leaf (CL), leaf (L) and root (R) explants were tested to induce callus on different PGRs. Organogenic calli were induced from CL and leaf explants whereas root explants failed to exhibit any response.

4.2.3.1 Cotyledonary leaf (CL) explant 4.2.3.1.1 Effect of explant age on callus production The cotyledonary leaf explants excised from seedlings of different age group (7, 14 and 21 days old) were cultured on MS medium without any hormone (control) as well as MS medium supplemented with 1.0, 2.5, 5.0, 7.5 and 10.0 µM of 2,4- D. Control treatment did not show any callus induction in all the three explants tested, although, augmentation of 2,4-D facilitated callus induction within 10-12 days of incubation. Initially callus differentiated from cut ends of the explant and gradually covered the entire surface. The MS medium comprised of 5.0 µM 2,4- D exhibited best response in 73.40 ± 0.45% cultures producing green, compact and nodular callus from CL explants of 14 days old seedlings after 6 weeks of inoculation. The callus was initially light green in colour and later on turned dark

131

Mean number of shoots/explant Mean shoot length (cm) 50 8.5

a a 8.0 40 b c ab c 7.5 b 30 d d bc 7.0

20 c 6.5

d (cm)length Mean shoot 10 shoots/explant Mean number of 6.0

0 5.5 I II III IV V VI Sbculture Passages

Figure 77. Effect of subculture passages on multiplication and proliferation of shoots through nodal explants of C. sophera on optimized regeneration medium*. [*Regeneration medium: I subculture passage - MS + BA (5.0 µM) + NAA (1.0 µM), II-VI subculture passage - MS + BA (5.0 µM) + NAA (1.0 µM) + AdS (20.0 µM)].

Explanation of Figure 78 Maintenance of cultures in C. sophera Cultures obtained through regenerative tissue of nodal explants after 4th subculture passage on optimal medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) + AdS (20.0 µM). (Bar = 1.50 cm)

Figure 78

green and brown. Beyond the optimal concentration (5.0 µM) of 2,4-D, a decline in the rate of callus induction (30.90 ± 0.37%) was noticed at 10.0 µM (Table 66). Explants excised from 7 and 21 days old seedlings showed moderate response and the calli induced were loose and friable from both the explants. Lower concentrations were less effective and moderate growth in callus was obtained. MS medium comprised of 5.0 µM 2,4-D exhibited an optimal growth of callus in 61.26 ± 0.50% and 56.40 ± 0.37% cultures from explants of 7 and 21 days old seedlings respectively. Higher concentrations showed inhibitory effects and reduced the callus growth from both the explants. Thus, the best growth in callus was obtained at 5.0 µM of 2,4-D from CL explants of 14 days old seedlings and consequently, such explants were used for further study (Table 66).

4.2.3.2 Leaf (L) explant 4.2.3.2.1 Effect of explant age on callus production Leaf explants excised from 20, 30 and 40 days old axenic seedlings were cultured on different concentrations of 2,4-D to select the best responsive age of the explant. MS medium without 2,4-D served as control, all the three different aged leaf explants failed to respond on control medium as well as on the medium comprised of lower concentrations (1.0 and 2.5 µM) of 2,4-D and died after 4 weeks of incubation. Conversely augmentation of 2,4-D at 5.0 µM onwards facilitated the induction of callus after 14-15 days of incubation, initially from the cut ends of the explant and later on covered the entire surface. Explants excised from 30 days old seedlings exhibited best response and produced green, compact and nodular calli at 7.5 µM 2,4-D in 53.53 ± 0.81% cultures after 6 weeks of inoculation, though, further increase in the concentration (10.0 µM) resulted in the formation of loose and friable calli. Explants of 20 and 40 days old seedlings were less responsive than that of 30 days old seedlings and provided moderate results at 7.5 µM 2,4-D in 48.26 ± 0.87% and 42.13 ± 1.02% cultures respectively (Table 67).

4.2.3.3 Effect of different auxins on callus induction from cotyledonary leaf (CL) and leaf (L) explants The CL explants (14 days old) and the leaf explants (30 days old) were selected and cultured on different concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of two

132

Table 66. Effect of age of cotyledonary leaf explant on callus induction in C. sophera cultured on MS medium containing different concentrations of 2,4-D.

PGR (µM) 7 days old explant 14 days old explant 21 days old explant

2,4-D % Rate of % Rate of % Rate of Response callus growth Response callus growth Response callus growth

0.0 00.00  0.00e NR 00.00  0.00e NR 00.00  0.00f NR 1.0 29.10  0.66d + 32.43  0.28f + 20.83  0.37d - 2.5 35.56  0.40c + 51.40  0.62d + + 31.56  0.52c - 5.0 61.26  0.50a + + + 73.40  0.45a + + + + 56.40  0.37a + 7.5 41.30  0.66b ++ 54.70  0.26c + + + 33.56  0.58b + + 10.0 34.80  0.46c + 30.90  0.37g + 18.90  0.58e +

Table 67. Effect of age of leaf explants on callus induction in C. sophera cultured on MS medium containing different concentrations of 2,4-D.

PGR (µM) 20 days old explant 30 days old explant 40 days old explant

2,4-D % Rate of % Rate of % Rate of Response callus growth Response callus growth Response callus growth

0.0 00.00  0.00d NR 00.00  0.00d NR 00.00  0.00d NR 1.0 00.00  0.00d NR 00.00  0.00d NR 00.00  0.00d NR 2.5 00.00  0.00d NR 00.00  0.00d NR 00.00  0.00d NR 5.0 31.23  0.63b + 33.36  0.37b + 25.86  0.35b + 7.5 48.26  0.87a ++ 53.53  0.81a + + + 42.13  1.02a + + 10.0 18.73  0.14c - 29.16  0.44c + 10.63  0.40c -

auxins i.e. 2,4-D and 2,4,5-T to induce organogenic callus. Between the two auxins tested 2,4-D delivered better response than 2,4,5-T at various concentrations and CL explants were proved to be more responsive than leaf explants. The lower concentrations (1.0 and 2.5 µM) of 2,4-D were failed to exhibit any response from leaf explants while CL explants at 1.0 µM 2,4-D induced callus in 32.43 ± 0.28% cultures after 6 weeks of inoculation. The medium comprised of MS + 2,4-D (5.0 µM) produced green and compact callus after 10-12 days of inoculation which later on turned dark brown in 73.40 ± 0.45% cultures through CL explants after 6 weeks, while, leaf explants were less responsive (33.36 ± 0.37%) at the same concentration, but showed optimal growth of callus in 53.53 ± 0.81% cultures at 7.5 µM of 2,4-D (Figure 79 A, B & C). The rate of callus growth reduced on further enhancement in the concentration of 2,4-D from 5.0 µM and produced loose and friable callus from CL explants (Table 68).

The explants cultured on various concentrations of 2,4,5-T exhibited poor response as compared to 2,4-D. At 1.0 and 2.5 µM of 2,4,5-T both the explants failed to induce any response, however, the rate of callus growth increased with an enhancement in the concentration of 2,4,5-T and an optimal growth of callus was obtained at 7.5 µM 2,4,5-T in 56.56 ± 0.34% and 41.33 ± 0.37% cultures from CL and leaf explants after 6 weeks of incubation. On further increasing the concentration of 2,4,5-T, reduction in the percent response as well as rate of callus growth was noticed (Table 68).

4.2.3.4 Effect of different cytokinins on callus induction from cotyledonary leaf (CL) and leaf (L) explants The CL and leaf explants were also cultured on various concentrations (1.0, 2.5, 5.0, 7.5 and 10.0 µM) of three different cytokinins (BA, Kn and TDZ) for the production of callus. The lower concentrations (1.0 and 2.5 µM) of BA and Kn failed to induce callus from both CL and leaf explants, however, an increase in the concentration of cytokinins triggered the induction of callus after 14-16 days from cut ends of the explants which was light green and friable in nature. Optimal growth of callus was recovered at 7.5 µM BA in 33.06 ± 0.60% and 21.40 ± 0.37% cultures from CL and leaf explants respectively after 6 weeks of culture.

133

Explanation of Figure 79 Production of callus and shoot differentiation in C. sophera. A. Production of compact and nodular callus from cut ends of the cotyledonary leaf (CL) explant on MS medium containing 5.0 µM 2,4- D - 4 weeks old culture. (Bar = 0.55 cm)

B. Production of loose and friable callus from cut ends of the leaf (L) explant on MS medium containing 5.0 µM 2,4-D - 4 weeks old culture. (Bar = 0.58 cm)

C. Callus induced through CL explant on 5.0 µM 2,4-D turned dark brown in colour - 6 weeks old culture. (Bar = 0.46 cm)

D. Dark brown callus showing differentiation of meristemoids on regeneration medium comprised of MS + BA (2.5 µM) - after 1 week of transfer. (Bar = 0.44 cm)

E. Differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (2.5 µM) - after 3 weeks of transfer. (Bar = 0.67 cm)

F. Differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (10.0 µM) - after 3 weeks of transfer. (Bar = 0.45 cm)

Figure 79

A B

C D

E F

Table 68. Effect of auxins on callus induction through cotyledonary leaf and leaf explants of C. sophera.

PGR (µM) Cotyledon explant Leaf explant

2,4-D 2,4,5-T % Rate of % Rate of Response callus growth Response callus growth

1.0 - 32.43  0.28f + 00.00  0.00g NR 2.5 - 51.40  0.62d + + 00.00  0.00g NR 5.0 - 73.40  0.45a + + + + 33.36  0.37c + 7.5 - 54.00  0.26c + + + 53.53  0.81a + + + 10.0 - 30.90  0.37g + 29.16  0.44c + - 1.0 00.00  0.00i NR 00.00  0.00g NR - 2.5 00.00  0.00i NR 00.00  0.00e NR - 5.0 34.53  0.26e + 23.40  0.45e + - 7.5 56.56  0.34b + + + 41.33  0.37e + + - 10.0 28.90  0.66h + 20.06  0.69f +

The explants cultured on Kn supplemented media produced soft, watery and yellowish calli at 7.5 µM concentration with only 26.20 ± 0.91% and 16.66 ± 0.44% response at CL and leaf explants respectively (Table 69). Amongst all the three cytokinins tested, TDZ was found to be the best for the induction of regenerative calli, but leaf explants were comparatively less responsive than CL explants on TDZ supplemented media and exhibited no response at 1.0 µM TDZ. However the CL explants initiated induction of callus within 14 days of culture and produced yellowish and loose calli in 11.26 ± 1.56% cultures at 1.0 µM TDZ. Optimal response (41.50 ± 1.30%) was achieved through CL explant at 7.5 µM of TDZ, initially; the callus was compact and greenish in colour later the surface turned dark brown while the inner tissue remained yellowish green in colour with healthy growth. The leaf explants produced loose and friable callus and at 7.5 µM TDZ, only 29.86 ± 2.08% response was obtained after 6 weeks of culture. Further increase in the level of TDZ reduced the rate of callus induction (Table 69).

4.2.3.5 Shoot differentiation from cotyledonary leaf derived callus 4.2.3.5.1 Effect of cytokinins on multiple shoot differentiation The compact and dark brown calli raised on MS medium comprised of 5.0 μM 2,4-D from 14 days old CL explants were selected for organogenesis and transferred to shoot regeneration medium comprised of different cytokinins (BA, Kn and TDZ) at various concentrations. The calli transferred to control medium did not induce shoot bud differentiation. While the dark brown and compact callus turned green with the development of shoot buds primordia on BA supplemented media after 10 days of transfer and shoot buds were induced after 15-17 days (Figure 79 D & E). BA was found to be better than Kn and TDZ and at 1.0 µM BA, 41.33 ± 0.72% response was obtained producing an average of 3.96 ± 0.14 shoots/explant after 6 weeks of culture. The rate of shoot differentiation further increased with an increase in the concentration of BA to 2.5 µM, where a maximum of 7.56 ± 0.23 shoots/explant were produced attaining highest shoot length of 4.30 ± 0.20 cm in 60.83 ± 0.32% cultures after 6 weeks of incubation. Further increase in the concentration of BA proved to be inhibitory and reduced the regeneration potential to 43.56 ± 0.78% and 30.63 ± 0.23% as

134

Table 69. Effect of different cytokinins on callus induction through cotyledonary leaf and leaf explants of C. sophera.

PGR (µM) Cotyledon explant Leaf explant

BA Kn TDZ % Rate of % Rate of Response callus growth Response callus growth

1.0 - - 00.00  0.00i NR 00.00  0.00h NR 2.5 - - 00.00  0.00i NR 00.00  0.00h NR 5.0 - - 27.56  0.60d - 14.90  0.20d - 7.5 - - 33.06  0.60b + 21.40  0.37b + 10.0 - - 20.73  0.68f + 11.20  0.51f + - 1.0 - 00.00  0.00i NR 00.00  0.00h NR - 2.5 - 00.00  0.00i NR 00.00  0.00h NR - 5.0 - 15.40  0.79g + 11.33  1.45f - - 7.5 - 26.20  0.91e + 16.66  1.15c + - 10.0 - 11.63  0.81h - 10.10  1.73g - - - 1.0 11.26  1.56h - 00.00  0.00h NR - - 2.5 21.96  2.30f + 12.76  1.45e - - - 5.0 31.30  0.96c + + 21.00  1.45b + - - 7.5 41.50  1.30a + 29.86  2.08a + - - 10.0 24.63  0.60e - 16.46  1.73c -

well as the number of shoots to 3.66 ± 0.20 and 1.56 ± 0.23 shoots/explant at 5.0 and 10.0 µM BA respectively (Figure 79 F, Table 70).

Augmentation of Kn exhibited poor response than BA but better than TDZ for shoot differentiation. Lower concentration (1.0 µM) of Kn was failed to induce shoot buds but at concentration of 5.0 µM, 35.90 ± 0.37% cultures differentiated multiple shoot buds producing an average of 3.86 ± 0.24 shoots/explant after 6 weeks of transfer. TDZ supplemented media resulted in further growth of callus mass and thus suppressed the shoot buds differentiation with the production of a maximum of 3.03 ± 0.27 shoots/explant on 5.0 µM TDZ with 31.06 ± 0.23% response after 6 weeks of transfer. Higher concentration of TDZ adversely affected the regeneration potential with heavy callusing and retarded the growth and development of new shoots (Table 70).

4.2.3.5.2 Effect of cytokinin-auxin combinations on multiple shoot differentiation The compact and brown calli obtained from 14 days old CL explants on 5.0 µM of 2,4-D were also transferred to cytokinin-auxin combinations. Augmentation of auxins at various concentrations (0.5, 1.0 and 2.0 µM) with the optimal concentration (2.5 µM) of BA enhanced the rate of shoots multiplication and shoot buds were differentiated within 7-8 days of incubation. Addition of 0.5 μM NAA along with BA (2.5 µM) to the medium enhanced the rate of shoot buds differentiation and produced a maximum of 14.63 ± 0.23 shoots/explant in 74.83 ± 0.32% cultures compared to the medium containing BA alone wherein only 7.56 ± 0.23 shoots/explant were produced in 60.83 ± 0.32% cultures after 6 weeks of transfer (Figure 80 A-C). Higher concentrations of NAA (1.0 and 2.0 μM) resulted in heavy callus production which adversely affected the regeneration process and thus retarded the growth and development of new shoots. Supplementation of IAA and IBA at various concentrations delivered moderate results and only a slight increase in the number of shoots was observed. A maximum of 9.73 ± 0.26 and 8.80 ± 0.26 shoots/explant with 63.26 ± 0.24% and 62.33 ± 0.20% response were obtained in the medium containing BA (2.5 µM) + IAA (1.0 µM) and BA (2.5 µM) + IBA (1.0 µM) respectively. Further

135

Table 70. Effect of different cytokinins on shoot differentiation from cotyledonary leaf derived callus of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA Kn TDZ

0.0 0.0 0.0 00.00  0.00i 0.00  0.00e 0.00  0.00g 1.0 - - 41.33  0.72c 3.96  0.14b 3.50  0.17bc 2.5 - - 60.83  0.32a 7.56  0.23a 4.30  0.20a 5.0 - - 43.56  0.78b 3.66  0.20b 3.83  0.20ab 10.0 - - 30.63  0.23e 1.56  0.23d 3.06  0.17cd - 1.0 - 00.00  0.00i 0.00  0.00e 0.00  0.00g - 2.5 - 21.40  0.58f 2.06  0.20d 2.93  0.26de - 5.0 - 35.90  0.37d 3.86  0.24b 4.10  0.20a - 10.0 - 22.60  0.30f 1.76  0.14d 2.70  0.17de - - 1.0 09.80  0.51h 1.43  0.26d 0.00  0.00g - - 2.5 21.56  0.63f 2.10  0.23d 2.50  0.17ef - - 5.0 31.06  0.23e 3.03  0.27c 3.00  0.11cde - - 10.0 18.06  0.23g 1.66  0.24d 2.03  0.20f

Explanation of Figure 80 Differentiation of shoots from cotyledonary leaf derived callus in C. sophera. A. Differentiation of multiple shoot buds on regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) - 3 weeks old culture. (Bar = 0.59 cm)

B. Multiplication and proliferation of shoots on MS medium containing BA (2.5 µM) and NAA (0.5 µM) - 4 weeks old culture. (Bar = 0.64 cm)

C. Proliferation and elongation of shoots on medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) - 6 weeks old culture. (Bar = 0.76 cm)

Figure 80

B C

increase in the concentration of IAA and IBA reduced the percent response as well as number of shoots/explant (Table 71).

4.2.3.6 Effect of subculture passages and maintenance of cultures The cotyledonary leaf derived callus regularly subcultured on to the fresh medium for continuous multiplication and proliferation of shoots after every 6 weeks. The elongated shoots were removed from the regenerative tissue and transferred to rooting media at the end of each subculture passage. The new shoots continued to develop up to 2nd subculture, where the maximum 20.60 ± 0.45 shoots/explant with highest shoot length of 6.43 ± 0.23 cm were obtained on the optimized medium containing MS + BA (2.5 µM) + NAA (0.5 µM) (Figure 81 & 82). Beyond 2nd subculture passage onwards the regeneration potential of the mother tissue decreased and thus the number of shoots reduced to 7.43 ± 0.23 shoots/explant at the end of 4th subculture passage.

4.2.4 Rooting in microshoots The development of healthy root system is required for the successful establishment of regenerated shoots in the external environment. Hence appropriate sized (4-5 cm) microshoots of C. sophera were excised from the regenerating cultures and transferred to different rooting media for the induction of roots either in vitro or ex vitro.

4.2.4.1 In vitro rooting The microshoots (4-5 cm long) were excised from the cultures and transferred to rooting media comprised of full and half strength MS media with or without auxins (IAA, IBA and NAA) at different concentrations. All the treatments applied for rooting showed induction of roots from microshoots within 2 weeks of transfer to the rooting media. Full and half strength MS media devoid of auxins induced relatively few and smaller roots in 42.33 ± 1.45% and 65.00 ± 1.73% of the microshoots respectively (Figure 83 A & B). However addition of different auxins in the medium significantly enhanced the rooting response. The best rooting response (93.66 ± 2.40%) was achieved on the medium comprised of half strength MS medium + IBA (1.0 µM), wherein roots were induced within 6-7 days of transfer and a maximum of 5.70 ± 0.47 roots/shoot with an average root length of 6.63 ± 0.49 cm were obtained after 4 weeks of transfer (Figure 83 C,

136

Table 71. Effect of optimal concentration of BA with different auxins on shoot differentiation from cotyledonary leaf derived callus of C. sophera.

PGR (M) Regeneration Mean number of Mean shoot (%) shoots/explant length (cm) BA NAA IAA IBA

2.5 0.5 - - 74.83  0.32a 14.63  0.23a 5.73  0.20a 2.5 1.0 - - 65.30  0.20b 11.63  0.20b 5.26  0.17ab 2.5 2.0 - - 62.10  0.37d 8.73  0.26d 4.76  0.14bc 2.5 - 0.5 - 61.46  0.67d 7.86  0.20e 3.76  0.14d 2.5 - 1.0 - 63.26  0.24e 9.73  0.26c 4.46  0.14c 2.5 - 2.0 - 58.56  0.23f 6.56  0.23f 3.33  0.24de 2.5 - - 0.5 60.13  0.23e 6.66  0.27f 2.96  0.14ef 2.5 - - 1.0 62.33  0.20cd 8.80  0.26d 3.23  0.14e 2.5 - - 2.0 55.06  0.34g 6.23  0.14f 2.53  0.14g

Mean number of shoots/explant Mean shoot length (cm) 25 10

a 9 20 b

8 15 c 7 a 10 a d 6 b Mean shootlength (cm) b 5

Mean numbershoots/explant of 5

0 4 I II III IV

Subculture Passage

Figure 81. Effect of subculture passages on shoot differentiation efficiency of cotyledon derived callus of C. sophera on optimal regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM).

Explanation of Figure 82 Subculturing and maintenance of CL derived cultures in C. sophera. Cultures obtained from cotyledonary leaf derived callus on optimal medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) - after 2nd subculture passage. (Bar = 1.81 cm)

Figure 82

Explanation of Figure 83 In vitro rooting on agar gelled medium in C. sophera. A. Development of thin short roots on MS basal medium - 4 weeks old culture. (Bar = 1.25 cm)

B. Development of thin long roots on half strength MS medium - 4 weeks old culture. (Bar = 1.38 cm)

C. Development of long healthy and highly branched roots on half strength MS medium supplemented with 1.0 µM IBA - 4 weeks old culture. (Bar = 1.56 cm)

D. Development of short stunted roots along with basal callusing on half strength MS medium supplemented with 2.5 µM IBA - 4 weeks old culture. (Bar = 1.13 cm)

E. Development of thin roots on half strength MS medium supplemented with 1.0 µM NAA - 4 weeks old culture. (Bar = 1.04 cm)

F. Development of thin delicate roots on half strength MS medium supplemented with 1.0 µM IAA - 4 weeks old culture. (Bar = 1.38 cm)

Figure 83

A B C

D E F

Table 72). Roots were healthy, thick and having well developed secondary branches. Further increase in IBA concentration resulted in the formation of basal callus and thus hampered the initiation and growth of roots. On half strength MS + IBA (2.5 µM) the rooting percentage was reduced to 80.33 ± 2.60% with only 4.03 ± 0.20 roots/shoot (Figure 83 D). NAA and IAA were found to be less effective as compared to IBA and an average of 4.16 ± 0.14 and 2.83 ± 0.20 roots/shoot were produced in half strength MS media supplemented with 1.0 µM NAA and 1.0 µM IAA respectively (Figure 83 E & F, Table 72).

In vitro regenerated microshoots were also rooted in phytagel gelled rooting medium containing three different auxins at the same concentrations. Induction of roots from the cut ends of the shoots took place within 10-12 days and thin, dark brown to black roots were produced in all the treatments (Figure 84 A & B). The best rooting (96.00 ± 2.08%) was achieved on half strength MS medium containing 1.0 µM IBA, wherein roots were induced after 7-8 days and the average number (7.63 ± 0.23) of roots/shoot was more than agar gelled medium, while the average root length (4.66 ± 0.35 cm) was less after 4 weeks of transfer (Figure 84 C, Table 73). Higher concentration (2.5 µM) of IBA reduced the rooting potential and resulted in the formation of short roots (Figure 84 D). NAA and IAA proved to be less effective than IBA for root induction (Figure 84 E & F).

4.2.4.2 Ex vitro rooting Ex vitro rooting is an alternative and economical method, saves time and requires less labor as ex vitro rooted plantlets do not need any additional acclimatization prior to their transfer to greenhouse or field conditions. Microshoots measuring 4-5 cm in length were dipped in different concentrations (50, 100, 150, 200, 250 and 300 µM) of IBA for about 30 min, subsequently transferred to thermocol cups containing sterile soilrite and covered with polythene bags to ensure high relative humidity. Shoots were irrigated with quarter strength MS salt solution for 2 weeks followed by tap water. After 6 weeks of treatment, shoots were taken out from the soilrite and healthy roots with lateral branches were observed from the cut ends of the shoots. Best rooting response of 90.66 ± 2.96% was detected in microshoots dipped in 200 µM IBA (Figure 85 & 86). These shoots produced a maximum of 5.06 ± 0.17

137

Table 72. Effect of different auxins on in vitro root induction in microshoots of C. sophera in agar gelled medium.

Treatment (M) Response Mean number of Mean root (%) roots/shoot length (cm)

MS 42.33  1.45f 1.76  0.14d 2.43  0.23e ½ MS 65.00  1.73de 2.43  0.23cd 2.66  0.17de ½ MS + IBA (0.5) 83.66  2.02b 3.80  0.20b 3.36  0.20cde ½ MS + IBA (1.0) 93.66  2.40a 5.70  0.47a 5.63  0.49a ½ MS + IBA (2.5) 80.33  2.60b 4.03  0.20b 5.20  0.28ab ½ MS + NAA (0.5) 72.33  1.45c 4.00  0.17b 4.96  0.39ab ½ MS + NAA (1.0) 78.33  1.20b 4.16  0.14b 4.23  0.44bc ½ MS + NAA (2.5) 67.00  1.73cde 3.60  0.23b 3.60  0.43cd ½ MS + IAA (0.5) 65.00  1.73de 2.70  0.15c 2.63  0.29de ½ MS + IAA (1.0) 70.00  1.15cd 2.83  0.20c 3.23  0.14cde ½ MS + IAA (2.5) 62.33  1.45e 2.46  0.17cd 2.50  0.17e

Explanation of Figure 84 In vitro rooting in phytagel gelled medium in C. sophera. A. Development of short roots on MS basal medium - 4 weeks old culture. (Bar = 1.19 cm)

B. Development of thin delicate roots on half strength MS medium - 4 weeks old culture. (Bar = 1.56 cm)

C. Development of long, thick and branched roots on half strength MS medium supplemented with 1.0 µM IBA - 4 weeks old culture. (Bar = 1.31 cm)

D. Development of thin stunted roots on half strength MS medium supplemented with 2.5 µM IBA - 4 weeks old culture. (Bar = 1.38 cm)

E. Development of thin delicate roots on half strength MS medium supplemented with 1.0 µM NAA - 4 weeks old culture. (Bar = 1.78 cm)

F. Development of thin delicate roots on half strength MS medium supplemented with 1.0 µM IAA - 4 weeks old culture. (Bar = 1.25 cm)

Figure 84

A B C

D E F

Table 73. Effect of different auxins on in vitro root induction in microshoots of C. sophera in phytagel gelled medium.

Treatment (M) Response Mean number of Mean root (%) roots/shoot length (cm)

MS 47.00  1.73h 3.70  0.58e 1.63  0.23f ½ MS 66.66  1.76g 4.23  0.14de 2.23  0.14ef ½ MS + IBA (0.5) 85.00  1.73b 4.90  0.20d 4.86  0.35a ½ MS + IBA (1.0) 96.00  2.08a 7.63  0.23a 4.66  0.35a ½ MS + IBA (2.5) 82.33  1.45bc 6.90  0.20ab 3.56  0.23bc ½ MS + NAA (0.5) 75.33  1.45de 4.50  0.28de 3.10  0.20cd ½ MS + NAA (1.0) 79.66  1.45cd 6.60  0.23bc 3.83  0.20b ½ MS + NAA (2.5) 69.-00  2.08fg 4.16  0.20de 2.50  0.17de ½ MS + IAA (0.5) 67.66  1.45fg 3.93  0.29e 2.43  0.17de ½ MS + IAA (1.0) 72.33  1.45ef 5.96  0.14c 3.23  0.14bc ½ MS + IAA (2.5) 65.33  1.45g 3.90  0.20e 2.23  0.14ef

% Response 100 a A 90 b 80 bc

c 70

% Response 50

40 e 30

20 0 50 100 150 200 250 300 350

IBA (µM)

Number of roots Root length (cm)

6 B a a 5 ab ab c abc bcd 4 cd d cd d d

roots/root length Number of 1

0 50 100 150 200 250 300 IBA (µM)

Figure 85. Effect of pulse treatment of different concentrations of IBA for half an hour to induce ex vitro rooting in microshoots of C. sophera. (A) Line represents percentage response (%) for root induction. (B) Bars represent average number of roots/shoot and average root length (cm).

Explanation of Figure 86 Ex vitro rooting in C. sophera. A. A group of ex vitro rooted plantlets through pulse treatment of 30 min with different concentrations (50-300 µM) of IBA - after 4 weeks of treatment. (Bar = 2.43 cm)

B. Development of short stunted roots through pulse treatment of 30 min with 150 µM IBA - after 5 weeks of treatment. (Bar = 1.50 cm)

C. Development of long healthy and branched roots through pulse treatment of 30 min with 200 µM IBA - after 5 weeks of treatment. (Bar = 1.50 cm)

Figure 86

B C

roots/shoot having maximum root length of 5.93 ± 0.17 cm after 6 weeks. Through ex vitro rooting technique healthy and longer roots were obtained.

4.2.5 Hardening and acclimatization Hardening and acclimatization of plantlets is essential for the survival of regenerated plantlets in the external environment. Regenerated plantlets with 4- 5 fully expanded leaves and well-developed root system were hardened off under aseptic conditions in three different planting substrates (garden soil, soilrite and vermiculite). Hardening of plantlets was done by the procedure described in the materials and methods. Maximum plantlets survived in the soilrite (97.66 ± 1.45%) followed by vermiculite which provided moderate response (90.66 ± 2.33%) while garden soil displayed minimum survival rate (70.66 ± 2.96%) (Figure 87 & 88 A-D). Almost 90% plantlets survived in the field conditions after successful acclimatization and exhibited normal growth and development pattern with no detectable morphological variations when compared with the in vivo grown plants (Figure 89).

4.2.6 Synthetic seeds The synthetic or artificial seeds of C. sophera were prepared by encapsulation of nodal segments excised from one month old microshoots to assess the regeneration potential of the explant under various culture conditions.

4.2.6.1 Effect of different concentrations of sodium alginate on beads formation

Different concentrations of Na2-alginate (1, 2, 3, 4 and 5%) were tested with 100 mM CaCl2· 2H2O were tested to evaluate the effect of gelling matrix on beads formation as depicted in Table 74. Lower concentrations (1 and 2%) of gelling matrix resulted in the formation of very soft and friable beads which were difficult to handle and such beads were failed to germinate or regenerate microshoots on MS basal medium. The optimum concentration of gelling matrix for the production of uniform, firm and clear beads was found to be 3% Na2-alginate with 100 mM CaCl2·2H2O exhibiting 67.33 ± 1.45% conversion response. The higher concentrations (4 and 5%) of gelling matrix formed hard beads which retarded the regeneration potential of the explant and revealed poor conversion

138

Garden soil Soilrite Vermiculite 120

100 a a

80 b

survival % 40

0 Garden soil Soilrite Vermiculite Planting substrate

Figure 87. Effect of different planting substrates on survival percentage (%) of regenerated plantlets of C. sophera during ex vitro acclimatization.

Explanation of Figure 88 Acclimatization and transferring of regenerated plantlets in C. sophera. A. Successfully acclimatized in vitro rooted plantlet in sterile soilrite - after 3 weeks of transfer. (Bar = 1.29 cm)

B. In vitro regenerated plantlet in soil - after 2 week of transfer from soilrite. (Bar = 8.33 cm)

C. Regenerated plantlet with normal inflorescence development – after 1 month of transfer from soilrite. (Bar = 11.25 cm)

D. In vitro raised plant – after 2 months of transfer from soilrite. (Bar = 12.50cm)

Figure 88

B C D

Explanation of Figure 89 A regenerated plant of C. sophera in field conditions with normal morphology and vigorous growth - 2 ½ months old.

Figure 89

responses i.e. 49.00 ± 2.08% and 33.00 ± 2.08% at 4% and 5% Na2-alginate respectively.

4.2.6.2 Effect of different concentrations of calcium chloride on beads formation Different concentrations (25, 50, 75, 100 and 200 mM) of calcium chloride were tested with 3% Na2-alginate to produce ideal beads. Lower concentrations (25 mM and 50 mM) of complexing agent produced very soft and fragile beads which were difficult to carry and indicated zero conversion response on MS medium.

Beads produced using 75 mM CaCl2·2H2O with 3% Na2-alginate exhibited 61.00 ± 2.08% conversion response, however, the beads were soft. The uniform and ideal synseeds with maximum conversion response of 67.33 ± 1.45% were produced with 100 mM CaCl2·2H2O and 3% gelling matrix. Higher concentration (200 mM) of complexing agent resulted in the production of hard beads which exhibited 44.00 ± 2.08% conversion response on MS basal medium (Table 75).

4.2.6.3 Effect of PGRs on conversion of synthetic seeds into plantlets

The optimized concentration of gelling matrix (3% Na2-alginate) and complexing agent (100 mM CaCl2·2H2O) was used for the production of uniform and ideal beads by encapsulating NS. These artificial seeds were cultured on MS medium with or without any PGR. The MS medium without any hormone showed 67.33 ± 1.45% conversion of beads into shoots (Figure 90 A & B). Addition of BA to MS medium enhanced the regeneration potential of the beaded explant and the shoots emerged out within 12 days of culture. The medium containing 2.5 µM of BA revealed 76.00 ± 2.30% conversion of beads into shoots after 6 weeks of culture (Figure 90 C). The regenerated microshoots were failed to develop roots on the same medium. Addition of NAA with the optimal concentration (2.5 µM) of BA also did not help in the induction of roots from microshoots, while increased the conversion response to 92.33 ± 1.45% at 0.5 µM NAA (Figure 90 D & E). The regenerated shoots of appropriate shoot length were isolated and transferred to optimized rooting media [half strength MS + IBA (1.0 µM)] for root induction and the development of complete plantlets (Table 76).

139 Table 74. Effect of different concentrations of sodium alginate with 100 mM calcium chloride on encapsulated nodal segments of C. sophera.

Na2-alginate Conversion response Texture of beads (% w/v) (%)

1.0 00.00  0.00d very soft and fragile beads 2.0 00.00  0.00d very soft and fragile beads 3.0 67.33  1.45a soft and uniform beads 4.0 49.00  2.08b hard beads 5.0 33.00  2.08c hard beads

Table 75. Effect of different concentrations of calcium chloride with 3% sodium alginate on encapsulated nodal segments of C. sophera.

CaCl2·2H2O Conversion response Texture of beads (mM) (%)

25 00.00  0.00d very soft and fragile beads

50 00.00  0.00d very soft and fragile beads

75 61.00  2.08b soft and fragile beads 100 67.33  1.45a soft and uniform beads

200 44.00  2.08c hard beads

Explanation of Figure 90 Synthetic seed production in C. sophera. A. Encapsulated aseptic nodal segments with 3% sodium alginate and 100 mM calcium chloride on MS basal medium - 1 day old culture. (Bar = 0.92 cm)

B. Germination of synseeds on MS basal medium - 2 weeks old culture. (Bar = 0.83 cm)

C. Germination of synseeds on MS medium supplemented with 2.5 µM BA - 2 weeks old culture. (Bar = 2.72 cm)

D. Development of multiple shoots from encapsulated nodal segments on medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) - 2 weeks old culture. (Bar = 0.45 cm)

E. -do- 3 weeks old culture. (Bar = 0.47 cm)

Figure 90

A B

D E

4.2.6.4 Low temperature storage The encapsulated NS as well as non-encapsulated NS were kept at 4ºC for different time periods (0, 1, 2, 4, 6 and 8 weeks) to check the reproducibility of the explants on optimized regeneration medium after different storage periods. The explants showed maximum conversion at 0 days of storage; however the conversion response decreased as the time period was increased. Encapsulated NS segments showed a gradual decrease in the conversion response from 92.33 ± 1.45% to 64.00 ± 3.05% at 0 days and 4 weeks of storage at 4ºC respectively, but the non-encapsulated NS showed a drastic decrease in the regeneration potential from 94.33 ± 2.33% to 25.00 ± 2.88% within 4 weeks of storage. Further increase in storage duration showed a sharp reduction in the regeneration potential of beads and at the end of 8 weeks of storage, 41.00 ± 2.08% beads showed conversion into microshoots (Table 77).

4.2.6.5 Ex vitro germination of synthetic seeds The encapsulated NS were failed to induce roots on optimized regeneration medium, therefore, synthetic seed were also sown into the sterilized soilrite for the induction of roots under ex vitro conditions. The regenerated plantlets showed 2-3 roots/shoot in 30% beads after 6 weeks of sowing in the soilrite.

4.2.7 Physiological studies Estimation of different photosynthetic pigments (Chlorophyll a, b, total chlorophyll and carotenoids) was carried out during different acclimatization periods. The rate of photosynthesis (PN ratio) was also measured at different acclimatization periods.

4.2.7.1 Chlorophyll a, b and total chlorophyll content The contents of chlorophyll a, b as well as total chlorophyll in acclimatized plantlets of C. sophera initially decreased and later on increased linearly with an increase in acclimatization periods from 0 to 4 weeks of transfer. During first week of transfer to ex vitro conditions, a decrease in all the photosynthetic pigments was observed. A decrease in the content of chlorophyll a from 0.53 to 0.38 mg/g, chlorophyll b from 0.39 to 0.32 mg/g and the total chlorophyll from 0.93 to 0.70 mg/g was observed. By the end of second week a sharp increase in

140

Table 76. Effect of different plant growth regulators on encapsulated nodal segments of C. sophera.

PGRs Conversion response

(µM) (%)

MS 67.33  1.45d MS + BA (1.0) 71.66  3.52c MS + BA (2.5) 76.00  2.30bc MS + BA (5.0) 69.00  3.78cd MS + BA (2.5) + NAA (0.5) 92.33  1.45a MS + BA (2.5) + NAA (1.0) 82.33  1.45b MS + BA (2.5) + NAA (2.0) 69.00  2.08cd

Table 77. Effect of storage at 4ºC for different time periods on conversion of encapsulated and non-encapsulated nodal segments of C. sophera on MS medium containing BA (2.5 µM) + NAA (0.5 µM).

Storage period Conversion response Conversion response (weeks) of encapsulated NS (%) of non-encapsulated NS (%)

0 92.33  1.45a 94.33  2.33a 1.0 89.00  2.08a 59.00  2.08b

2.0 75.66  3.48b 39.66  1.45c

4.0 64.00  3.05c 25.00  2.88d 6.0 52.00  1.15c 19.00  2.08d 8.0 41.00  2.08d 12.33  1.45e

pigments content was noticed but after that increased slowly. By the end of fourth week the content of chlorophyll a (1.20 mg/g), chlorophyll b (1.16 mg/g) and the total chlorophyll (2.36 mg/g) got almost stabilized with a very gradual increase (Figure 91 & 92).

4.2.7.2 Carotenoids content Similar to the chlorophyll pigments, carotenoids also showed the same trend during different acclimatization periods. Initially the carotenoids content was reduced to 0.20 mg/g from 0.22 mg/g during first week of transfer to the ex vitro conditions. Later on the content was increased to 0.46 mg/g after second week and by the end of fourth week the carotenoids content was recorded as 0.53 mg/g (Figure 93).

4.2.7.3 Net photosynthetic rate (PN ratio)

The amount of CO2 absorbed by per unit area of the plant per second is calculated as Net photosynthetic rate (PN ratio). It was estimated during different acclimatization periods of in vitro raised plantlets. Initially a decrease in the -2 -1 amount of CO2 absorbed per unit area per second (4.16 to 3.33 µmol CO2m s ) was recorded when plantlets were transferred to the external environment, during first week. As soon as the plantlets get adapted to the external environment, the net photosynthetic rate increased linearly and 8.70 µmol CO2 -2 -1 -2 -1 m s and 10.23 µmol CO2 m s were recorded by the end of third and fourth week of acclimatization respectively (Figure 94).

4.2.8 Histological studies The histological analysis of regenerating tissue of C. sophera was performed to confirm the mode of shoot organogenesis via direct regeneration through nodal segment (NS) or callus formation through cotyledonary leaf (CL).

The nodal segments cultured on medium comprised of MS + BA (5.0 µM) were fixed at various stages of shoot morphogenesis for histological analysis. One week old regenerating tissue showed only enlargement and swelling of the explant without any differentiation of meristematic zone (Figure 95 A). However, meristematic zones were clearly visible in the sections of two weeks old cultures; these meristematic regions differentiate to produce multiple shoot buds (Figure

141 Chlorophyll a Chlorophyll b 1.4 a ab 1.2 a a b 1.0

0.8 b

0.6 c

Pigment contents (mg/g) c 0.4 c

0.2 0 1 2 3 4 Acclimatization period (weeks)

Figure 91. Change in chlorophyll a and b contents (mg/l) of in vitro raised C. sophera plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

Total chlorophyll 2.6

2.4 a a 2.2 2.0 1.8 b 1.6

1.4

1.2 c

Pigment content (mg/g) content Pigment 1.0

c 0.8 0.6 0.4 0 1 2 3 4 Acclimatization period (weeks)

Figure 92. Change in total chlorophyll content (mg/l) of in vitro raised C. sophera plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

carotenoids (mg/g) 0.6 a

a 0.5 a

0.4

0.3 b b

Carotenoids content (mg/g) content Carotenoids 0.2

0.1 0 1 2 3 4 Acclimatization period (weeks)

Figure 93. Change in carotenoid content (mg/l) of in vitro raised C. sophera plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

Net photosynthetic rate 12

-1 )

s 10

-2

2 b

8 c

d 4 d

Netphotosynthetic rate (µmolCO 2 0 1 2 3 4 Acclimatization period (weeks) Figure 94. Change in Net Photosynthetic rate (μmol CO2 m-2 s-1) of in vitro raised C. sophera plantlets during acclimatization period. The line represents Mean ± SE of three repeated experiments. Line denoted by the same letter within weeks variables are not significantly different (P=0.05) using DMRT.

95 B). After several anticlinal and periclinal divisions of cells, complete shoot buds were formed having well distinct apical dome and leaf primordia which later on develop into shoots (Figure 95 C & D). The nodal explants cultured on TDZ supplemented MS medium showed various abnormal structures and resulted in the formation of deformed shoots. The prolonged exposure to TDZ (5.0 µM) resulted in the formation of highly abnormal shoot buds having deformed apical dome with multiple or single leaf primordia (Figure 96 A & B). Cultures on higher concentration of TDZ (10.0 µM) also produced abnormalities with deformed apical dome having abnormal structures of leaf primordial (Figure 96 C). Such shoot buds developed and produced abnormal and stunted shoots.

The callus tissue produced through CL explants on MS medium containing 5.0 µM 2,4-D showed well differentiated outer and inner rings of meristematic zones or meristemoids embedded in parenchymatous cells of the callus tissue (Figure 97 A). Most of the meristemoids were situated at the peripheral region and frequently differentiated into shoot buds on transferring to the regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) (Figure 97 B & C). The embedded meristemoids also differentiated to produce shoot buds having apical dome and well-marked leaf primordia but exhibited slower growth compared to peripheral meristemoids (Figure 97 D).

142

Explanation of Figure 95 Histological sections showing differentiation pattern in nodal segment (NS) culture of C. sophera.

A. A portion of the section showing swelling and enlargement of the explant on MS + BA (5.0 µM). (Bar = 100 µm)

B. Differentiation of multiple shoot buds (sb) from the explant. (Bar = 100 µm)

C. A well-developed shoot bud having apical dome (ap) flanked with a pair of leaf primordia (lp). (Bar = 250 µm)

D. A developing shoot bud at later stages of development. (Bar = 100 µm)

Figure 95

A B lp lp

ap lp ap lp

C D

Explanation of Figure 96 Histological sections showing differentiation pattern in NS explants of C. sophera cultured on TDZ supplemented medium.

A. An abnormal shoot bud showing deformed apical dome (ap) having multiple leaf primordia (lp) on prolonged exposure to TDZ (5.0 µM) supplemented MS medium. (Bar = 150 µm) B. An abnormal shoot bud with deformed apical dome (ap) and single leaf primordium (lp) on the same medium. (Bar = 120 µm) C. An abnormal shoot bud with deformed apical dome (ap) and double leaf primordia (lp) on same side on MS medium supplemented with 10.0 µM TDZ. (Bar = 80 µm)

Figure 96

lp ap lp

lp ap

A B

Explanation of Figure 97 Histological sections showing differentiation pattern in cotyledonary leaf (CL) culture of C. sophera.

A. Organization of meristemoids in the form of a ring embedded in callus tissue. (Bar = 120 μm) B. Differentiation of shoot buds (db) from the meristemoids in the peripheral region of the callus tissue. (Bar = 50 μm) C. -do- (Bar = ) D. Development of a deep seated shoot bud in callus tissue showing apical dome (ap) surrounded by single leaf primordia (lp). (Bar = 50 μm)

Figure 97

sb A B

lp sb ap

lp sb C D

Chapter 5 Discussion

Chapter 5

DISCUSSION

All the living cells within the plant body possess the potential to regenerate the entire plant i.e. to express their totipotency. This potentiality has been exploited through the culture of cells, tissues and organs in vitro and provided the opportunity to micropropagate specific, and often elite, plant clones by the stimulation of axillary buds and latterly through the production of somatic embryos. Furthermore, manipulation of culture conditions has facilitated the adventitious regeneration of plants from whole organs, explants, callus and isolated protoplasts. Advances achieved during the 1970s and 1980s in the application of in vitro techniques to the genetic manipulation of members of plant families, such as the Solanaceae, stimulated interest in extending such procedures to other economically important crop plants, including legumes (Davey et al. 1994). The micropropagation of legumes was first reported by Kartha and Gamborg (1978) who cultured excised meristems of pea, Pisum sativum (0.2-0.3 mm in diameter) on a B5 based medium supplemented with BA and NAA. Since then a large number of herbaceous and woody leguminous plants including many grain legumes, forage legumes, timber and fuel legumes, forest tree species, ornamental legumes as well as medicinally valuable legumes have been successfully propagated exploiting the in vitro techniques (Gharyal and Maheshwari 1983; Maheswaran and Williams 1984; Dhanalakshmi and Lakshmanan 1992; Kaneda et al. 1997; Beck et al. 1998; Chand and Singh 2004a; 2004b; Barik et al. 2005; Husain et al. 2008; Vibha et al. 2009; Cheruvathur et al. 2010; Dhabhai and Batra 2010; Ochatt et al. 2010; Parveen and Shahzad 2010; 2011; Parveen et al. 2010; 2012 and Singh and Tiwari 2010).

The in vitro techniques offer an alternative means for the propagation of medicinally important plants through the development of simple and reliable protocols which are efficient, reproducible, cost effective and adaptable and also offer a platform for genetic manipulation (Jusekutty et al. 1993; Roy et al. 1994; Bhattacharyya and Bhattacharyya 2001; Olmos et al. 2002; Chaturvedi et al.

143

2004 and Sathyanarayana et al. 2008). The present study was carried out to explore the morphogenetic potential of two medicinally important leguminous plant species belonging to genus Cassia i.e. C. angustifolia and C. sophera. In both the plant species various explants (CN, NS, ST, CL, L and R) were collected from in vitro raised seedlings of different age group and cultured on different combinations and concentrations of PGRs (BA, Kn, 2iP, TDZ, IAA, IBA, NAA, 2,4-D and 2,4,5-T) to develop efficient and reproducible protocols and obtain multiple shoots via different modes of regeneration viz.: direct, indirect and somatic embryogenesis.

5.1 Source of explants Explant excised from seedlings or younger plants have more regeneration capacity than explants collected from mature plants. This could be due to fewer amounts of differentiation and more number of juvenile tissues present in seedling explants (Devi et al. 2011). In vitro derived explants are free from contaminations and easy to handle and inoculate than the explants collected from in vivo grown plants. Altaf et al. (1998) concluded that the growth responses of explants from younger seedlings (3-4 days) were much better than that of the older seedlings. Moreover, one of the main advantages of using seedling derived explants for in vitro regeneration is availability of uniform explant source all through the year (Yadav et al. 2010). That is why many workers used seedling derived explants to establish in vitro cultures in different plant species (Kim et al. 1990; Distanbanjong and Geneve 1997; Vengadesan et al. 2000; Tiwari et al. 2004; Shahzad et al. 2007; Barik et al. 2007; Rathore et al. 2008; Prakash and Gurumurthy 2010 and Yadav et al. 2010).

5.2 In vitro seed germination Seeds of both the plant species showed poor germination rate i.e. 30% (C. angustifolia) and 40% (C. sophera) in soil under natural conditions (Data not shown), therefore, seeds were allowed to germinate in vitro for the collection of different explants. Rate of seed germination enhanced considerably under controlled conditions in both the species and about 62.33 ± 1.45% seeds of C. sophera germinated on MS basal medium after 4 weeks of inoculation, however, germination started after 10.90 ± 0.20 days of inoculation. Reduction in nutrient

144 strength to half further improved the rate of seed germination (90.33 ± 1.45%) and reduced the time required for the emergence of radicle (6.56 ± 0.23 days) in C. sophera. This is in consonance with the earlier reports of Kishore et al. (2006) and Pandey and Agrawal (2009), in Ascocenda species and Spilanthes acmella respectively, in which the seeds were germinated on half strength MS medium. Barik et al. (2007) also reported that 96-98% seeds of a medicinal legume Clitoria ternatea germinated on half strength MS medium without any hormone.

However, the seeds of C. angustifolia failed to germinate on full as well as half strength MS media under controlled conditions and remained dormant even after 3 weeks of inoculation. The presence of growth inhibitors such as abscisic acid (ABA) in the seeds might be responsible for the onset of dormancy and maintaining the dormant state (Berry and Bewley 1992). However, augmentation of GA3 at various concentrations considerably facilitated seed germination in C. angustifolia and a maximum of 77.00 ± 2.31% seeds germinated on half strength

MS medium supplemented with 5.0 µM GA3 after 4 weeks, however, germination started within 3.20 ± 0.24 days of inoculation. In C. sophera also, addition of GA3 (1.0 µM) in half strength MS medium, enhanced the rate of seed germination to a maximum of 99.33 ± 0.66% and germination started within 4.46 ± 0.24 days of inoculation. GA3 is known to break the seed dormancy and enhanced the rate of seed germination via synthesis of α-amylase and other hydrolases (Shepley et al. 1972). It is also known to eliminate the requirement of seed for various environmental factors as it promotes germination and counteracts the inhibitory effects of ABA (Bewly and Black 1994). The role of GA3 in breaking seed dormancy is in agreement with earlier studies in Prunus avium (Çetinbas and Koyuncu 2006), Clitoria ternatea (Shahzad et al. 2007), Pterocarpus marsupium (Husain et al. 2007b), Cassia siamea (Parveen et al. 2010), Prunus mahaleb (Al- Absi 2010) and Bixa orellana (Joseph et al. 2011).

5.3 Direct regeneration It is the most common and highly efficient mode of in vitro regeneration for the production of large number of plants within very short span of time. It can be achieved either through preformed meristem containing explants or through differentiated plant tissues lacking natural meristem which is referred as

145 adventitious regeneration. In the present study, only meristem containing explants were able to induce multiple shoot buds via direct regeneration while the non meristematic explants resulted in the production of multiple shoots via indirect organogenesis i.e. through intermediate callus phase.

5.3.1 Type of explant The success of a regeneration protocol depends largely on the selection of a suitable plant part, called as ‘explant’, which is used as the starting material for the establishment of aseptic cultures (Barik et al. 2007) and play a critical role in development in in vitro culture (Vasil 1987). Steward et al. (1964) were the first to demonstrate the importance of explant selection in tissue culture. The extensive survey of literature revealed that the explant type had a great influence on multiple shoot induction in various plants such as Morus alba (Bhau and Wakhlu 2001), Lycopersicon esculentum (Gubis et al. 2003), Linum usitatissimum (Blinstrubiene et al. 2004), Pterocarpus marsupium (Anis et al. 2005), Scrophularia yoshmurae (Tsay et al. 2006), Jatropha curcas (Kaewpoo and Te-chato 2009) and Macadamia spp. (Gitonga et al. 2010).

Cotyledonary node (CN) explants of C. angustifolia cultured on regeneration medium containing different PGRs singly or in combination provided the highest number (39.16 ± 0.14) of shoots with significant growth and development compared to those induced from nodal segment (30.33 ± 0.24) and shoot tip (22.46 ± 0.49) explants on the same composition of medium. This is in agreement with the earlier report of Agrawal and Sardar (2003) in C. angustifolia, wherein they also demonstrated that among various seedling derived explants, CN explants showed highest multiplication rate (2.4 shoots) than NS (1.5 shoots) and ST explants (1.08 shoots). Similarly, CN explants have widely been used for in vitro shoot proliferation of many medicinal plants such as Lupinus texensis (Upadhyay et al. 1992a), Dalbergia sissoo (Pradhan et al. 1998a), Albizia chinensis (Sinha et al. 2000), Acacia sinuata (Vengadesan et al. 2002), Sesbania rostrata (Jha et al. 2004), Pterocarpus marsupium (Husain et al. 2007a) and Cassia siamea (Parveen et al. 2010). Conversely, in C. sophera, NS explants showed better morphogenic response (25.36 shoots) for multiple shoot development than CN (19.50 shoots) and ST (10.56 shoots) explants on the

146 same composition of media. The present findings have been found in corroboration with the previous report of Pandeya et al. (2010) in micropropagation of Clitoria ternatea, where nodal explants produced largest number (11.1) of shoots followed by cotyledonary node (9.8 shoots) and shoot tips (7.1 shoots) on MS medium supplemented with 2.0 mg/l BA. Nodal explants were proved better option for significant multiplication of shoots in several other plants (Kannan and Jasrai 1998; Thakur et al. 1998; Tiwari et al. 2004; Rani et al. 2006; Khalekuzzaman et al. 2008; Husain et al. 2008 and Sujana and Naidu 2011).

5.3.2 Age of explant The age of the explant played an important role on the onset of morphogenesis in in vitro culture (Distanbanjong and Geneve 1997). As the results indicated the CN explants of C. angustifolia obtained from 14 days old seedlings were more suitable than those of 7 and 21 days old seedlings. While, the NS and ST explants obtained from 21 days old seedlings provided best response than those obtained from 14 and 28 days old seedlings. However, in C. sophera, it was found that only 21 days old seedlings provided the most responsive explants of all type viz.: CN, NS and ST for direct shoot regeneration than those of 14 and 28 days old seedlings. Similarly, Jeyakumar and Jayabalan (2002) used CN explants from 20 days old aseptic seedlings of Psoralea corylifolia for in vitro regeneration. Husain et al. (2008) also suggested that NS excised from 18 days old axenic seedlings of Pterocarpus marsupium responded better for shoot induction as compared to 6, 12 and 24 days old seedling explants. Likewise, there are so many reports available, where the age of the explant affected the rate of shoot regeneration (Gitonga et al. 2010; Karuppusamy and Kalimuthu 2010; Devi et al. 2011 and Mohebodini et al. 2011).

5.3.3 Effect of cytokinins and auxins on multiple shoot regeneration Explants containing axillary/apical buds have quiescent or active meristems depending upon the physiological stage of the plant. These buds have the potential to develop into complete plantlets. But, in nature these buds remain dormant for a specific period of time depending on the growth pattern of the plant (Rani and Rana 2010). Nevertheless, with tissue culture techniques, the rate of

147 shoot multiplication can be greatly enhanced by culturing these explants on a nutrient medium containing suitable combinations and concentrations of different plant growth regulators. Generally, cytokinins have a stimulatory effect on production of multiple shoots via direct regeneration without any intermediate callus phase.

In the present study, the entire three meristem containing seedling derived explants (CN, NS and ST) of both C. angustifolia and C. sophera failed to respond on hormone free MS medium. However, augmentation of different cytokinins (BA, Kn and 2iP) to the MS medium triggered the explants to respond and multiple shoots were produced at appropriate concentrations. Amongst three cytokinins tested, BA was found superior to other cytokinins (Kn and 2iP) for multiple shoot productions in both the plants. Of all the concentrations of BA tested, 5.0 µM BA proved to be the optimal concentration for inducing multiple shoots irrespective of the explants type, however, the number of shoots/explant and shoot length differ with the type of explants in both the plants. The results described that cotyledonary nodes excised from 14 days old axenic seedlings of C. angustifolia produced the maximum number of shoots compared to nodal segments and shoot tip explants. The best response reported in CN explants was evaluated as the mean number of 25.33 ± 0.20 shoots/explants and the shoot length of 4.33 ± 0.20 cm in 75.40 ± 0.30% cultures. Whereas, on the same concentration (5.0 µM) of BA, nodal and shoot tip explants were less responsive and provided an average of 20.20 ± 0.11 and 12.56 ± 0.34 shoots/explant respectively. Although the regenerated shoots in NS and ST explants were less in number than CN explants but their number was quite satisfactory for mass multiplication of the plant and these two explants might also be used as a good source of explant for the micropropagation of C. angustifolia. The present findings are much more significant regarding multiplication of shoots than the previous report of Agrawal and Sardar (2003) in C. angustifolia, wherein they reported only 2.4 shoots/explant on BA (1.0 µM) supplemented MS medium through seedling derived CN explants compared to NS and ST explants.

In case of C. sophera maximum regeneration was obtained through nodal segments (21 days old) compared to CN and ST explants cultured on MS

148 medium containing optimal 5.0 µM BA, producing the highest 16.46 ± 1.21 shoots/explant with maximum shoot length of 4.80 ± 0.37 cm in 90.33 ± 3.17% cultures. The CN and ST explants produced an average of 12.43 ± 0.29 and 6.10 ± 0.26 shoots/explant on the same concentration of BA respectively. The superiority of BA on bud break and multiple shoot production has also been reported earlier in several medicinal and aromatic plants including Pogostemon cablin (Kukreja et al. 1990), Piper spp. (Bhat et al. 1995), Holarrhena antidysenterica (Raha and Roy 2001), Decalepis hamiltonii (Anitha and Pullaiah 2002b), Eclipta alba (Husain and Anis 2006b), Mucuna pruriense (Sathyanarayana et al. 2008), Clitoria ternatea (Singh and Tiwari 2010), Justicia gendarussa (Thomas and Yoichiro 2010) and Veronica anagallis-aquatica (Shahzad et al. 2011).

A decline in regeneration potential and multiple shoot induction was observed at higher concentrations of BA beyond the optimal level (5.0 µM) in both C. angustifolia and C. sophera. Increase in the concentration of BA resulted in basal callusing which might be one of the reason for reduction in the regeneration potential of the explants and hampered the growth and development of new shoots which ultimately reduced the average number of shoots/explant as well as shoot length. These findings substantiate with the earlier reports of Withania somnifera (Sen and Sharma 1991), Ocimum spp. (Pattnaik and Chand 1996), Gymnema sylvestre (Reddy et al. 1998), Entada phaseoloids (Rao and Vishnupriya 2002) and Saussurea obvallata (Joshi and Dhar 2003).

Explants cultured on MS medium supplemented with various concentrations of Kn and 2iP also produced multiple shoots, but the number of shoots was lower than that in BA and was not encouraging in both C. angustifolia and C. sophera. Cotyledonary nodes of C. angustifolia produced an average of 8.96 ± 0.20 shoots/explant with shoot length of 4.23 ± 0.14 cm in 54.60 ± 0.23% cultures at 7.5 µM Kn. While, explants on medium containing an optimal 7.5 µM 2iP produced only 6.10 ± 0.20 shoots/explant. Similarly, the nodal explants of C. sophera exhibited optimal response at 7.5 µM Kn inducing an average of 6.56 ± 0.34 shoots/explant having 3.56 ± 0.29 cm of shoot length in 60.33 ± 0.15%

149 cultures. In this plant also 2iP proved to be the least effective cytokinin and exhibited optimal response at 7.5 µM. Our results are in accordance with the previous studies on various plant species by other workers who also determined that Kn and 2iP are less efficient cytokinins than BA (Mao et al. 1995; Pattnaik and Chand 1996; Patil 1998; Jeong et al. 2001; Loc et al. 2005; Hiregoudar et al. 2006 and Shahzad et al. 2011).

In general higher concentration of cytokinin and lower concentration of auxin are required in a medium to promote multiplication and proliferation of shoots (Kohlenbach 1997; Reddy et al. 1998 and Beena et al. 2003). This was found true in the present study also irrespective of the explant types in both the plants. When the CN explants of C. angustifolia were cultured on MS medium containing optimal concentration (5.0 µM) of best cytokinin (BA) with lower concentrations of three auxins, an enhanced regeneration was resulted. Amongst various cytokinin-auxin combinations, BA-NAA combination exhibited best morphogenic potential compared to BA-IAA and BA-IBA combinations. The CN explants on MS medium with optimal BA (5.0 µM) and NAA (0.6 µM) produced the highest 39.16 ± 0.14 shoots/explant having maximum shoot length of 5.63 ± 0.20 cm in 85.26 ± 0.17% cultures. Augmentation of IAA and IBA at various concentrations with 5.0 µM BA provided acceptable results but the best auxin was proved to be NAA. Similar results were obtained through nodal explants of C. sophera cultured on different cytokinin-auxin combinations. Amongst three auxins tested, NAA at 1.0 µM along with 5.0 µM BA exhibited optimal response and produced the maximum 19.50 ± 0.51 shoots/explant with an average shoot length of 5.23 ± 0.14 cm in 86.00 ± 2.08% cultures. IAA and IBA proved to be less responsive than NAA in C. sophera also. The higher concentrations of NAA beyond optimal level resulted in the production of heavy basal callusing and exerted inhibitory effects on multiplication of shoot buds in both the plant species since callus formation acts as a mechanical barrier to nutrient and water uptake (Thorpe et al. 1991 and De Klerk 2002). The synergistic effects of BA-NAA combination have also been reported in other plant species such as Psoralea corylifolia (Saxena et al. 1997), Mentha arvensis (Shahzad et al. 1999), Echinacea pallida (Koroch et al. 2003), Rauvolfia

150 serpentina (Baksha et al. 2007), Tylophora indica (Faisal et al. 2007) and Cassia siamea (Parveen et al. 2010).

These results indicated that synergism of BA and NAA in proper concentration was extremely favourable for the multiplication of different plants. However, there are certain reports which revealed that exogenous auxin (NAA) was not essential for the induction of multiple shoot buds, but in turn, produced basal callusing and reduced the number of shoots (Fracaro and Echeverrigaray 2001; Vengadesan et al. 2002; Malik et al. 2005; Khalafalla and Daffalla 2008 and Shahzad et al. 2011). In contrast in Stevia rebaundiana (Anbazhagan et al. 2010) and Adhatoda beddomei (Sudha and Seeni 1994), best regeneration was obtained on BA + IAA combination, whereas, in Curcuma zedoria (Loc et al. 2005) and Rotula aquatica (Martin 2003b), IBA was found to be the best auxin among various auxins (IAA, IBA and NAA) used in combination with BA.

The synergistic effect of Kn and 2iP with different auxins (IAA, IBA and NAA) was also evaluated through all the three explants of C. angustifolia and C. sophera respectively. Amongst various combinations tested, Kn + NAA and 2iP + NAA proved to be the ideal combinations in both the plants. It was observed that CN explants cultured on medium comprised of MS + Kn (7.5 µM) + NAA (0.6 µM) produced a maximum of 15.20 ± 4.32 shoots/explant in 70.00 ± 2.88% cultures of C. angustifolia, whereas, an average of 10.90 ± 0.20 shoots/explant in 72.33 ± 1.45% cultures were produced by the nodal explants of C. sophera on MS + Kn (7.5 µM) + NAA (1.0 µM). The average number of shoots produced by the explants on 2iP + NAA combination was less than BA and Kn supplemented media in both the plants.

5.3.4 Effect of TDZ on multiple shoot regeneration Besides adenine type cytokinins (BA, Kn and 2iP) the thidiazolylurea derivative, thidiazuron (TDZ) also possess intrinsic cytokinin like activity (Mok et al. 1982). TDZ may be involved in the synthesis or in accumulation of cytokinins in plant tissues and exhibited stronger effect than other conventionally used cytokinins in a wide range of species (Capelle et al. 1983; Huettman and Preece 1993; Thomas 2003; Thomas and Puthur 2004 and Husain et al. 2007a). It is well established that the concentration of TDZ is highly sensitive for the induction of

151 multiple shoots and their proper growth and development as compared to other cytokinins (Parveen and Shahzad 2010). Therefore, all the three explants (CN, NS and ST) of C. angustifolia and C. sophera were cultured on a wide range of concentrations (0.1-10.0 µM) of TDZ. It was observed that amongst all the explants tested, cotyledonary node explants of C. angustifolia and nodal explants of C. sophera provided best response at an optimal 2.5 µM TDZ. The medium comprised of MS + TDZ (2.5 µM) exhibited highest regeneration potential (94.33 ± 2.33%) and produced a maximum of 28.53 ± 0.99 shoots/explant with shoot length of 4.03 ± 0.14 cm through CN explants of C. angustifolia. Our results are in agreement with the previous reports of other workers who also cultured CN explants of different plants on TDZ supplemented media (Dewan et al. 1992; Purohit and Dave 1996; Jha et al. 2004; Husain et al. 2007a; Parveen and Shahzad 2010 and Devi et al. 2011). In C. angustifolia, it was observed that TDZ at optimal concentration (2.5 µM) found to exhibit better regeneration potential than single cytokinin (5.0 µM BA), but less effective compared to cytokinin-auxin combination [BA (5.0 µM) + NAA (0.6 µM)] treatment.

However, the nodal explants of C. sophera, cultured on TDZ supplemented media exhibited lesser regeneration potential than the single cytokinin (5.0 µM BA) as well as cytokinin-auxin combination [BA (5.0 µM) + NAA (1.0 µM)] treatment. Thus, the results suggested that TDZ found to be less effective in C. sophera and produced an average of 13.76 ± 0.38 shoots/explant in 95.00 ± 1.15% cultures having shoot length of 3.26 ± 0.14 cm after 6 weeks. However, contrary to these results the nodal explants of Cannabis sativa (Lata et al. 2009b) cultured on MS medium supplemented with 0.5 µM TDZ exhibited highest (100%) shoot regeneration with the production of maximum 12.6 shoots/explants, furthermore, the quality and quantity of regenerants was better with TDZ than with BA or Kn. Similar observations were also made by Karuppusamy and Kalimuthu (2010) in Andographis nissiana, in which amongst all the cytokinins tested, TDZ proved to be more potent. The nodal explants taken from 30 days old aseptic seedlings provided highest shoot multiplication on 10.0 µM TDZ with the production of an average of 34 shoots in 94% cultures. Similarly, in Psoralea corylifolia (Faisal and Anis 2006), Vitex negundo (Ahmad

152 and Anis 2007), Andrographis nessiana (Karuppusamy and Kalimuthu 2010), Hypericum perforatum (Murch et al. 2000) and Arachis correntina (Mroginski et al. 2004) TDZ proved to be a more potent cytokinin. It was further observed that the higher concentrations of TDZ beyond optimal level resulted in reduction of number of shoots/explant as well as shoot length and produced heavy callusing in both the plants. Similar inhibitory effects of higher concentration of TDZ have also been reported in Adhatoda beddomei (Sudha and Seeni 1994), Pelargonium x hortorum (Hutchinson and Saxena 1996), Murraya koenigii (Bhuyan et al. 1997), Oroxylum indicum (Dalal and Rai 2004) and Cassia sophera (Parveen and Shahzad 2010).

Although a high rate of shoot multiplication was observed on TDZ supplemented media, the regenerated shoots failed to elongate and exhibit abnormal growth. Moreover, prolonged exposure of cultures on TDZ containing media resulted in the distortion, fasciation and stunting of shoots in both the plants. These results are in consonance with some reports that also revealed the deleterious effects of continued presence of TDZ in Pisum sativum (Bohmer et al. 1995), Cicer arietinum (Murthy et al. 1996), Dalbergia sissoo (Pradhan et al. 1998a), Hypericum perforatum (Murch et al. 2000) and Rauvolfia tetraphylla (Faisal et al. 2005b). The inhibition of shoot elongation by TDZ may be attributed to its high cytokinin activity as cytokinins are generally known to inhibit the shoot elongation, whereas, the presence of phenyl group in TDZ may be the possible cause of shoot bud fasciation (Huettman and Preece 1993). In order to avoid these deleterious effects of TDZ, the two-step culture procedure was adopted which include the transferring of regenerated cultures (induced on 2.5 µM TDZ supplemented medium) to TDZ free MS basal medium as well as MS medium containing lower concentrations of BA. MS medium without BA was not very much effective for further growth and proliferation of shoots while addition of BA proved beneficial and improved the growth in shoots, facilitated multiplication and proliferation of shoots.

The medium comprised of 2.5 µM BA enhanced the number of shoots to the maximum 45.50 ± 0.51 shoots/explant, attaining an average shoot length of 6.20 ± 0.11 cm in C. angustifolia, although in C. sophera maximum response was

153 obtained on medium containing 1.0 µM BA, where 19.83 ± 0.02 shoots/explant with 6.00 ± 0.11 cm of shoot length were obtained. These results are in agreement with the earlier findings of Huettman and Preece (1993) that the problem of shoot elongation could be resolved by using a two-step culture procedure consisting of TDZ containing primary medium for the induction of multiple shoots followed by a secondary medium with another cytokinin that facilitated further development and proliferation of induced shoots. Similar strategy of two-step culture procedure has also been implemented by several workers in other plant species such as Cajanus cajan (Eapen et al. 1998), Nothapodites foetida (Satheeshkumar and Seeni 2000), Rosa damascene (Kumar et al. 2001), Acacia sinuata (Vengadesan et al. 2002), Morus alba (Thomus 2003), Pterocarpus marsupium (Husain et al. 2007a), Bacopa monneiri (Ceasar et al. 2010) and Cassia sophera (Parveen and Shahzad 2010).

5.3.5 Effect of subculture passages on proliferation of shoots Subculturing was done at a regular interval of 6 weeks each on to the fresh medium containing best hormonal supplements in both the plants. Shoot tip necrosis, yellowing and abscission of leaves was observed in the regenerated shoots during the first sub culture stage in C. angustifolia as well as in C. sophera. Similar anomalies were also reported in Celastrus paniculatus, which might have been cause by nutritional deficiencies (Nair and Seeni 2001). Thus, the regeneration medium was supplemented with an additional nitrogen source in the form of adenine sulphate (AdS) at various concentrations (10-50 µM) and it was found that the optimal 30 µM concentration of AdS in C. angustifolia and 20 µM in C. sophera proved to be effective to overcome these adverse effects. AdS can stimulate cell growth and greatly enhance the shoot multiplication; it also provides an available source of nitrogen to the cell that can generally be taken up more rapidly than inorganic nitrogen (Murashige 1974 and Thom et al. 1981). Consequently, addition of AdS at appropriate concentrations proved to be fruitful and facilitated the development of healthy shoots with vigorous growth. AdS is reported to exhibit synergistic effect with other plant hormones and it has been used as an adjuvant by several workers in many woody plant species including Acacia nilotica (Singh et al. 1993), Tectona grandis (Devi et al. 1994), Bauhinia vahlii (Dhar and Upreti 1999), Acacia sinuata (Vengadesan et al.

154

2003b), Murraya koenigii (Rout 2005b), Pterocarpus marsupium (Husain et al. 2008), Melia azedarach (Husain and Anis 2009) and Plumbago zeylanica (Sivanesan and Jeong 2009).

The rate of shoot multiplication enhanced during each subculture passage and continued up to 4th subculture passage in both the plants. In C. angustigolia, number of shoots became stable at 5th subculture passage and beyond that a decline in regeneration potential was observed, whereas, in C. sophera a sharp decline in regeneration rate was observed after 4th subculture passage. The highest shoot multiplication was achieved through cotyledonary node explants of C. angustifolia on best hormonal supplement consisting of MS + BA (5.0 µM) + NAA (0.6 µM) + AdS (30.0 µM) producing the highest 64.90 ± 0.32 shoots/explant with shoot length of 6.56 ± 0.23 cm by the end of 4th subculture passage. Similar responses have also been noticed through nodal explants of C. sophera, where multiplication and proliferation of new shoots continued up to 4th subculture passage producing a maximum of 42.33 ± 0.32 shoots/explant with 7.76 ± 0.14 cm shoot length on MS medium containing BA (5.0 µM) + NAA (1.0 µM) + AdS (20.0 µM). The elongated shoots were regularly excised from the mother tissue at the end of each subculture passage and transferred to rooting media for the development of roots.

The enhanced shoot multiplication with subsequent subculture passages substantiate the previous report of Siddique and Anis (2007a) in C. angustifolia and Phulwaria et al. (2012) in Terminalia bellirica who also observed that the number of shoots increased up to 4th subculture passage and decline afterwards. But the average number of shoots obtained by Siddique and Anis (2007a) was much less (21.7 ± 1.54 shoots/explant) than the present study (64.90 ± 0.32 shoots/explant). The increase shoot number due to successive transfer of mother tissue may be owing to suppression of apical dominance during subculture that induced basal dominant meristematic cells to form new shoots (Tripathi and Kumari 2010 and Shekhawat and Shekhawat 2011). In contrast to the present results Rout (2005b) concluded that the regeneration frequency in Murraya koenigii increased up to 10 subcultures and was stable thereafter up to 16 subcultures without the loss of the morphological response. Amplified

155 regeneration rates might be attributed to the adaptation of the explants to the in vitro environment which is in corroboration to the earlier reports (Rout et al. 1999a; Arockiasamy et al. 2000; Borthakur et al. 2000; Raha and Roy 2001; Rout 2005a; Husain and Anis 2006b; Prakash et al. 2006; Bohidar et al. 2008 and Sharma and Shahzad 2011). However, in certain species like Ocimum (Pattnaik and Chand 1996) repeated subculturing did not enhance the rate of shoot multiplication, therefore, it seems that the effect of subculturing on multiplication is species-specific.

5.4 Effect of different media, pH and sucrose concentrations Different factors like nutrients composition, pH of the culture medium and sucrose concentration have an influence on the induction and proliferation of multiple shoots. Thus, in the present investigation all these three factors were standardized for the optimum regeneration of shoots via different explants in C. angustifolia as well as C. sophera. Some species give similar response in all the media while others show preference for a specific medium for the establishment and proliferation of cultures (McCown and Sellmer 1987). Amongst four different media tested (B5, L2, MS and WPM), MS medium found to be the best for optimal regeneration in both the plants followed by WPM, L2 and B5 media.

Multiple shoot buds were differentiated on WPM, L2 and B5 media but showed limited growth and development even if they were maintained for longer period in culture. Similar results were obtained by Faisal et al. (2006a) in Mucuna pruriens with different media. The explants on B5 medium exhibited poor response which might be due to the presence of high ammonium and nitrate content which inhibit the induction and multiplication of shoots (Constable 1984). Thus, the degree of growth and differentiation varied considerably with the medium composition (Shekhawat et al. 1993 and Das et al. 1996b). In vitro propagation of several other plants has also been shown to have optimum regeneration on MS medium than B5, L2, SH or WPM media (Pattnaik and Debata 1996; Komalavalli and Rao 2000; Catapan et al. 2002; Tiwari et al. 2002; Baskaran and Jayabaln 2005; Husain et al. 2008 and Abbas et al. 2010). The need of MS salts for maximum shoot induction and proliferation showed the high salt requirement for the growth of Cassia species. However, in contrast to our results Bhatt and Dhar (2004), concluded that WPM showed better response compared to MS and B5 media for

156 the micropropagation of Myrica esculanta, similarly, Raghu et al. (2006a) also suggested the superiority of WPM over MS medium for the induction of multiple shoots in Tinospora cordifolia.

Hydrogen ion concentration (pH) of the culture medium is one of the important factors of physico-chemical environment during development of plant tissues under in vitro conditions (Williams et al. 1990). The optimal pH of the medium varies according to the different phases of morphogenesis viz: establishment of cultures, shoot proliferation and induction of roots (Saborio et al. 1997 and Ostrolucká et al. 2004). In the present study, it was found that among different pH levels tested with MS medium containing optimized concentration of PGRs, pH value of 5.8 was the optimum and provided maximum regeneration in both the plants. It is known that medium pH has effects not only on the uptake of nutrients but also on chemical reactions especially those catalysed by enzymes (Thorpe et al. 2008). Increase or decrease in the pH value from optimal level reduced the regeneration potential of the explants and ultimately affected the multiplication rate in both the plants. Our findings substantiate with earlier reports on C. angustifolia (Siddique and Anis 2007a) and several other plants where pH 5.8 was found to be the optimum value (Nair and Seeni 2003 and Faisal et al. 2007).

Plant cell, tissue or organ culture normally require the incorporation of a carbon or energy source to the culture medium (George 1993 and Karhu 1997) and sucrose has been used as the major carbon source in plant tissue culture (Fuentes et al. 2000). Plants growing under in vitro culture conditions are semi autotrophic, moreover, they have limited accessibility to CO2 inside culture vessels. Therefore sucrose is supplemented to maintain an adequate supply of carbon source for the multiplication and growth of regenerated tissue (Hazarika 2003). The optimal concentration of sucrose varied with the type of species, thus it is essential to standardize the amount of sucrose needed for a particular species. In the present study all the explants were tested for multiple shoot regeneration at different concentrations of sucrose on MS medium supplemented with optimized PGRs at pH 5.8 in both the plants. Addition of 3% sucrose was found most suitable for multiplication and proliferation of shoots

157 through various explants in both the plants. Lower and higher concentrations of sucrose beyond optimal 3% did not provide satisfactory results and reduced the regeneration efficacy of the explants. Similar to our results, Anitha and Pullaiah (2002a) also obtained optimal regeneration at 3% sucrose in comparison to other concentrations of sucrose. However in Eucomis autumnalis, maximum regeneration was obtained at 4% sucrose and the lower concentrations decreased the regeneration rate (Taylor and van Staden 2001), similarly, in Amygdalus communis shoot multiplication was obtained only at 5 and 6% sucrose (Gürel and Gülsen 1998) which is contrary to our results. On the other hand majority of micropropagation protocols generally utilize 3% sucrose as per recommendation of Murashige and Skoog (1962). Exogenous supply of sugar increases starch and sucrose reserves in micropropagated plants and could favour ex vitro acclimatization and speed up physiological adaptations (Pospíšilová et al. 1999).

5.5 Indirect organogenesis In vitro organogenesis via callus production may be quicker and easier than conventional breeding technique to induce morphological and chemical variations (Gao and Bjork 2000). Interestingly, it has been observed that in the same plant, different explants revealed differential response for callus induction which may be explained on the basis that different explants were at different biochemical or physiological stage at the time of collection (Paterson and Everett 1985).

5.5.1 Type of explant In the present study three different types of explants, lacking preformed meristems viz.: cotyledonary leaf (CL), leaf (L) and root (R) excised from axenic seedlings were chosen for the production of callus in both the plants. Amongst all the three explants tested, root explants provided best response followed by cotyledon and leaf explants in C. angustifolia. Root explants are advantageous over other explants as they can easily be manipulated and maintained, possess higher regeneration potential and excellent susceptibility for Agrobacterium transformation (Morton and Browse 1991; Knoll et al. 1997; Franklin et al. 2004 and Parveen and Shahzad 2011). Moreover, plants regenerated from the root

158 segments are suggested to be genetically identical (Sharma et al. 1993). The root explant has been used as a source material for mass multiplication of several other plants such as Cephalis ipecacuanha (Yoshimatsu and Shimomura 1994), Albizzia julibrissin (Sankhla et al. 1995), Populus tremula (Vinocur et al. 2000), Robinia pseudoacacia (Hosseini-Nasr and Rashid 2003/2004), Solanum melongena (Franklin et al. 2004), Tylophora indica (Chaudhuri et al. 2004 and Sahai et al. 2010b), Clitoria ternatea (Shahzad et al. 2007) and Picrorhiza kurroa (Sood and Chauhan 2009).

Whereas, surprisingly in C. sophera root explants were not able to induce callus on various hormonal supplements and remain as such even after 4 weeks of incubation. While cotyledonary leaf and leaf explants induced calli and the best response was exhibited by the cotyledon explants. Cotyledon explants have also been proved superior over other explants in several earlier studies conducted for organogenesis using different plant growth regulators (Saafi and Borthakur 2002 and Shahzad et al. 2006). So our findings revealed that the type of explant had a great impact on the morphogenesis and it is found to be species dependent. Savita et al. (2010) also concluded that response to callus induction was influenced by the type of explant as well as the concentration and type of PGRs used. Similar results showing variations among explant types with respect to callus induction have also been observed in other plants such as Albizzia lebbeck (Lakshmanan Rao and De 1987), Lonicera japonica (Georges et al. 1993) and Holarrhena antidysenterica (Raha and Roy 2003).

5.5.2 Age of explant The rate of callus induction also varied depending on the age of the explants revealing a decrease with the increasing or decreasing age of the explants. Cotyledonary leaf explants of 14 days old axenic seedlings proved to be more efficient than 7 and 21 days old explants in C. angustifolia (Parveen et al. 2012) as well as C. sophera, while, root segments of C. angustifolia (Parveen and Shahzad 2011) and leaf explants of C. sophera, excised from 30 days old seedlings provided best response compared to that from 20 and 40 days old seedlings. Nef-Campa et al. (1996) also used root segments excised from 30 days old aseptically grown seedlings of a tropical legume Aeschynomene

159 sensitiva for regeneration which is in consonance with our results. Whereas, in earlier reports of C. angustifolia, callus was induced through cotyledon and leaflet explants excised from 20 days old aseptic seedlings (Agrawal and Sardar 2006) and petiole explants excised from 21 days old axenic seedlings (Siddique et al. 2010). Similar results have also been described by other workers where age of the explant affected the response to callus induction in different plant species (Dhar and Joshi 2005; Prakash and Gurumurthy 2010 and Mohebodini et al. 2011).

5.5.3 Effect of cytokinins and auxins on callus induction A comparative study was done between root and cotyledonary leaf explants of C. angustifolia and cotyledonary leaf and leaf explants of C. sophera on various concentrations of cytokinins and auxins to obtain the best organogenic callus for shoot morphogenesis. Among all the three cytokinins (BA, Kn and TDZ) tested, TDZ provided best response followed by BA and Kn in both the plants irrespective of explants type. In C. angustifolia, root explants cultured on medium comprised of MS + TDZ (1.0 µM) produced nodular and organized callus mass with optimal growth and high regeneration potential (90.66 ± 1.45%) than cotyledon explants at the same concentration, which produced loose and friable callus in only 49.00 ± 2.08% cultures. Whereas, the CL explants of C. sophera produced an optimal growth of compact and greenish callus at higher concentration of TDZ (7.5 µM) in 41.50 ± 1.30% cultures compared to leaf explants exhibiting merely 29.86 ± 2.08% regeneration response at the same concentration of TDZ. Shahzad et al. (2006) also reported to induce highly organogenic callus using TDZ from green cotyledons of Acacia sinuata. TDZ has also been described to induce callus through various explants in other plants (Chang and Chang 1998; Manjula et al. 2000; Chen et al. 2000; Lata et al. 2002 and Wang et al. 2003). However, Agrawal and Sardar (2006) were successful to induced organogenic callus from cotyledon and leaflet explants of C. angustifolia on MS medium segmented with BA (1.0 µM) + 2,4-D (1.0 µM) which is in contrast to our results.

Usually the induction of organogenic callus is promoted by an auxin, especially 2,4-D (Bennici and Bruschi 1999 and Kamo et al. 2005). In the present study

160 also, it was observed that between the two auxins tested, 2,4-D was more efficient for the induction of callus than 2,4,5-T in both the plants. In C. angustifolia, greenish, compact and nodular callus was obtained from cotyledon explants at 5.0 µM 2,4-D with 84.66 ± 2.60% response, whereas, yellowish, loose and friable callus was obtained from root explants at the same concentration with 57.66 ± 1.45% response. Similarly, in C. sophera, 2,4-D at 5.0 µM produced greenish, compact and nodular callus from CL explants with 73.40 ± 0.45% response, while, leaf explants produced yellowish, loose and friable callus with 53.53 ± 0.81% response at 7.5 µM 2,4-D. Such type of variations in response to different explant type has been reported earlier in Astragalus adsurgens (Luo and Jia 1998), Plumbago zeylanica (Rout et al. 1999b) and Arnebia euchroma (Manjkhola et al. 2005). The calli produced by root and cotyledonary leaf explants were initially green but later on the upper surface turned brown in colour which is in agreement with the findings of Ignacimuthu et al. (1999) and Irvani et al. (2010), who reported that browning of callus may occurred due to activation of secondary metabolite synthesis from the explants. In an earlier report by Siddique et al. (2010), callus was induced on 5.0 µM 2,4-D along with 0.5 µM TDZ through petiole explants of C. angustifolia. Similarly, 2,4- D has been found to induce organogenic callus in a number of plant species either alone or in combination with a cytokinin (Srivastava and Rajani 1999; Shahzad et al. 1999; 2007; 2009; Saafi and Borthakur 2002; Faisal et al. 2006c; Roy et al. 2008 and Nikam and Savant 2009). However, Faisal and Anis (2005) reported the production of organogenic callus on 10.0 µM 2,4,5-T through stem explant in Tylophora indica.

5.5.4 Effect of cytokinins and auxins on differentiation of shoots from callus The dark brown, compact and nodular callus obtained through root explants at 1.0 µM TDZ in C. angustifolia and through cotyledon explants at 5.0 µM 2,4-D in both the plants, turned green and meristematic on transferring to regeneration medium composed of different cytokinins and auxins singly or in combinations. Similar observations have been recorded in other legumes like Macroptilium atropurpureum (Ezura et al. 2000) and Lathyrus sativus (Zambre et al. 2002), where callus turned green on regeneration medium and this is considered a

161 prerequisite for regeneration. However, the dark brown and friable callus produced from leaf explants of C. sophera remained as such and failed to exhibit shoot differentiation on all the treatments tested. The regeneration frequency and the number of shoots/explant varied in callus produced from various explants and the highest regeneration of shoots was obtained from callus cultures derived from root explants than cotyledon explants in C. angustifolia, while, in C. sophera only cotyledonary leaf explants provided maximum shoot differentiation. Variation between explants within a plant is a common phenomenon observed in some medicinal plants (Martin 2004c and Thomas and Maseena 2006). Amongst three cytokinins (BA, Kn and TDZ) tested, BA provided maximum differentiation of shoot buds from the callus followed by Kn and TDZ in both the plants. Superiority of BA for shoot induction has been described due to the ability of plant tissue to metabolise natural hormones more readily than artificial growth regulators or due to the ability of BA to induce production of natural hormones such as zeatin within the tissue and thus work through natural hormone system (Sharma and Wakhlu 2003).

In C. angustifolia a maximum of 24.56 ± 1.97 shoots/explant were obtained through root derived callus at 2.5 µM BA, whereas the CL derived callus produced an average of 10.73 ± 0.95 shoots/explant at 5.0 µM BA. Similarly, in C. sophera the highest differentiation of shoots was observed at 2.5 µM BA producing a maximum of 7.56 ± 0.23 shoots through CL derived callus. The differential response of explants to callus production and shoot regeneration may be due to the concentrations of endogenous levels of cytokinins and auxins in different explants and has been reported in several plants like Psoralea corylifolia (Saxena et al. 1997), Centella asiatica (Patra et al. 1998), Daucus carota (Jiménez et al. 2005) and Picrorhiza kurroa (Sood and Chauhan 2009). The results obtained in this study showed consistency with other studies, where the use of BA proved to be more potent than other cytokinins for the differentiation of multiple shoots from calli, such as Sesbania bispinosa (Kapoor and Gupta 1986), Citrus aurantifolia (Bhat et al. 1992), Tamarindus indica (Sonia et al. 1998), Leucaena leucocephala (Saafi and Borthakur 2002) and Clitoria ternatea (Shahzad et al. 2007).

162

In most of the study it was found that the cytokinin-auxin combinations triggered significant shoot differentiation from the callus than the single cytokinin (Castillo et al. 2000; Saxena et al. 2000; Zambre et al. 2002 and Wei et al. 2006). Similarly, in the present study the differentiation of shoots from the callus further enhanced when the optimal concentration of BA was used in combination with different auxins at lower concentrations in both the plants. It was observed that the addition of NAA facilitated better morphogenesis and enhanced the rate of shoot buds differentiation. In C. angustifolia the root callus (Parveen and Shahzad 2011) produced the highest 35.63 ± 0.75 shoots/explant on the medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM) compared to 23.16 ± 1.44 shoots/explant through cotyledon derived callus (Parveen et al. 2012) on MS + BA (5.0 µM) + NAA (0.4 µM). However, in earlier reports on C. angustifolia (Agrawal and Sardar 2006 and Siddique et al. 2010), merely 12.00 ± 1.00 and 12.5 ± 1.10 shoots were obtained through cotyledonary leaf and petiole derived calli respectively, thus, the present study reveals a much better and efficient regeneration system in this species. Similarly, in C. sophera the maximum 14.63 ± 0.23 shoots/explant were produced through CL derived callus at MS + BA (2.5 µM) + NAA (0.5 µM). These findings clearly reveals that NAA exhibited synergistic effect with BA which is in consonance with the previous studies in Psoralea corylifolia (Saxena et al. 1997), Coleus forskohlii (Reddy et al. 2001), Tylophora indica (Faisal and Anis 2003; 2005), Eleucine indica (Yemets et al. 2003), Hypericum perforatum (Wojcik and Podostolski 2007) and Sansevieria cylindrica (Shahzad et al. 2009).

5.5.5 Effect of subculture passages and maintenance of cultures For further multiplication and continuous induction of shoot buds in C. angustifolia, the regenerating callus was subculture continuously on to the fresh regenerating medium. A sharp decline was noticed in the regeneration potential of CL derived callus just after first subculturing which ultimately reduced the growth and development of new shoots. While the root derived callus showed an increase in the number of shoots after each subculture passage on optimized medium i.e. MS + BA (2.5 µM) + NAA (0.6 µM) up to 4th subculturing at an interval of 6 weeks each. The frequency of shoot regeneration reached its maximum at the end of 4th subculture passage where the highest 42.66 ± 1.47

163 shoots/explant were obtained and then it became stable at 5th subculture passage but declined thereafter in subsequent passages (Parveen and Shahzad 2011). In every subculture, elongated shoots were regularly removed and transferred to rooting media. However, no significant difference was recorded in shoot length among the subcultures in both the plants. Our results substantiate with the reports of Rout et al. (1999b) in Plumbago zeylanica, where the rate of regeneration of shoot buds per callus continued up to eight subculture passages, similarly in Coleus forskohlii (Reddy et al. 2001) multiplication of shoot buds continued up to sixth subculture passage.

Nevertheless, in case of C. sophera, among four subculture passages at 6 week intervals, multiplication and differentiation of new shoots from CL derived callus continued up to 2nd subculture passage only, producing the highest 20.60 ± 0.45 shoots/explant on optimal regeneration medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM). After third subculture passage, the regeneration efficacy of the callus reduced drastically, which affected the production of new shoots and a decline was also observed in average number of shoots/explant in subsequent passages. These results showed consistency with the study of Saxena et al. (1997) in Psoralea corylifolia, wherein the maximum regeneration potential was detected in the first subculture and thereafter resulted in the loss of organogenic potential.

5.6 Somatic embryogenesis Somatic embryogenesis is a process where a bipolar structure resembling a zygotic embryo develops from a non-zygotic cell without vascular connection with the original tissue (Arnold et al. 2002). Embryogenic callus was induced through immature green cotyledons of C. angustifolia excised from green seeds of semi mature pods, while the cotyledon explants obtained from in vitro derived seedlings did not exhibit any response towards embryogenesis. However, in C. sophera, no embryogenic respose was obtained through any of the explants tested on various treatments. The mature cotyledons of C. angustifolia excised from the mature and dried seeds failed to induce embryogenic response on the same treatments. These observations revealed that the age of the explant plays an important role in the induction of somatic embryos. Callus production followed

164 by somatic embryogenesis has been reported in several plants using different explants such as cotyledon explants in Juglans nigra (Neuman et al. 1993), leaf explants in Holostemma ada-kodien (Martin 2003a) and stem petiole and leaflet explants in Swainsona formosa (Sudhersan and Abo El-Nil 2002). However, immature and young explants are generally more responsive as in the case of Hardwickia binata (Chand and Singh 2001) and Dalbergia sissoo (Singh and Chand 2003). The developmental stage of the explant has been reported to have a critical influence on the ability to induce somatic embryogenesis in woody legumes (Trigiano et al. 1992) and transition of somatic cells into embryogenic cells (Ammirato 1983 and Conger et al. 1983).

Generally an auxin like 2,4-D is considered essential for the induction and maintenance of embryogenic cultures (Chang 1991; Choi et al. 1999 and Joseph et al. 2000), however, a combination of auxin and cytokinin also found to induce embryogenic calli in several plants (Jayanthi and Mandal 2001; Martin 2004b and Varshney et al. 2009). In the present investigation, various auxins were tried and amongst them 2,4-D provided best response. The medium containing 10.0 µM 2,4-D exhibited highest 83.90 ± 1.70% response with the production of 9.23 ± 0.67 embryoids/explant. Similarly in Eleutherococcus sessiliflorus and Holostemma ada-kodien, the role of 2,4-D in the production of embryogenic callus has been emphasized (Choi et al. 2002 and Martin 2003a) and the same is further corroborated by our study. The capability of 2,4-D in activating embryogenic pathway may be related to its capacity to induce stress genes, which have been shown to contribute to the cellular reprogramming of the somatic cells towards embryogenesis (Kitamiya et al. 2000). All other auxins tested proved to be less effective than 2,4-D, while the use of NAA (5.0 µM) found to be efficient for the germination of embryoids on the same medium although produced lesser number of embryoids (6.26 ± 0.40 embryoids/explant) in 42.96 ± 1.43% cultures. According to Peeters et al. (1991), uptake and utilization of NAA is faster, so conversion of somatic embryos may be due to the faster uptake and utilization of NAA. Similarly, in the earlier report of somatic embryogenesis by Agrawal and Sardar (2007) in C. angustifolia, the somatic embryos produced in 54.16% cultures on 10.0 µM NAA underwent germination (2.63%) on the same medium.

165

The presence of cytokinin in the induction medium proved to be crucial for the high frequency of somatic embryos. Accordingly, augmentation of different cytokinins with optimal concentration of 2,4-D (10.0 µM) enhanced the rate of embryogenesis and facilitated germination of embryoids in the present study. The maximum of 22.80 ± 1.59 embryoids/explant were obtained on medium containing 2,4-D (10.0 µM) and BA (1.0 µM) in 90.56 ± 1.88% cultures. However, Agrawal and Sardar (2007) reported only 14.36 ± 2.26 somatic embryos in 70% cultures on MS medium supplemented with 10.0 µM 2,4-D and 2.5 µM BA. The combined favourable influence of auxin and cytokinins observed in the present study is in accordance with the reports on other plants like Rauvolfia caffra (Upadhyay et al. 1992b), Echinochloa colona (Samantaray et al. 1997) and Arachis hypogea (Venkatachalam et al. 1999b). Conversely, the addition of 2,4-D alone or with BA was not favourable for somatic embryos formation in Rauvolfia micrantha (Sudha and Seeni 2006), similarly, the inefficiency of 2,4-D for somatic embryogenesis has also been reported by Zhou et al. (1992) in lettuce, which is in contrast to our results.

Globular, torpedo and cotyledonary staged embryoids were clearly visible, while the heart shaped embryoids were observed under histological sections. The mature somatic embryos started germination on the cytokinin-auxin supplemented medium and exhibited maximum 35.33 ± 2.90% germination on optimal medium comprised of MS + 2,4-D (10.0 µM) + BA (1.0 µM). However, Xie and Hong (2001b) reported callus induction in Acacia mangium in MS medium supplemented with 2,4-D and Kn from cotyledon explants of mature zygotic embryos, which is in contrast to the present study. Transferring of 8 days old germinating embryoids to hormone free MS basal and half strength MS medium helped in the development of complete plantlets with proper shoot and root formation within 3 weeks. Although, the survival percentage of embryioids on MS basal and half strength MS medium was quite low i.e. 20 and 30% respectively. Similar to our results, Sahai et al. (2010a) reported successful development of complete plantlets in Tylophora indica, when 10 days old germinating somatic embryoids were transferred to half strength MS medium, within 3 weeks of transfer. However, Purohit et al. (2002) reported the germination and conversion of somatic embryos of Quercus floribunda into

166 complete plantlets on a combined SH and MS medium supplemented with 0.44- 2.20 µM BA.

5.7 Rooting in microshoots Rooting in regenerated microshoots is an important step in micropropagation which is essential for the development of complete plantlets and it involves three distinct phases namely: induction, initiation and expression (Kevers et al. 1997 and De Klerk et al. 1999). In the absence of proper root system, plantlets will not be able to survive under external or ex vitro conditions and losses at this stage have vast economic consequences (De Klerk 2002). Rooting can be induced via in vitro or ex vitro methods and in the present investigation both the strategies were adopted in C. angustifolia as well as C. sophera. During the study, it was found that rooting in microshoots of C. angustifolia was often problematic and resulted in the production of basal callusing in addition to yellowing and abscission of leaves. Therefore, efforts were taken to reduce these problems and a considerable success has been achieved through the adoption of ex vitro mode of rooting and by the incorporation of phloroglucinol (PG) in the in vitro rooting medium.

5.7.1 In vitro rooting Strength of the MS medium played an important role in rooting of microshoots as it was observed that in C. angustifolia, full strength MS medium without any auxin failed to induce rooting while reducing the strength of MS medium to half proved to be beneficial and showed induction of roots in 11.00 ± 2.08% microshoots with 1.33 ± 0.20 roots/shoot. Whereas, in C. sophera full strength MS medium without auxin found to be quite effective for the induction of roots in 42.33 ± 1.45% microshoots with 1.76 ± 0.14 roots/shoot, furthermore the half strength MS without auxin enhanced the rooting percentage (65.00 ± 1.73%) with the production of 2.43 ± 0.23 roots/shoot. Rooting of microshoots on auxin free media might be due to high endogenous levels of auxins in in vitro raised shoots. Rooting on auxin free MS basal medium has also been reported by Reddy et al. (1998) in Gymnema sylvestre and Pyati et al. (2002) in Dendrobium macrostachium. Superiority of half strength MS in rooting over full strength MS

167 medium has been well documented in the literature (Nabi et al. 2002; Faisal and Anis 2005; Shahzad et al. 2006; Parveen et al. 2010 and Shahzad et al. 2012).

Incorporation of three different auxins (IAA, IBA and NAA) with half strength MS medium facilitated better rhizogenesis and amongst three auxins tested, IBA at 1.0 µM proved to be the ideal for in vitro rooting in both the plants. While NAA and IAA provided less response. In C. angustifolia, the maximum 3.23 ± 0.52 roots/shoot with 2.66 ± 0.35 cm of root length in 59.00 ± 7.81% microshoots were produced on half strength MS medium containing 1.0 µM IBA. However, Agrawal and Sardar (2006) used 10.0 µM IBA with half strength MS medium for the induction of roots in C. angustifolia. Microshoots of C. sophera also exhibited the highest rooting percentage (93.66 ± 2.40%) on medium comprised of half strength MS + IBA (1.0 µM) with the production of an average 5.70 ± 0.47 roots/shoot having root length of 5.63 ± 0.49 cm. Our results are in accordance with various findings where IBA proved to be more efficient for rooting than other auxins in number of plants such as Cunila galoides (Fracaro and Echeverrigaray 2001), Embelia ribes (Raghu et al. 2006b), Clitoria ternatea (Shahzad et al. 2007), Cassia siamea (Parveen et al. 2010) and C. sophera (Parveen and Shahzad 2010). Another reason for being more potent auxin is that IBA is comparatively lesser degraded by autoclaving than IAA and is generally considered to be more stable in the light than IAA, which is rapidly photooxidized (Nissen and Sutter 1990; Epstein and Müller 1993 and De Klerk et al. 1999).

The incorporation of different concentrations of phloroglucinol (PG) with the optimized rooting medium in C. angustifolia enhanced the rooting percentage significantly. The best response observed in the medium comprised of half strength MS + IBA (1.0 µM) + PG (5.0 µM), where the highest 4.80 ± 0.17 roots/shoot with an average root length of 4.36 ± 0.14 cm were obtained in 82.00 ± 6.42% microshoots. PG, a phenolic compound is responsible for the suppression of peroxidase activity in the culture and thus protects the endogenous auxin from peroxidase-catalysed oxidation which facilitates healthy root formation (De Klerk et al. 1999 and Parveen et al. 2012). The promotive effect of PG on rooting was identified in several plant species including Prunus avium (Hammatt and Grant 1996), Malus pumila (Zanol et al. 1998), Decalepis

168 hamiltonii (Giridhar et al. 2005) and Pterocarpus marsupium (Husain et al. 2007a and 2008). In all these reports PG enhanced rooting in the presence of auxin, which is in consonance with our findings.

The gelling substance used in rooting medium also had a great impact on in vitro rooting. In case of C. angustifolia, in vitro rooting in liquid medium (without agar) proved to be more efficient than agar gelled medium. The half strength liquid MS medium without any auxin induced rooting in 20.33 ± 2.02% microshoots with the production of 1.60 ± 0.20 roots/shoot after 4 weeks of transfer. Addition of three different auxins at various concentrations further enhanced the rooting response and the best rooting response (87.33 ± 2.33%) was obtained on half strength liquid MS medium containing IBA (1.0 µM) and PG (5.0 µM) with a maximum of 5.56 ± 0.23 roots/shoot having average root length of 6.23 ± 0.14 cm after 4 weeks. Roots were healthy, thicker and longer and more in number compared to agar gelled medium at the same concentration of hormones. The superiority of liquid medium in rooting has also been observed in different plant species (Gangopadhyay et al. 2002; 2004 and Sutheer et al. 2011). The filter paper bridge/support provided in liquid medium gave better anchorage owing to its porosity that facilitated better absorption throughout its surface area. While the rooting medium comprised of phytagel (0.25%) proved to be least effective in C. angustifolia and produced short, stunted roots along with basal callusing. On the other hand in C. sophera, in vitro rooting in phytagel gelled medium provided satisfactory results, although the number of roots (7.63 ± 0.23) produced on optimal rooting medium comprised of half strength MS + IBA (1.0 µM) was more than the agar gelled medium but the average root length (4.66 ± 0.35 cm) was less. Faisal et al. (2006a) also reported that rooting medium solidified by agar was more suitable than phytagel or gelrite and provided more branched and thicker roots in Mucuna pruriens.

5.7.2 Ex vitro rooting In vitro rooting in C. angustifolia, involved several problems like necrosis of shoot tips and yellowing or abscission of leaves on transferring to rooting media and the formation of callus at cut end of the microshoots also prevented the development of roots in auxin-supplemented medium (Parveen and Shahzad

169

2011). Thus, to rectify the problems of in vitro rooting, an alternative method (ex vitro rooting) was adopted to induce rooting in C. angustifolia. Microshoots were given pulse treatment with higher concentrations of IBA for short duration and the best results obtained through pulse treatment of 200 µM IBA for 30 min. Treated microshoots were subsequently transferred to sterile soilrite and kept under controlled conditions. An average of 4.83 ± 0.24 roots/shoot were obtained in 90.33 ± 2.60% microshoots attaining the root length of 5.80 ± 0.23 cm. Through ex vitro rooting technique, necrosis and the leaf abscission has been minimized considerably and healthy developmental pattern was observed. Ex vitro rooting has also been achieved in C. sophera and similar responses were noticed. The optimal response was obtained through 200 µM IBA producing the highest 5.06 ± 0.17 roots/shoots with root length of 5.93 ± 0.17 cm in 90.66 ± 2.96% microshoots. Our results substantiate with the report of Pandeya et al. (2010) in Clitoria ternatea, in which ex vitro rooting was obtained through pulse treatment with 250 mg/l IBA for half an hour.

Further, it was found that ex vitro rooted shoots attained longer root length compared to in vitro rooted shoots within same time period in C. sophera which facilitated in the adaptation and survival of plantlets in the field conditions. Similarly, Bozena (2001) suggested that the plantlets of strawberry developed after ex vitro rooting have better developed root system than the ones raised through in vitro rooting, which is in agreement with our findings. Rooting in the external environment is an aid for simultaneous hardening and acclimatization of plantlets and decreased the micropropagation cost as well as the time from laboratory to field conditions (Pruski et al. 2000). Ex vitro rooting proved to be more advantageous over in vitro rooting as the later requires utmost care while planting out and also more labour and time. This is in corroboration with the earlier studies in several other plant species such as Lagerstroemia parviflora (Tiwari et al. 2002), Prunus fruticosa (Kris et al. 2005), Celastrus paniculatus (Martin et al. 2006); Aegle marmelos (Raghu et al. 2007), Holarrhena antidysenterica (Mallikarjuna and Rajendrudu 2007) and Siratia grossvinorii (Yan et al. 2010b).

170

5.8 Acclimatization of regenerated plantlets The success of any micropropagation protocol depends on the acclimatization of regenerated plantlets in the external environment at low cost and with high survival rate. During this period of transition from in vitro to ex vitro conditions, plants have to compete many adverse conditions as they were cultured under aseptic conditions, with low light intensity on medium containing ample sugar and nutrients to allow heterotrophic growth in the atmosphere of high relative humidity. These conditions result in the plantlets with altered morphology, anatomy and physiology (Kozai 1991; Desjardins 1995 and Pospíšilová et al. 1999). Therefore, after ex vitro transplantation plantlets usually need few weeks of acclimatization and gradually overcome these inadequacies to adapt in the external environment. The survival of plantlets during acclimatization also depends on the use of suitable planting substrate. Thus, in the present study three different planting substrates i.e. garden soil, soilrite and vermiculite were used to standardize the best planting substrate for the regenerated plantlets of both the species. It was observed that amongst three substrates used, soilrite provided maximum survival rate in C. angustifolia (89.33 ± 2.33%) as well as C. sophera (97.66 ± 1.45%) followed by vermiculite and garden soil. Faisal et al. (2006a and 2006b) also obtained the same responses with these three planting substrates in Mucuna pruriens which are in corroboration with our study. However, in contrast to our results, vermicompost (Chandramu et al. 2003 and Rani and Rana 2010) and vermiculite (Swamy et al. 2004; Rajeswari and Paliwal 2006 and Faisal et al. 2007) have been used for different plant species.

After successful acclimatization, in vitro raised plantlets of C. angustifolia and C. sophera were transferred to earthen pots containing sterilized soil and manure (1:1) and kept under green house for 2 weeks and then finally transferred to the field with 90% survival rate. These plants did not show any detectable variation in morphological or growth characteristics when compared to the control plants in both the species.

5.9 Synthetic seeds The encapsulation technique is an important application of micropropagation that offers the potential of easy handling, exchange of germplasm between

171 laboratories, efficient short or long term storage and improves delivery of in vitro regenerated plantlets to the field or to the green house (Piccioni and Standardi 1995; Chand and Singh 2004b and Rai et al. 2009). Synthetic, artificial or somatic seed is an analogous to the true or botanical seed and consists of a somatic embryo surrounded by one or more artificial layers forming a capsule (Pintos et al. 2008). Production of synthetic seeds by encapsulating somatic embryos has been largely favoured since somatic embryos possess both shoot and root primordial and are usually able to develop directly into complete plantlets (Ara et al. 2000). However, there are several reports which have used meristematic shoot tips or axillary buds for the production of synthetic seeds (Faisal et al. 2006d; Rai et al. 2008; Sharma et al. 2009; Ahmad and Anis 2010; Mishra et al. 2011 and Sharma and Shahzad 2012).

In the present investigation synthetic seeds were prepared through encapsulation of nodal segments excised from one month old in vitro microshoots of both the plants to assess the regeneration potential of the explants under different culture conditions and storage at 4ºC. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) tested to prepare ideal beads with uniform texture and the best results were obtained through the combination of 3% Na2- alginate with 100 mM CaCl2·2H2O in C. angustifolia as well as C. sophera as described for many other species (Faisal and Anis 2007; Rai et al. 2008; Singh et al. 2010 and Ahmad and Anis 2010). Lower concentrations of Na2-alginate below 3% resulted in the formation of soft and fragile beads which were difficult to handle whereas, concentrations above 3%, produced isodiametric beads which were hard enough to cause considerable delay in germination. Lower concentrations of CaCl2·2H2O, prolonged the complexion time whereas, higher concentrations adversely affected the bead quality. Nevertheless an encapsulation matrix of 5% Na2-alginate with 50 mM CaCl2·2H2O was found most suitable for the formation of ideal beads in Cannabis sativa (Lata et al. 2009a) which is contrary to our results.

The most desirable property of the encapsulated explants is their ability to retain viability in terms of regrowth and conversion abilities after encapsulation (Adriani

172 et al. 2000 and Micheli et al. 2007). In the present study, the encapsulated explants of both the plants were transferred to regeneration medium for the conversion of beads into plantlets, but unfortunately instead of complete plantlets production, multiple shoots were induced which lack roots. Maximum conversion (94.06 ± 1.56%) was obtained on medium comprised of MS + BA (2.5 µM) + NAA (0.4 µM) in C. angustifolia and in C. sophera 92.33 ± 1.45% beads were converted into shoots on MS + BA (2.5 µM) + NAA (0.5 µM). The synergistic influence of the combination of cytokinin and auxin on synseed germination has also been demonstrated in other plants like Dalbergia sissoo (Chand and Singh 2004b), Withania somnifera (Singh et al. 2006b) and Spilanthes acmella (Sharma et al. 2009). Emergence of single or multiple shoots from the encapsulated explants has also been reported in other medicinal plants (Mandal et al. 2000; Lata et al. 2009a and Shrivastava et al. 2009).

For the production of roots, the shoots were excised and rooted either through in vitro or ex vitro methods which have already been standardised for both the plants. Similar to our results, Mishra et al. (2011) also described that merely 21.43% encapsulated explants of Picrorhiza kurroa exhibited simultaneous production of shoots and roots while rest of the non-rooted shoots were transferred to root induction medium for the development of roots. Direct sowing of encapsulated nodal segments to sterile soilrite provided simultaneous induction of roots from the shoots, although with a small success i.e. 20% and 30% germination in C. angustifolia and C. sophera respectively. Ex vitro conversion of synthetic seeds has also been performed previously in other plants (Mandal et al. 2000; Soneji et al. 2002 and Naik and Chand 2006).

Synseeds of both the plant species were stored at 4ºC for short term preservation and the rate of germination was examined after different storage periods. In C. angustifolia the encapsulated nodal segments retained sufficient regeneration potential (72.30 ± 1.21%) up to 4 weeks of cold storage compared to non-encapsulated nodal segments which exhibited only 33.33 ± 1.35% germination on optimal medium. Thereafter, a sharp decline was observed in the rate of germination and after 8 weeks merely 43.9 ± 1.49% synseeds were germinated. Similar results were obtained with the synseeds of C. sophera,

173 where after four weeks of cold storage, 64.00 ± 3.05% germination was recorded in encapsulated nodal segments compared to 25.00 ± 2.88% germination in non- encapsulated explants. The conversion percentage decreased with an increase in storage period and temperature and commonly cold temperature is more effective than room temperature for post storage survival and germination of encapsulated explants (Mohanraj et al. 2009 and Ikhlaq et al. 2010). The alginate matrix supplemented with the necessary ingredients, served as an artificial/synthetic endosperm, thereby providing resistance to manipulation and transport and preserves viability and conversion (Prewein and Wilhelm 2003). These findings suggested that storage of encapsulated explants for a considerable period of time allows the preservation of germplasm and could be used efficiently for regeneration of plantlets. Our results are in corroboration with the earlier findings of Lulsdorf et al. (1993) who also reported that encapsulated and non-encapsulated somatic embryos of interior and black spruce survived one month of storage at 4ºC with no loss in conversion capacity. Similarly, Rao and Bapat (1993) reported that encapsulated embryos of Santalum album retained their germinability (18%) after storage at 4ºC for 45 days. Cold temperature (4ºC) is generally used for storing encapsulated explants in several plant species (Chand and Singh 2004b; Tsvetkov and Hausman 2005; Faisal et al. 2006d; Singh et al. 2006b and 2010). However, contrary to these reports, storage of Khaya senegalensis capsule was much less effective at 4ºC (28-84%) compared to 25ºC (84-92%).

5.10 Physiological studies In vitro raised plantlets usually exhibit abnormal morphology, anatomy and physiology (Desjardins 1995 and Kozai and Smith 1995). In many plants, these abnormal characters affected the acclimatization of in vitro plantlets to the external environment. The plants growing under heterotrophic conditions in vitro have low photosynthetic rate due to low light intensities, low CO2 concentrations (Infante et al. 1989) and inhibition of photosynthesis by high sugar concentration in the medium (Sheen 1990; Lees et al. 1991 and Reuther 1991). Therefore, the physiology of micropropagated plantlets, in particular the photosynthetic performance during in vitro and ex vitro growth has been the subject of several investigations (Masmoudi et al. 1999 and Van Huyelenbroeck et al. 2000).

174

Nevertheless, after transfer to ex vitro conditions, most plantlets develop a functional photosynthetic apparatus (Kozai 1991). In the present study photosynthetic competence of regenerated plantlets of C. angustifolia and C. sophera was evaluated by the estimation of different pigment contents

(chlorophyll a, b and carotenoids) and net photosynthetic rate (PN ratio) during different periods of acclimatization under ex vitro conditions.

The total chlorophyll content (chlorophyll a and b) and carotenoids showed a decreasing trend during initial days of acclimatization of plantlets of C. angustifolia and C. sophera from in vitro to ex vitro conditions, but later on a linear increase was observed in all the pigments content. Lu and Zhang (1998) and Sopher et al. (1999) suggested loss of chlorophyll during acclimatization means that the leaves were damaged due to photoinhibition. By the end of fourth week, the highest contents of Chl a, Chl b, total Chl and carotenoids were recorded in both the plant species suggesting that the plants are now fully acclimatized and adapted to the external environment. Similar trend was observed for chlorophyll content 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 observed thereafter. Our results substantiate with the earlier reports of Pospíšilová et al. (1998); Amâncio et al. (1999) and Jeon et al. (2005). An increase in carotenoid level 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 and Van Huylebroeck et al. 2000).

Net photosynthetic rate (PN ratio) is affected by irradiance, CO2 concentration in the vessels and type and concentration of sugar in the medium; it usually decreased in the first days after transplantation and increased thereafter (Pospíšilová et al. 2009). Similar observations were recorded in the present study also, PN ratio decreased during first week of acclimatization in both the plant species and increased thereafter. The decline in photosynthetic rate indicated that climatic conditions create stress in regenerated plants, while,

175 increase in PN ratio after ex vitro transfer is usually associated with the formation of new leaves (Amâncio et al. 1999 and Slavtcheva and Dimitrova 2001). Similar results were also observed earlier in different plant species (Van Huylenbroeck and Debergh 1996; Kadleček et al. 2001 and Guan et al. 2008).

5.11 Histological studies Histological studies have contributed significantly to the understanding of in vitro culture systems. A good histological study based on histological changes provides insight into cellular processes and also provides clues that allow for the proposal of the hypotheses for further experimentation (Yeung 1999). Structural analysis is an important first step in the study of the organization and changes in the plant body, and it is an extremely useful approach in the study of plant morphogenesis (Wetmore and Wardlaw 1951). Histological analysis of regenerating tissue helped in determining the exact origin and mode of regeneration of the developing shoot buds and embryoids through various explants of C. angustifolia and C. sophera.

Histology of the regenerating tissue obtained on BA (5.0 µM) supplemented MS medium through CN explant of C. angustifolia showed that shoot buds were formed successively from actively dividing cells at the base of the axillary buds as well as from the basal swollen region of the cotyledonary node without any intervening callus formation. The pattern of bud origin and development was similar to the cotyledonary nodes of Pisum culinaris (Malik and Saxena 1992b), Cajanus cajan (George and Eapen 1994), Vigna radiata (Avenido and Hattori 2001) and Vicia faba (Aly and Hattori 2007). NS explant of C. sophera on BA supplemented medium showed direct origin of shoot buds from the swollen region of the nodal explant through meristemoids formation. Meristemoids were differentiated around the node junction of the explant as several small aggregates of cells with prominent nuclei and later on developed into shoot buds having apical meristem and leaf primordial. Similar developmental pattern of shoot buds has also been observed in nodal segment culture of Veronica anagallis-aquatica (Shahzad et al. 2011). The explants (CN and NS) cultured on TDZ supplemented medium produced deformed and abnormal shoots on prolonged exposure as well as at higher concentrations in both the plant species.

176

This was confirmed by the histological analysis of the regenerating tissues at various stages of development on TDZ supplemented media in both the plants. Histological sections revealed the formation of abnormal shoot buds having deformed apical dome with single, double or multiple abnormal leaf primordia.

The histological analysis of CL derived callus tissue in C. angustifolia revealed that after transferring to the regeneration medium, most of the peripheral cells of the callus tissue underwent divisions leading to the formation of meristemoids similar to those described by Saravitz et al. (1993). Further it was observed that the peripheral meristemoids frequently developed into shoot buds, however, the meristemoids which were deep seated into the callus tissue remain suppressed and showed slower growth and development. Similar observations were also made in the histology of CL derived callus of C. sophera, wherein the peripheral meristemoids enlarged in size developed apical dome surrounded by the leaf primordia and showed faster growth and development compared to deep seated meristemoids. Our findings are similar to the earlier reports of Das et al. (1996b) and Vengadesan et al. (2000) in Acacia catechu and Acacia sinuata respectively. The application of hormones within MS medium leads the parenchymatous cells to dedifferentiate, subsequently forming a meristemoid and finally an organ (Thorpe 1980). The combining effect of cytokinin (BA) and auxin (NAA) enhanced shoot regeneration from organogenic calli as it has been evident in several earlier studies (Gonzalez-Benito and Alderson 1990 and Jethwani and Kothari 1996). In histological study of root derived callus tissue of C. angustifolia, shoot buds development was clustered in several pockets (nodules) rather than solitary as in most of the cases wherein generally meristematic zones are organized separately and converted into shoot buds. Nodular proliferation and subsequent shoot bud formation from root explant has also been observed in Acacia albida (Ahee and Duhoux 1994) and Brassica napus (Sharma et al. 1993), whereas, in A. julibrissin (El Maataoui et al. 1998) buds developed from callus tissue originated from some pericycle cells. In our view this type of indirect multiplication would be least prone to somaclonal variations as a stock regenerative tissue was formed which continued to proliferate and regenerate shoot buds for long-term culture establishment (Parveen and Shahzad 2011).

177

Histology of embryogenic callus produced through immature cotyledon explants of C. angustifolia revealed the mode of differentiation of somatic embryos via indirect embryogenesis. Certain cells of the callus differentiate to produce proembryos which later developed into globular embryos having distinct boundaries and no vascular connection with the parent tissue. This criterion was one suggested by Haccius (1978) to distinguish somatic (non-zygotic) embryos from shoot buds. Globular embryos underwent several divisions and grew in size to become heart shaped embryo having cotyleonary initials and distinct apical meristem region. A single celled row of suspensor-like structure or rudimentary suspensor was also observed at heart shaped stage confirming the unicellular origin of somatic embryo development. There are only a few reports which mention the appearance of suspensor or suspensor like structure during somatic embryogenesis (Nonohay et al. 1999; Quiroz-Figueroa et al. 2002 and Sharma and Millam 2004). The developing heart shaped embryos further progressed towards late heart shaped or torpedo shaped embryos and finally attained the cotyledonary stage with well-developed mature cotyledons having differentiating apical meristem clearly visible under histological sections.

178

Chapter 6 Summary & Conclusions

Chapter 6

SUMMARY AND CONCLUSIONS

In recent years there has been an increased interest in plant tissue culture technique which offers a viable tool for mass multiplication and conservation of species especially those having medicinal or pharmacological value. Most of the medicinal plants are collected from wild population which involve destructive harvesting because of the use of plant parts like root, stem, wood, bark and the whole plant in case of herbs and only a few medicinal plants are commercially cultivated, thus challenging their existence in the nature. Therefore, to meet the future demands and conservation of valuable medicinal plant species, development of in vitro regeneration protocols, exploiting techniques of plant tissue culture is mandatory. In the present study two medicinally important species of genus Cassia i.e. C. angustifolia Vahl. and C. sophera Linn. have been selected to develop reproducible and efficient in vitro protocols via different modes of regeneration for mass propagation and conservation of these plant species.

6.1 Cassia angustifolia Vahl. Seeds of C. angustifolia germinated under in vitro culture conditions to raise aseptic seedlings for the collection of different explants for shoot morphogenesis via direct or indirect organogenesis. Maximum seed germination (77.00 ± 2.31%) was obtained on medium comprised of half strength MS supplemented with 5.0

µM GA3 after 4 weeks of inoculation, while, germination started within 3.20 ± 0.24 days of inoculation. Three different explants viz.: CN, NS and ST excised from axenic seedlings at different age were cultured on various concentrations of cytokinins (BA, Kn and 2iP) or auxins (IAA, IBA and NAA) singly or in combinations. CN explants excised from 14 days old seedlings showed early and enhanced regeneration compared to those from 7 and 21 days old seedlings, similarly NS and ST explants taken from 21 days old seedlings provided better response than explants of 14 and 28 days old seedlings.

179

CN explant proved to be the best explant amongst all the three explants tested for direct shoot regeneration in C. angustifolia on various hormonal treatments. Amongst three cytokinins tested, 5.0 µM BA found to be the optimal concentration producing the highest 25.33 ± 0.20 shoots/explant attaining a maximum shoot length of 4.33 ± 0.20 cm in 75.40 ± 0.30% cultures through CN explants after 6 weeks of inoculation. Explants cultured on Kn supplemented media, provided optimal response at higher concentration i.e. 7.5 µM, producing an average of 8.96 ± 0.20 shoots/explant with shoot length of 4.23 ± 0.14 cm in 54.60 ± 0.23% cultures. While, 2iP proved to be the least effective cytokinin in C. angustifolia providing merely 6.10 ± 0.20 shoots/explant having shoot length of 3.26 ± 0.17 cm in 42.40 ± 0.30% cultures. Thus, BA proved to be the best cytokinin followed by Kn and 2iP (BA > Kn > 2iP).

Addition of lower concentrations of auxins with cytokinin enhanced the shoot multiplication rate and the explants cultured on BA (5.0 µM) + NAA (0.6 µM) combination produced a maximum of 39.16 ± 0.14 shoots/explant with shoot length of 5.63 ± 0.20 cm in 85.26 ± 0.17% cultures after 6 weeks. Cultures were maintained and transferred to fresh medium of the same composition up to 6 subculture passages of 6 weeks interval. Addition of adenine sulphate (AdS) at various concentrations to the regeneration medium after first subculturing, found effective to overcome the adverse effects of leaf abscission and shoot tip necrosis. Hence, the medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) + AdS (30.0 µM) proved to be the best regeneration medium for multiplication and proliferation of shoots in C. angustifolia. Differentiation and multiplication of shoots continued up to 4th subculture passage, where the highest 64.90 ± 0.32 shoots/explant having shoot length of 6.56 ± 0.23 cm were obtained. The emergence of shoots became constant at 5th subculture passage and thereafter a decline in regeneration potential was observed.

The CN explants cultured on a wide range of TDZ concentrations responded similar as the single BA treatment on shoot morphogenesis. Optimal response was recorded at 2.5 µM TDZ with the production of maximum 28.53 ± 0.99 shoots/explant having shoot length of 4.03 ± 0.14 cm in 94.33 ± 2.33% cultures after 6 weeks. However, prolonged exposure of TDZ resulted in certain anomalies and retarded the elongation in shoots. Therefore, a two-step culture

180 procedure was adopted in which the shoots regenerated on 2.5 µM TDZ were further transferred to MS medium devoid of TDZ as well as to the medium containing lower concentrations of BA after 6 weeks. The regenerated shoots showed growth and development of new shoots on BA containing medium and the highest 45.50 ± 0.51 shoots/explant having shoot length of 6.20 ± 0.11 cm were obtained at 2.5 µM BA after 6 weeks. The regenerative tissue was regularly subcultured on to the fresh medium containing 2.5 µM BA at an interval of 6 weeks but the number of shoots remained constant and 45.50 ± 0.51 shoots/explant were obtained at the end of each subculture passage up to second subculturing and beyond that a decline in regeneration efficacy was recorded. Thus, TDZ induced cultures could not be maintained for long term multiplication and proliferation of shoots.

The augmentation of 5.0 µM BA proved to be optimal for nodal explants, where a maximum of 20.20 ± 0.11 shoots/explant with shoot length of 4.20 ± 0.11 cm was obtained in 66.00 ± 1.15% cultures after 6 weeks of inoculation. Regeneration efficacy of the explant further enhanced on cytokinin-auxin combination treatments and reached to the maximum 30.33 ± 0.24 shoots/explant having shoot length of 5.26 ± 0.14 cm in 79.33 ± 1.79% cultures on MS + BA (5.0 µM) + NAA (0.6 µM) in 6 weeks of incubation. TDZ at an optimal concentration of 2.5 µM exhibited maximum regeneration efficiency (86.00 ± 2.30%), producing an average of 21.70 ± 0.62 shoots/explant with shoot length of 3.80 ± 0.17 cm after 6 weeks. TDZ induced cultures were further transferred to BA containing medium for growth and proliferation. The medium comprised of 2.5 µM BA enhanced the number of shoots to 32.03 ± 0.31 shoots/explant attaining maximum shoot length of 5.66 ± 0.17 cm after 6 weeks.

Like CN and NS explants, ST explants also exhibited best regeneration on cytokinin-auxin combinations, producing the maximum 22.46 ± 0.29 shoots/explant with shoot length of 4.00 ± 0.11 cm on medium comprised of MS + BA (5.0 µM) + NAA (0.6 µM) after 6 weeks. ST explants were also cultured on different concentrations of TDZ and the same strategy of two-step culture procedure was adopted in which TDZ (2.5 µM) induced cultures (15.06 ± 0.1 shoots/explant) were transferred to medium containing lower concentrations of BA. Medium containing 2.5 µM BA improved the regeneration efficacy and the

181 shoot number increased to 25.90 ± 0.20 shoots/explant with shoot length of 4.56 ± 0.23 cm after 6 weeks.

Various factors like composition of culture medium, pH of the medium and concentration of sucrose affected the shoot morphogenesis and vary from species to species. In the present study all these factors were optimized for different explants (CN, NS and ST) for maximum shoot regeneration and it was found that among different media tested (B5, L2, MS and WPM), MS medium at pH value of 5.8 with 3% sucrose provided highest response for shoot multiplication and proliferation in C. angustifolia.

For indirect organogenesis, three different explants namely cotyledonary leaf (CL), leaf (L) and root (R) were taken from seedlings of different age group and cultured on different concentrations of cytokinins and auxins. Among these three explants, CL explants (14 days old) and root (30 days old) explants found to be efficient for the production of multiple shoots via callus formation, while leaf derived callus failed to induce shoot differentiation. Root explant produced compact and nodular organogenic callus on medium supplemented with TDZ (1.0 µM) with 90.66 ± 1.45% regeneration response, while the cotyledonary leaf explants produced regenerative callus on 2,4-D (5.0 µM) containing medium suggesting that the efficiency of callus induction may vary within the same plant species depending on the type of explants used.

Callus derived through root explants on TDZ (1.0 µM) was transferred to different concentrations of cytokinins and the optimal response was obtained at 2.5 µM BA with the production of 24.56 ± 1.97 shoots/explant having shoot length of 4.70 ± 0.26 cm after 6 weeks. Incorporation of auxins with optimal BA concentration facilitated better shoot differentiation from the callus and a maximum of 35.63 ± 0.75 shoots/explant having shoot length of 5.43 ± 0.20 cm were produced in 90.33 ± 1.45% cultures on MS + BA (2.5 µM) + NAA (0.6 µM) after 6 weeks. Similarly, cotyledonary leaf derived callus showed maximum shoot differentiation (23.16 ± 1.44 shoots/explant) with an average shoot length of 5.00 ± 0.26 cm in 96.33 ± 1.45% cultures on medium comprised of MS + BA (5.0 µM) + NAA (0.4 µM), although the number of shoots was less compared to root explant.

182

Cotyledonary leaf derived callus could not be used for long term maintenance and proliferation of shoots, as a sudden decline in the regeneration potential of the callus was observed just after first subculture passage. However, on the other hand, root derived callus was regularly subcultured on to the fresh medium of optimal hormonal composition [MS + BA (2.5 µM) + NAA (0.6 µM)] at an interval of 6 weeks. Elongated shoots were isolated and transferred to rooting media at the end of each subculture passage. Differentiation of shoot buds with multiplication and proliferation of shoots continued up to 4th subculture passage where the highest 42.66 ± 1.47 shoots/explant with shoot length of 6.46 ± 0.12 cm were obtained and thereafter decline in regeneration potential was observed.

Immature cotyledons excised from green seeds of semi mature pods found ideal for the production of embryogenic callus in C. angustifolia. A number of treatments containing different auxins were tested for the induction of somatic embryogenesis. Best response (83.90 ± 1.70%) was obtained at 10.0 µM 2,4-D with the production of 9.23 ± 0.67 embryoids/explant after 6 weeks of culture. The embryogenic response further enhanced (90.56 ± 1.88%) with the addition of 1.0 µM BA along with optimal 10.0 µM 2,4-D, wherein a maximum 22.80 ± 1.59 embryoids/explant was obtained with 35.33 ± 2.90% germination on the same medium.

Regenerated microshoots of 3-4 cm length were excised from the cultures and rooted either in vitro or ex vitro for the development of complete plantlets. The gelling substance used to solidify the rooting medium had great impact on in vitro rooting in C. angustifolia. Best rooting response was obtained in liquid rooting medium (without agar) compared to agar or phytagel gelled rooting medium. Half strength liquid MS medium supplemented with IBA (1.0 µM) found to be optimal for in vitro root induction in 67.33 ± 2.60% microshoots with maximum of 3.93 ± 0.29 roots/shoot having root length of 3.00 ± 0.28 cm after 4 weeks. Rooting percentage further enhanced by the incorporation of phloroglucinol (PG) at 5.0 µM with optimal rooting medium and produced maximum 5.56 ± 0.23 roots/shoot having root length of 6.23 ± 0.14 cm after 4 weeks.

Ex vitro rooting provided an alternative way for the induction of roots in sterile soilrite through pulse treatment with an auxin. In the present study, microshoots

183 were best rooted through pulse treatment with 200 µM IBA for 30 min, producing an average of 4.83 ± 0.24 roots/shoot having 5.80 ± 0.23 cm of root length after 4 weeks. Ex vitro rooting proved to be beneficial, cost effective and required less time compared to in vitro rooting as hardening and acclimatization of plantlets took place simultaneously.

In vitro rooted plantlets were hardened and acclimatized under controlled conditions on three different planting substrates namely garden soil, soilrite and vermiculite. Among all the three planting substrates tested, maximum survival percentage was recorded in soilrite (89.33 ± 2.33%) followed by vermiculite (64.66 ± 2.60%) and garden soil (54.66 ± 2.90%).

Acclimatized plantlets were successfully transferred to the green house under shade and then finally to the field conditions with 90% survival. No detectable phenotypic variations were observed in the in vitro raised plantlets when compared with in vivo grown plants.

For the production of synthetic or artificial seeds, the in vitro derived nodal segments excised from one month old microshoots were encapsulated for short term storage and conservation of propagules. Encapsulation was best achieved with 3% sodium alginate as gelling matrix and 100 mM calcium chloride as complexing agent for the production of ideal beads with uniform texture. Conversion or regrowth of the shoots from the beads was obtained on optimal medium comprised of MS + BA (2.5 µM) + NAA (0.4 µM) with highest 94.06 ± 1.56% conversion response after 6 weeks. Microshoots were isolated from the cultures and rooted either in vitro or ex vitro through optimized protocols. However, direct sowing of synseeds in sterilized soilrite or ex vitro sowing resulted in simultaneous production of shoots as well as roots in 20% beads within 6 weeks.

Encapsulated as well as non-encapsulated nodal segments were stored at 4ºC for different time periods and then germinated on optimal medium to assess the reproducibility of the explant after cold storage. After 4 weeks of storage at 4ºC encapsulated nodal explants showed better conversion response (72.30 ± 1.21%) compared to non-encapsulated nodal explants (33.33 ± 1.75%) on medium containing MS + BA (2.5 µM) + NAA (0.4 µM). However, further

184 increase in the storage duration reduced the reproducibility of the explant and after 8 weeks of storage, only 43.90 ± 1.79% and 12.80 ± 1.32% conversion was recorded in encapsulated and non-encapsulated explants.

Different physiological parameters like content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were evaluated at different acclimatization periods of regenerated plantlets. All the pigments showed an initial drop in the content during first week of acclimatization but during subsequent weeks a linear increase in pigments content was recorded. Net photosynthetic rate (PN ratio) was also evaluated and it showed the same trend, decreased during the first week of acclimatization and as soon as the plantlets get acclimatized to the external environment, PN ratio increased linearly.

Histological observations at different developmental stages of regenerative tissue obtained through CN explants revealed the origin of shoot buds directly from the mother explant without any intervening callus tissue, while the CL and root derived callus revealed the mode of differentiation of shoot buds in callus tissue, confirming indirect organogenesis. Histology of embryogenic callus at various developmental stages confirmed indirect origin of somatic embryos through immature cotyledons.

6.2 Cassia sophera Linn. In C. sophera also, seeds were germinated in vitro to collect various explants for shoot morphogenesis via different modes of regeneration. Maximum seed germination (99.33 ± 0.66%) was achieved on half strength MS medium containing 1.0 µM GA3 after 4 weeks of inoculation and the germination started within 4.46 ± 0.14 days of inoculation. Different explants namely cotyledonary node (CN), nodal segment (NS) and shoot tip (ST) were taken from aseptic seedlings of different age group (14, 21 and 28 days old) for direct shoot regeneration. All the three different types of explants collected from 21 days old seedlings found to be more responsive compared to 14 and 28 days old seedlings. Among three explants tested, NS proved to be the best explant for shoot morphogenesis in this plant species.

Nodal explants were cultured on different concentrations of three cytokinins (BA, Kn and 2iP) singly or in combination with various auxins at lower concentrations 185 for direct shoot induction. Amongst various concentrations of cytokinins, optimal response (90.33 ± 3.17%) was induced at 5.0 µM BA with the production of an average 16.46 ± 1.21 shoots/explant having shoot length of 4.80 ± 0.37 cm after 6 weeks. Kn and 2iP provided optimal regeneration at higher concentration i.e. 7.5 µM. The explants cultured on 7.5 µM Kn produced 7.60 ± 0.34 shoots/explant with the shoot length of 3.66 ± 0.20 cm in 62.33 ± 1.45% cultures. While, 2iP provided least number of shoots (5.16 ± 0.49 shoots/explant) having an average shoot length of 3.10 ± 0.20 cm in 53.33 ± 2.02% cultures. Thus, amongst three cytokinins tested, BA proved to be the best cytokinin in C. sophera also.

The regeneration potential of the explant further increased when cultured on cytokinin-auxin combination treatments and the highest frequency (97.33 ± 1.45%) of shoot production (25.36 ± 0.34 shoots/explant) having maximum shoot length (6.23 ± 0.24 cm) was obtained on MS + BA (5.0 µM) + NAA (1.0 µM) after 6 weeks. Regenerative tissues were subcultured on to the fresh medium of same composition and maintained up to 6 subculture passages of 6 weeks interval. At the end of each subculture passage elongated shoots were harvested and transferred to rooting medium. The regeneration medium was supplemented with AdS at different concentrations to prevent shoot tip necrosis and yellowing and abscission of leaves. In this regard the medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM) + AdS (20.0 µM) proved to be the best for culture multiplication and proliferation on which differentiation of shoots continued up to 4th subculture passage and the highest 42.33 ± 0.32 shoots/explant having shoot length of 7.76 ± 0.14 cm were obtained. Beyond 4th subculture passage reduction in regeneration frequency was observed and the number shoots declined afterwards.

The treatment of TDZ at various concentrations, although induced multiple shoots through nodal segments, but the shoots were stunted and showed abnormal features on prolonged culture incubation. Optimal response (95.00 ± 1.15%) was obtained at 2.5 µM TDZ with the production of an average 13.76 ± 0.38 shoots/explant having shoot length of 3.26 ± 0.14 cm after 6 weeks. To avoid the deleterious effects of TDZ, regenerating cultures were subsequently transferred on to fresh medium devoid of TDZ as well as to medium containing

186 lower concentrations of BA. The number of shoots increased to the maximum 19.83 ± 0.02 shoots/explant with shoot length of 6.00 ± 0.11 cm when the medium was supplemented with 1.0 µM BA. Thus, the two-step culture procedure proved effective in this plant species also but the overall production of shoots and regeneration potential of the explant was better in cytokinin-auxin combination medium compared to TDZ supplemented medium.

CN explants cultured on different concentrations of cytokinins also provided optimal response at 5.0 µM BA with the production of an average 12.43 ± 0.29 shoots/explant with shoot length of 4.23 ± 0.26 cm in 78.66 ± 2.33% cultures after 6 weeks. The combination of cytokinin-auxin treatments showed synergistic effect on shoot multiplication and the highest number of shoots (19.50 ± 0.51 shoots/explant) having shoot length of 5.23 ± 0.14 cm were obtained in 86.00 ± 2.08% cultures on MS + BA (5.0 µM) + NAA (1.0 µM) through CN explant after 6 weeks.

The CN explants cultured on TDZ supplemented media showed a poor response with the production of only 6.73 ± 0.17 shoots/explant having shoot length of 2.26 ± 0.14 cm in 93.00 ± 1.52% of cultures on MS + TDZ (2.5 µM) after 6 weeks. Again it was required to transfer the regenerative tissue to TDZ free medium to retrieve the regenerability of the tissue and for that lower concentrations of BA (1.0 µM) proved to be effective and improved the number of shoots to 14.90 ± 1.35 shoots/explant with shoot length of 5.76 ± 0.23 cm after 6 weeks.

Similar to C. angustifolia, ST explant of C. sophera also exhibited least response on various hormonal supplements. Maximum regeneration was observed in cytokinin-auxin combination with the production of 10.56 ± 0.23 shoots/explant having shoot length of 4.80 ± 0.11 cm in 69.66 ± 1.45% cultures on MS + BA (5.0 µM) + NAA (1.0 µM) after 6 weeks. The cultures induced at 2.5 µM TDZ showed an increase in shoot regeneration from initial 4.70 ± 0.20 shoots/explant to a maximum of 8.56 ± 0.23 shoots/explant when transferred to MS + BA (1.0 µM) with shoot length of 5.20 ± 0.11 cm in 6 weeks of incubation.

Different factors like nutrient composition of the culture medium, pH of the medium and concentration of sucrose were optimized through all the three explants for maximum regeneration. Amongst all the four media tested (B5, L2,

187

MS and WPM), MS medium supplemented with 3% sucrose at pH 5.8 provided optimal response through all the explants and thus used throughout the study.

For the production of callus, three different explants viz.: cotyledonary leaf (CL), leaf (L) and root (R) excised from axenic seedlings of different age were cultured on various concentrations of cytokinins and auxins. Among these three explants, CL explants excised from 14 days old seedlings provided best regenerative callus compared to leaf explants (30 days old), while, root explant failed to induce callus on all the concentrations of hormones. The optimal growth of callus (73.40 ± 0.45%) was obtained through CL explants on medium containing 5.0 µM 2,4-D with the production of green compact and nodular callus. Whereas, leaf explants showed an average growth (53.53 ± 0.81%) of green compact and nodular callus on 7.5 µM of 2,4-D.

Multiple shoots were differentiated from cotyledonary leaf derived callus on MS medium containing cytokinin and auxins singly or in combination, while leaf derived callus failed to differentiate shoots. The highest 14.63 ± 0.23 shoots/explant was produced from CL derived callus on MS + BA (2.5 µM) + NAA (0.5 µM) with shoot length of 5.73 ± 0.20 cm in 74.83 ± 0.32% cultures after 6 weeks of transfer. The regenerative tissue of CL explants regularly subcultured on to the fresh medium after 6 weeks interval on optimal medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM). Multiple shoots continued to differentiate up to 2nd subculture passages where a maximum of 20.60 ± 0.45 shoots/explant with shoot length of 6.43 ± 0.23 cm were obtained and thereafter decline in the regeneration potential was observed.

Microshoots were rooted through both in vitro and ex vitro methods. In vitro rooting was best achieved on agar gelled half strength MS medium supplemented with 1.0 µM IBA producing a maximum of 5.70 ± 0.47 roots/shoot having root length of 5.63 ± 0.49 cm after 4 weeks. Phytagel gelled rooting medium although provided more number of roots/shoot (7.63 ± 0.23) on optimal rooting medium, but, the average root length (4.66 ± 0.35 cm) was less than agar gelled medium. Ex vitro rooting was obtained through pulse treatment with 200 µM IBA for 30 min and subsequently transferring the microshoots to sterile

188 soilrite with the production of maximum 5.06 ± 0.17 roots/shoot having root length of 5.93 ± 0.17 cm in 90.66 ± 2.96% microshoots after 6 weeks.

Rooted plantlets were hardened and acclimatized in three different planting substrates before transplanting in the external environment. Maximum survival of plantlets was observed in soilrite (97.66 ± 1.45%) followed by vermiculite (90.66 ± 2.33%) and garden soil (70.66 ± 2.96%). After acclimatization period of 4 weeks under controlled conditions, plantlets were successfully transferred to field conditions with 90% survival rate.

Synthetic seeds were prepared through encapsulation of in vitro derived nodal segments of one month old microshoots. Ideal beads with uniform texture were obtained through 3% sodium alginate and 100 mM calcium chloride. Maximum conversion of beads (92.33 ± 1.45%) into shoots was observed on optimal medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) after 6 weeks. The microshoots were rooted either in vitro or ex vitro on optimized rooting media. Ex vitro sowing of synseeds in sterilised soilrite produced shoots as well as roots simultaneously in 30% beads after 6 weeks of transfer.

The effect of cold storage for short term conservation and reproducibility of the explant was tested by storing encapsulated and non-encapsulated nodal segments at 4ºC for different time period. Encapsulated explants exhibited an average of 64.00 ± 3.05% conversion on optimal regeneration medium after a storage period of 4 weeks on 4ºC, while, the non-encapsulated explants showed only 25.00 ± 2.88% conversion on the same concentration after 4 weeks of cold storage. The conversion percentage further decreased on increasing the storage duration of both encapsulated and non-encapsulated explants.

Estimation of different pigment contents was made during different acclimatization periods of regenerated plantlets. Contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids decreased during the first week of acclimatization but later on increased linearly in subsequent weeks of acclimatization. Net photosynthetic rate (PN ratio) also showed the same trend and decreased during first week while later on as the plantlets get adapted to the ex vitro conditions, PN ratio increased linearly.

189

Histology of the regenerative tissue obtained through NS explant confirmed the direct origin of shoot buds from the explant without callus formation, while CL derived callus revealed the formation of numerous meristemoids which lead to the development of shoot buds and thus confirming indirect mode of organogenesis.

Conclusions The following conclusions have been drawn from the present study:

1. In vitro raised aseptic seedlings were used for the collection of various explants in both C. angustifolia and C. sophera.

2. In C. angustifolia maximum seed germination was recorded on half

strength MS medium supplemented with 5.0 µM GA3, whereas in C. sophera highest seed germination was obtained on half strength MS

medium containing 1.0 µM GA3.

3. MS medium supplemented with 3% sucrose and pH adjusted at 5.8 was found optimum for shoot morphogenesis in both the plant species.

4. Direct shoot regeneration was achieved through cotyledonary node (CN), nodal segment (NS) and shoot tip (ST) explants on various cytokinins and auxins singly or in combination in both the plants.

5. Age of the explant showed a great impact on shoot multiplication rate, thus different explant were excised from most responsive stage of the seedlings.

6. In C. angustifolia, CN explant excised from 14 days old axenic seedlings was found most responsive compared to NS and ST explants. While, in C. sophera, NS (21 days old) proved to be the best explant in comparison to CN and ST explants.

7. BA at 5.0 µM found to be the best concentration of cytokinin for induction of multiple shoots in both the plants irrespective of the explant type.

190

8. Augmentation of lower concentrations of auxins (IAA, IBA and NAA) with optimal BA concentration (5.0 µM) found to be highly efficient for the production of maximum shoots in both the plant species.

9. In C. angustifolia maximum shoots/explant were produced on MS medium supplemented with BA (5.0 µM) + NAA (0.6 µM) through CN explants, whereas, in C. sophera, NS explants produced maximum shoots/explant on medium comprised of MS + BA (5.0 µM) + NAA (1.0 µM).

10. Adenine sulphate (AdS) at a concentration of 30 µM and 20 µM was added to the optimal regeneration medium in C. angustifolia and C. sophera respectively, to prevent yellowing or abscission of leaves and shoot tip necrosis during subculture passages.

11. TDZ at 2.5 µM proved optimal for maximum shoot regeneration through CN and NS explants in C. angustifolia and C. sophera respectively.

12. Indirect organogenesis has also been achieved in both the plant species through various explants viz.: cotyledonary leaf (CL), leaf (L) and root (R) explants.

13. In C. angustifolia, compact and nodular callus was induced through root explant (30 days old) at 1.0 µM TDZ, while, CL explant (14 days old) produced compact and nodular callus at 5.0 µM 2,4-D in both the plants.

14. Medium comprised of MS + BA (2.5 µM) + NAA (0.6 µM) proved efficient for maximum shoot differentiation from root derived callus in C. angustifolia. Whereas, in C. sophera, maximum differentiation of shoots was achieved through CL derived callus on MS + BA (2.5 µM) + NAA (0.5 µM).

15. Embryogenesis was induced in C. angustifolia through immature cotyledons excised from semi mature seeds, while in C. sophera no embryogenic response was obtained on any treatment from various explants tested.

16. Best embryogenic response was obtained on MS medium supplemented with 2,4-D (10.0 µM) and BA (1.0 µM).

191

17. Microshoots were rooted through both in vitro and ex vitro modes of rooting in both the plants.

18. In vitro rooting was best achieved on half strength liquid MS medium supplemented with 1.0 µM IBA and 5.0 µM PG in C. angustifolia. Whereas in C. sophera, agar gelled rooting medium comprised of half strength MS + 1.0 µM IBA found effective for maximum root induction.

19. Ex vitro rooting through pulse treatment with 200 µM IBA for 30 min found beneficial in both the plants as hardening and acclimatization took place simultaneously with rooting.

20. Synseed production with 3% sodium alginate and 100 mM calcium chloride proved to be ideal for the formation of uniform beads in both the plants.

21. Maximum conversion of beads was recorded on MS + BA (2.5 µM) + NAA (0.4 µM) in C. angustifolia, while, in C. sophera medium comprised of MS + BA (2.5 µM) + NAA (0.5 µM) provided maximum conversion of synseeds.

22. Synseeds were successfully stored at 4ºC for 8 weeks for short term conservation and provided satisfactory conversion into microshoots after 4 weeks of storage.

23. Amongst different planting substrates tested (garden soil, soilrite and vermiculite), soilrite was found to be the best planting substrate for maximum survival of regenerated plantlets in both the plants.

24. During acclimatization of regenerated plantlets increase in pigment contents and net photosynthetic rate was observed from an initial decline during first week of acclimatization in both the plants.

25. Histological sections clearly revealed the mode of regeneration via direct or indirect organogenesis as well as somatic embryogenesis.

192

References

Aasim M, Khawar KM, Ozcan S (2009). In vitro micropropagation from plumular apices of Turkish cowpea (Vigna unguiculata L.) cultivar Akkiz. Sci Hort 122: 468-471

Abbas H, Qaiser M (2010). In vitro conservation of Cadaba heterotricha stocks, an endangered species in Pakistan. Pak J Bot 42: 1553-1559

Abbas H, Qaiser M, Naqvi B (2010). Rapid in vitro multiplication of Acacia nilotica subsp. hemispherica, a critically endangered endemic taxon. Pak J Bot 42: 4087-4093

Abdi G, Khosh-Khui M (2007). Shoot regeneration via direct organogenesis from leaf segments of valerian (Valerina officinalis L.). Int J Agric Res 2: 877-882

Adriani M, Piccioni E, Standardi A (2000). Effect of different treatments on the conversion of ‘Hayward’ kiwifruit synthetic seeds to whole plants following encapsulation of in vitro derived buds. New Zeal J Crop Hort 28: 59-67

Agastian P, Lincy W, Ignacimuthu S (2006). In vitro propagation of Justicia gendarussa Burm f. - a medicinal plant. Indian J Biotechnol 5: 246-248

Agrawal V, Sardar PR (2003). In vitro organogenesis and histomorphological investigations in senna (Cassia angustifolia) - a medicinally valuable shrub. Physiol Mol Biol Plants 9: 131-140

Agrawal V, Sardar PR (2006). In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli. Biol Plant 50: 118-122

Agrawal V, Sardar PR (2007). In vitro regeneration through somatic embryogenesis and organogenesis using cotyledons of Cassia angustifolia Vahl. In Vitro Cell Dev Biol-Plant 43: 585-592

Aguilar ML, Espadas FL, Coello J, Maust BE, Trejo C, Robert ML, Santamarfa JM (2000). The role of abscisic acid in controlling leaf water loss, survival and growth of micropropagated Tagetes erecta plant when transferred directly to the field. J Exp Bot 51: 1861-1866

193

Ahee J, Duhoux E (1994). Root culturing of Faidherbia = Acacia albida as a source of explants for shoot regeneration. Plant Cell Tiss Organ Cult 36: 219- 225

Ahmad N, Anis M (2007). Rapid clonal multiplication of a woody tree, Vitex negundo L. through axillary shoots proliferation. Agroforest Syst 71: 195-200

Ahmad N, Anis M (2010). Direct plant regeneration from encapsulated nodal segments of Vitex negundo. Biol Plant 54: 748-752

Ahmad N, Faisal M, Anis M, Aref IM (2010). In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L. S Afr J Bot 76: 597-600

Ahmed MB, Salahin M, Karim R, Razvy MA, Hannan MM, Sultana R, Hossain M, Islam R (2007). An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni.) in Bangladesh. Am Eur J Sci Res 2: 121-125

Ahmed R, Gupta SD, De DN (1996). Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean (Psophocarpus tetragonolobus L.). Plant Cell Rep 15: 531-535

Akbaş F, Işikalan C, Namli S, Ak BE (2009). Effect of plant growth regulators on in vitro shoot multiplication of Amygdalus communis L. cv. Yaltsinki. Afr J Biotechnol 8: 6168-6174

Al-Absi KM (2010). The effects of different pre-sowing seed treatments on breaking the dormancy of mahaleb cherries, Prunus mahaleb L. seeds. Seed Sci Tech 38: 332-340

Alam I, Sharmin SA, Mondal SC, Alam MJ, Khalekuzzaman M, Anisuzzaman M, Alam MF (2010). In vitro micropropagation through cotyledonary node culture of castor bean (Ricinus communis L.). Aust J Crop Sci 4: 81-84

Altaf N, Iqbal J, Ahmad MS (1998). Influence of explant source and medium ingredients on culture responses of Microsperma Lentil cultivar Masoor-85. Pak J Biol Sci 1: 321-325

194

Aly HMA, Hattori K (2007). Histological observations on plant regeneration in faba bean cotyledon (Vicia faba L.) cultured in vitro. Asian J Plant Sci 6: 723-731

Amâncio S, Rebordão JP, Chaves MM (1999). Improvement of acclimatization of micropropagated grapevine: photosynthetic competence and carbon allocation. Plant Cell Tiss Organ Cult 58: 31-37

Amin MN, Rahman MM, Hossain MF (2003). In vitro culture and plantlet regeneration from the explants of Thankuni (Centella asiatica Linn.). Bangladesh J Genet Biotechnol 4: 49-51

Ammirato PV (1983). Embryogenesis. In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds). Handbook of plant cell cultures, Vol 1 Macmillan Publishing Co, New York pp 82-123

Amoo SO, Ayisire BE (2005). Induction of callus and somatic embryogenesis from cotyledon explants of Parkia biglobosa (Jacq.) Benth. Afr J Biotechnol 4: 68-71

Anand PHM, Hariharan M (1997). In vitro multiplication of greater galangal [Alpinia galanga (L.) Wild.] a medicinal plant. Phytomorph 47: 45-50

Anand SP, Jeyachandran R (2004). In vitro multiple shoot regeneration from nodal explants of Zehneria scabra (L.f.) Sonder - an important medicinal climber. Plant Tiss Cult 14: 101-106

Anand SP, Nandagopalan V, Doss A (2011). In vitro direct plant regeneration from shoot tip explants of Clitoria ternatea - an important medicinal plant. Int J Biol Technol 2: 61-63

Anand Y, Bansal YK (2002). Synthetic seeds: a novel approach of in vitro plantlet formation in Vasaka (Adhatoda vasica Nees.). Plant Biotechnol 19: 159- 162

Anbazhagan M, Kalpana M, Rajendran R, Natarajan V, Dhanavel D (2010). In vitro production of Stevia rebaundiana. Emir J Food Agric 22: 216-222

195

Anbazhagan VR, Ganapathi A (1999). Somatic embryogenesis in cell suspensions of pigeonpea (Cajanus cajan L.). Plant Cell Tiss Organ Cult 56: 179-184

Andrea-Kodym F, Zapata-Arias J (2001). Low cost alternatives for the micropropagation of banana. Plant Cell Tiss Organ Cult 66: 67-71

Anis M, Husain MK, Shahzad A (2005). In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr Sci 88: 861-863

Anitha S, Pullaiah T (2002a). Shoot regeneration from hypocotyl and shoot tip explants of Sterculia foetida L. derived from seedlings. Taiwania 47: 62-69

Anitha S, Pullaiah T (2002b). In vitro propagation of Decalepis hamiltonii. J Trop Med Plants 3: 227-232

Anonymous (1992a). Cassia. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products (Vol III), CSIR, New Delhi, pp 354-363

Anonymous (1992b). Cassia. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products (Vol III), CSIR, New Delhi, pp 366-367

Anuradha M, Pullaiah T (1999). In vitro seed culture and induction of enhanced axillary branching in Pterocarpus santalinus and Pterocarpus marsupium: a method for rapid multiplication. Phytomorph 49: 157-163

Anwar MH, Hossain MT, Raihanali M, Rahman SMM (2005). Effect of different carbon sources on in vitro regeneration of Indian Penny wort (Centella asiatica L.). Pak J Biol Sci 8: 963-965

Ara H, Jaiswal U, Jaiswal VS (1999). Germination and plantlet regeneration from encapsulated somatic embryos of mango (Mangifera indica L.). Plant Cell Rep 19: 166-170

Ara H, Jaiswal U, Jaiswal VS (2000). Synthetic seed: prospects and limitations. Curr Sci 78: 1438-1444

196

Arndt FR, Rusch R, Stillfried HV, Hanisch B, Martin WC (1976). SN 49537, A new defoliant. Plant Physiol 57: s-99 (Abstr)

Arnold SV, Sabala I, Bozhkov P, Dyachok J, Filonova L (2002). Developmental pathways of somatic embryogenesis. Plant Cell Tiss Organ Cult 69: 233-249

Arockiasamy S, Ignacimuthu S, Melchias G (2000). Influence of growth regulators and explant type on in vitro shoot propagation and rooting of red sandalwood (Pterocarpus santalinus L.). Indian J Exp Bot 38: 1270-1273

Arockiasamy S, Prakash S, Ignacimuthu S (2002). Direct organogenesis from mature leaf and petiole explants of Eryngium foetidum L. Biol Plant 45: 129-132

Arora R, Bhojwani SS (1989). In vitro propagation and low temperature storage of Saussarea lappa C.B. Clarke - an endangered medicinal plant. Plant Cell Rep 8: 44-47

Arrillaga L, Tobolski JJ, Merkie SA (1994). Advances in somatic embryogenesis and plant production of black locust (Robinia pseudoacacia L.). Plant Cell Rep 13: 171-175

Arya V, Shekhawat NS, Singh RP (2003). Micropropagation of Leptadenia reticulata - a medicinal plant. In Vitro Cell Dev Biol-Plant 39: 180-185

Atta-Alla H, van Staden J (1997). Micropropagation and establishment of Yucca aloifolia. Plant Cell Tiss Organ Cult 48: 209-212

Avenido RA, Hattori K (2001). Benzyladenine-preconditioning in germinating mung bean seedling stimulates axillary buds in cotyledoanry nodes resulting in multiple shoot regeneration. Breed Sci 51: 137-142

Babber S, Mittal K, Ahlawat R, Varghese TM (2001). Micropropagation of Cardiospermum halicacabum. Biol Plant 44: 603-606

Badji S, Maitone Y, Ndiaye I, Merlin G, Danthu P, Palma B, Callona JP (1993). In vitro propagation of the gum Arabic tree (Acacia senegal L. Willd.) developing a rapid method for producing plants. Plant Cell Rep 12: 629-633

197

Bajaj YPS (1988). Cryopreservation and the retention of biosynthetic potential in cell cultures of medicinal and alkaloid producing plants. In: Bajaj YPS (ed). Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants I, Vol 4 Berlin, Springer, Verlag

Bajaj YPS, Furmanowa M, Olszzowska (1988). Biotechnology of the micropropagation of medicinal and aromatic plants. In: Bajaj YPS (ed). Biotechnology in Agriculture and Forestry, Medicinal and Aromatic Plants I, Vol 4 Berlin, Springer, Verlag

Baksha R, Jahan MAA, Khatun R, Munshi JL (2007). In vitro rapid clonal propagation of Rauvolfia serpentina (Linn.) Benth. Bangladesh J Ind Res 42: 37‐44

Balaraju K, Agastian P, Ignacimuthu S (2009). Micropropagation of Swertia chirata Buch.-Hams. ex Wall.: a critically endangered medicinal herb. Acta Physiol Plant 31: 487-494

Ball E (1946). Development in sterile culture of stem tips and subjacent regions of Tropaeolum majus L. and Lupinus albus L. Am J Bot 33: 301-318

Banerjee M, Shrivastava S (2006). In vitro regeneration of Jatropha curcas (Ratanjyot): prospects for biofuel production and medicines. Indian J Bot Res 2: 195-200

Bapat VA, Rao PS (1990). In vivo growth of encapsulated axillary buds of mulberry (Morus indica L.). Plant Cell Tiss Organ Cult 20: 69-70

Bapat VA, Rao PS (1992). Plantlet regeneration from encapsulated and non- encapsulated dessiccated somatic embryos of a forest tree Sandalwood (Santalum album L.). Plant Biochem Biotechnol 1: 109-113

Barik DP, Mohapatra U, Chand PK (2005). High frequency in vitro regeneration of Lathyrus sativus L. Biol Plant 49: 637-639

198

Barik DP, Naik SK, Mudgal A, Chand PK (2007). Rapid plant regeneration through in vitro axillary shoot proliferation of Butterfly Pea (Clitoria ternatea L.) - a twinning legume. In Vitro Cell Dev Biol-Plant 43: 144-148

Barna KS, Wakhlu AK (1993). Somatic embryogenesis and plant regeneration from callus cultures of chickpea (Cicer arietinum L.). Plant Cell Rep 12: 521-524

Baroja ME, Aguirreolea J, Sánchez-Díaz M (1995). CO2 exchange of in vitro and acclimatizated potato plantlets. In: Carre F, Chagvardieff P (ed). Ecophysiology and Photosynthetic In Vitro Cultures. CEA Centre ďÉtudes de Cadarache, Saint- Paul-lez-Durance pp 187-188

Baskaran P, Jayabalan N (2005). An efficient micropropagation system for Eclipta alba - a valuable medicinal herb. In Vitro Cell Dev Biol-Plant 41: 532-539

Baskaran P, Jayabalan N (2008). Effect of growth regulators on rapid micropropagation and psoralen production in Psoralea corylifolia L. Acta Physiol Plant 30: 345-451

Baskaran P, Jayabalan N (2009a). In vitro regeneration of Melothria maderaspatana via indirect organogenesis. In Vitro Cell Dev Biol-Plant 45: 407- 413

Baskaran P, Jayabalan N (2009b). An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L. Sci Hort 123: 283-286

Basu P, Chand S (1996). Regeneration of plantlets from root derived callus of Egyptian Henbane. Cell Chromo Res 19: 31-34

Bates S, Preece JE, Navarrete NE, Van Sambeek JW, Gaffney GR (1992). Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.). Plant Cell Tiss Organ Cult 3: 21-29

Beck SL, Dunlop R, Van Staden J (1998). Rejuvenation and micropropagation of adult Acacia mearnsii using coppice material. Plant Growth Reg 26: 149-153

199

Beena MR, Martin KP (2003). In vitro propagation of the rare medicinal plant Ceropegia candelabrum L. through somatic embryogenesis. In Vitro Cell Dev Biol-Plant 39: 510-513

Beena MR, Martin KP, Kirti PB, Hariharan M (2003). Rapid in vitro propagation of medicinally important Ceropegia candelabrum. Plant Cell Tiss Organ Cult 72: 285-289

Belkengren RO, Miller PW (1962). Culture of apical meristems of Fragaria vesca strawberry plants as a method of excluding latent A virus. Plant Dis Reptr 46: 119-121

Bennici A, Bruschi P (1999). Stability of habituation in Nicotiana bigelovii var. bigelovii shoots regenerated from habituated callus. Plant Sci 143: 195-199

Bensal YK, Pandey P (1993). Micropropagation of Sesbania acculeata by adventitious organogenesis. Plant Cell Tiss Organ Cult 32: 315-355

Berger K, Schaffner W (1995). In vitro propagation of the leguminous tree Swartzia madagascariensis. Plant Cell Tiss Organ Cult 40: 289-291

Berry TA, Bewley JD (1992). A role for the surrounding fruit tissues in preventing germination of tomato (Lycopersicon esculentum) seeds. A consideration of the osmotic environment and abscisic acid. Plant Physiol 100: 951-957

Bettaieb T, Mhamdi M, Hajlaoui I (2008). Micropropagation of Nolina recurvata Hemsl.: b-Cyclodextrin effects on rooting. Sci Hort 117: 366-368

Bewly JD, Black M (1994). Seeds: Physiology of development and germination. Plenum Press, New York

Bhagyalakshmi N, Singh NS (1988). Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of Oleoresin. J Hortic Sci 63: 321-327

Bhat SR, Chandel KPS, Malik SK (1995). Plant regeneration from various explants of cultivated Piper species. Plant Cell Rep 14: 398-402

200

Bhat SR, Chitralekha P, Chandel KPS (1992). Regeneration of plants from long term root culture of lime Citrus aurantifolia (Christm.) Swing. Plant Cell Tiss Organ Cult 29: 19-25

Bhatia NP, Bhatia P, Ashwath N (2002). Ex vitro rooting of micopropagated shoots of Stackousia tryonii. Biol Plant 45: 441-444

Bhatia P, Ashwath N (2005). Effect of medium pH on shoot regeneration from the cotyledonary explants of tomato. Biotechnol 4: 7-10

Bhatt ID, Dhar U (2000). Micropropagation of Indian wild strawberry. Plant Cell Tiss Organ Cult 60: 83-88

Bhatt ID, Dhar U (2004). Factors controlling micropropagation of Myrica esculenta buch.-Ham. ex D. Don: a high value wild edible of Kumaun Himalaya. Afr J Biotechnol 3: 534-540

Bhattacharyya R, Bhattacharya S (2001). In vitro multiplication of Coleus forskohlii Briq.: an approach towards shortening the protocol. In Vitro Cell Dev Biol-Plant 37: 572-575

Bhau BS, Wakhlu AK (2001). Effect of genotype, explant type and growth regulators on organogenesis in Morus alba. Plant Cell Tiss Organ Cult 66: 25-29

Bhojwani SS, Evans PK, Cocking EC (1977). Protoplast technology in relation to crop plants: progress and problems. Euphytica 26: 343-360

Bhuyan AK, Pattnaik S, Chand PK (1997). Micropropagation of curry leaf tree [Murraya koenigii (L.) Spreng.] by axillary proliferation using intact seedlings. Plant Cell Rep 16: 779-782

Biahoua A, Bonneau L (1999). Control of in vitro somatic embryogenesis of the spindle tree (Euonymus europaeus L.) by the sugar type and the osmotic potential of the culture medium. Plant Cell Rep 19: 185-190

Bilal A, Khan NA, Ghufran A, Inamuddin (2005). Pharmacological investigation of Cassia sophera Linn. var, purpurea Roxb. Med J Islamic World Acad Sci 15: 105-109

201

Bingham ET, Hurley LV, Kaatz DM, Saunders JW (1975). Breeding alfalfa which regenerates from callus tissue in culture. Crop Sci 15: 719-721

Biswas, Bari MA, Roy M, Bhadra SK (2009). Clonal propagation through nodal explant culture of Boerhaavia diffusa L. - a rare medicinal plant. Plant Tiss Cult Biotechnol 19: 53-59

Blinstrubiene A, Sliesaravicius A, Burbulis N (2004). Factors affecting morphogenesis in tissue culture of linseed flax (Linum isitatissimum L.). Acta Universitatis Latviensis Biol 676: 149-152

Bohidar S, Thirunavoukkarasu, Rao TV (2008). Effect of plant growth regulators on in vitro micropropagation of ‘Garden Rue’ (Ruta graveolens L.). Int J Integ Biol 3: 36-43

Bohmer P, Meyer B, Jacobson HJ (1995). Thidiazuron induced high frequency of shoot induction and plant regeneration in protoplast derived pea callus. Plant Cell Rep 15: 26-29

Borad VP, Barve DM, Macwan SJ, Meheta AR (2001). Regeneration of plantlets in Sapindus trifoliatus L. Indian J Exp Biol 39: 1288-1292

Borthakur M, Dutta K, Nath SC, Singh RS (2000). Micropropagation of Eclipta alba and Eupatorium adenophorum using a single-step nodal cutting technique. Plant Cell Tiss Organ Cult 62: 239-242

Bouhouche N, Ksiksi T (2007). An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Bioss. Plant Biotechnol Rep 1: 179- 184

Boxus P, Quoirin M, Laine JM (1977). Large scale propagation of strawberry plants from tissue culture. In: Reinert J, Bajaj YPS (eds). Applied and fundamental aspects of plant cell, tissue and organ culture. Berlin, Springer pp 130-143

202

Bozena B (2001). Morphological and physiological characteristics of micropropagated strawberry plants rooted in vitro or ex vitro. Sci Hort 89: 195- 206

Britto SJ, Natarajan E, Arockiasamy DI (2003). In vitro flowering and shoot multiplication from nodal explants of Cerpegia bulbosa Roxb. var. bulbosa. Taiwania 48: 106-111

Brown DCW, Leung DMW, Thorpe TA (1979). Osmotic requirement for shoot formation in tobacco callus. Physiol Plant 46: 36-41

Buendía-González L, Orozco-Villafuerte J, Cruz-Sosa F, Chávez-Ávila VM, Vernon-Carter EJ (2007). Clonal propagation of mesquite tree (Prosopis laevigata Humb. Bonpl. ex Willd. M.C. Johnston) via cotyledonary nodes. In Vitro Cell Dev Biol-Plant 43: 260-266

Bunn E (2005). Development of in vitro methods for ex situ conservation of Eucalyptus impensa, an endangered mallee from south west Western Australia. Plant Cell Tiss Organ Cult 83: 97-102

Burdyn L, Luna C, Tarrago J, Sansberro P, Dudit N, GonzaLez N, Mroginski L (2006). Direct shoot regeneration from leaf and internode explants of Aloysia polystachya (Gris.) Mold. (Verbenaceae). In Vitro Cell Dev Biol-Plant 42: 235- 239

Cao H, Yang J, Peng ZS, Kang CY, Chen DC, Gong ZC, Tan X (2007). Micropropagation of Penthorum chinense through axillary bud. In Vitro Cell Dev Biol-Plant 43: 149-153

Capelle SC, Mok DWS, Kirchner SC, Mok MC (1983). Effects of thiadiazuron on cytokinin autonomy and metabolism of N6-(2-isopentyl) [8-14C] adenosine in callus tissue of Phaseolus lunatus L. Plant Physiol 73: 796-802

Capuano G, Piccioni E, Standardi A (1998). Effect of different treatments on the conversion of M-26 apple root stock synthetic seeds obtained from encapsulated apical and axillary micropropagated buds. J Hort Sci Biotechnol 73: 299-305

203

Casado JP, Navarro MC, Utrilla MP, Martinez A, Jiménez J (2002). Micropropagation of Santolina canescens lagasca and in vitro volatiles production by shoot explants. Plant Cell Tiss Organ Cult 68: 73-78

Castillo B, Smith MA L, Yadava UL (1998). Plant regeneration from encapsulated somatic embryos of Carica papaya L. Plant Cell Rep 17: 172-176

Castillo P, Márquez J, Rubluo A, Hernández G, Lara M (2000). Plant regeneration from callus and suspension cultures of Valeriana edulis ssp. procera via simultaneous organogenesis and somatic embryogenesis. Plant Sci 151: 115-119

Catapan E, Luis M, da Silva B, Moreno FN, Viana AM (2002). Micropropagation, callus and root culture of Phyllanthus urinaria (Euphorbiaceae). Plant Cell Tiss Organ Cult 70: 301-309

Ceasar SA, Maxwell SL, Prasad KB, Karthigan M, Ignacimuthu S (2010). Highly efficient shoot regeneration of Bacopa monnieri (L.) using a two stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD. Acta Physiol Plant 32: 443-452

Cellarova E, Hocariv R (2004). The influence of N-(2-isopentenyl) adenine on shoot differentiation in Digitalis purpurea L. tissue cultures. Acta Biotechnol 11: 331-334

Çetinbas M, Koyuncu F (2006). Improving germination of Prunus avium L. seeds by gibberellic acid, potassium nitrate and thiourea. Hort Sci 33: 119-123

Chalupa V (1987). Effect of benzylaminopurine and thidiazuron on in vitro shoot proliferation of Tilia cordata Mill, Sorbus aucuparia L. and Robinia pseudoacacia L. Biol Plant 29: 425-429

Chand S, Sahrawat AK (2002). Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L. an endangered medicinally important plant. In Vitro Cell Dev Biol-Plant 38: 33-38

204

Chand S, Singh AK (2001). Direct somatic embryogenesis from zygotic embryos of a timber yielding leguminous tree, Hardwickia binata Roxb. Curr Sci 80: 882- 887

Chand S, Singh AK (2004a). In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, Pterocarpus marsupium Roxb. In Vitro Cell Dev Biol-Plant 40: 167-170

Chand S, Singh AK (2004b). Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber yielding leguminous tree species. J Plant Physiol 161: 237-243

Chandra S, Bandopadhyay R, Kumar V, Chandra R (2010). Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnol Lett 32: 1199-1205

Chandramu C, Rao DM, Reddy VD (2003). High frequency induction of multiple shoots from nodal explants of Vitex negundo L. using sodium sulphate. J Plant Biotechnol 5: 107-113

Chang C, Chang WC (1998). Plant regeneration from callus cultures of Cymbidium ensifolium var. misericors. Plant Cell Rep 17: 251-255

Chang WC (1991). Bamboos. In: Bajaj YPS (ed). Biotechnology in agriculture and forestry, Vol 16, Berlin, Springer, Verlag pp 211-237

Chaturvedi HC, Jain M, Kidwai NR (2007). Cloning of medicinal plants through tissue culture - a review. Indian J Exp Biol 45: 937-948

Chaturvedi R, Razdan MK, Bhojwani SS (2004). In vitro clonal propagation of an adult tree of neem (Azadirachta indica A. Juss.) by forced axillary branching. Plant Sci 166: 501-506

Chaudhuri KN, Ghosh B, Jha S (2004). The root: a potential new source of competent cells for high frequency regeneration in Tylophora indica. Plant Cell Rep 22: 731-740

205

Chaudhuri RK, Amita P, Timir BJ (2007). Production of genetically uniform plants from nodal explants of Swertia chirata Buch-Ham ex Wall - an endangered medicinal herb. In Vitro Cell Dev Biol-Plant 43: 467-472

Cheepala SB, Sharma NC, Sahi SV (2004). Rapid in vitro regeneration of Sesbania drummondii. Biol Plant 48: 13-18

Chen JT, Chang C, Chang WC (1999). Direct somatic embryogenesis from leaf explants of Oncidium ‘Gower Ramsey’ and subsequent plant regeneration. Plant Cell Rep 19: 143-149

Chen JT, Chang WC (2006). Direct somatic embryogenesis and plant regeneration from leaf explants of Phalaenopsis amabilis. Biol Plant 50: 169-173

Chen UC, Hsia CN, Yeh MS, Agrawal DC, Tsay HS (2006). In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal plant native to Taiwan. In Vitro Cell Dev Biol-Plant 42: 128-133

Chen YC, Chang C, Chang W (2000). A reliable protocol for plant regeneration from callus cultures of Phalaenopsis. In Vitro Cell Dev Biol-Plant 36: 420-423

Cheruvathur MK, Britto J, Thomas TD (2010). Callus induction and shoot regeneration from epicotyl explants of ethnomedicinally important Caesalpinia bonducella (L.) Roxb. Iranian J Biotechnol 8: 263-269

Chhabra G, Chaudhary D, Varma M, Sainger M, Jaiwal PK (2008). TDZ induced direct shoot organogenesis and somatic embryogenesis on cotyledoray node explants of lentil (Lens culinaris Medik.). Physiol Mol Biol Plants 14: 347-353

Chithra M, Martin KP, Sunandakumari C, Madhusoodan PV (2005). Somatic embryogenesis, encapsulation and plant regeneration of Rotula aquatica Lour., a rare rheophytic woody medicinal plant. In Vitro Cell Dev Biol-Plant 41: 28-31

Choi JY, Kim HJ, Lee CH, Bae JM, Chung YS, Shin JS, Hyung NI (2001). Efficient and simple plant regeneration via organogenesis from leaf segment cultures of permission (Diospyros kaki Thumb.). In Vitro Cell Dev Biol-Plant 37: 274-279

206

Choi YE, Kim JW, Soh WY (1997). Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai. Plant Cell Rep 17: 84-88

Choi YE, Kim JW, Yoon ES (1999). High frequency of plant production via somatic embryogenesis from callus or cell suspension cultures in Eleutherococcus senticosus. Ann Botany 83: 309-314

Choi YE, Ko SK, Lee KS, Yoon ES (2002). Production of plantlets of Eleutherococcus sessiliflorus via somatic embryogenesis and successful transfer to soil. Plant Cell Tiss Organ Cult 69: 35-40

Chu IYE, Kurtz SL (1989). Commercialization of plant micropropagation. In: Ammirato PV, Evans DA, Sharp WR, Bajaj YPS (eds). Handbook of plant cell culture Vol 5, New York, McGraw-Hill pp 126-164

Chuenboonngarm N, Charoonsote S, Bhamarapravati S (2001). Effect of BA and 2iP on shoot proliferation and somaclonal variation of Gardenia jasminoides Ellis in vitro culture. Sci Asia 27: 137-141

Chung HH, Chen JT, Chang WC (2007). Plant regeneration through direct somatic embryogenesis from leaf explants of Dendrobium. Biol Plant 51: 346- 350

Conger BV, Hanning GE, Grey DJ, McDaniel JK (1983). Direct embryogenesis from mesophyll cells of orchad grass. Science 221: 850-851

Constabel F (1984). Callus culture: Induction and maintenance. In: Vasil IK (ed). Cell culture and somatic cell genetics of plants, Vol 1 Academic Press, New York

Cristina M, dos Santos F, Esquibel MA, dos Santos VP (1990). In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr. Plant Cell Tiss Organ Cult 21: 75-78

Daigny G, Paul H, Sangwan RS, Sangwan-Norreel BS (1996). Factor influencing secondary somatic embryogenesis in Malus x domestica Borkh. (cv. Gloster 69). Plant Cell Rep 6: 153-157

207

Dalal NV, Rai RV (2004). In vitro propagation of Oroxylum indicum Vent: a medicinally important forest tree. J For Res 9: 61-65

Das P, Palai, SK, Patra A, Samantaray S, Rout GR (1999). In vitro somatic embryogenesis in Typhonium trilobatum Schott. Plant Growth Reg 27: 193-197

Das P, Sammtaray S, Rout GR (1996b). In vitro propagation Acacia catechu, a xerophylous tree. Plant Tiss Cult 6: 117-126

Das S, Timir BJ, Sumita J (1996a). In vitro propagation of cashewnut. Plant Cell Rep 15: 615-619

Dave A, Purohit SD (2002). Factors influencing in vitro culture of ‘Safed Musli’ (Chlorophytum borivilianum). Oikoassay 15: 19-27

Davey MR, Kumar V, Hammatt N (1994). In vitro culture of legumes. In: Vasil IK, Thorpe TA (eds). Plant Cell and Tissue Culture, Kluwer Academic Publishers, Dordrecht, Netherlands pp 313-329

De Fossard RA (1976). Tissue Culture for Plant Propagators. University of New England, Armidale pp 409

De Klerk GJ, (2002). Rooting of microcuttings: theory and practice. In Vitro Cell Dev Biol-Plant 38: 415-422

De Klerk GJ, Vander Krieken W, de Jong JC (1999). The formation of adventitious roots: new concepts, new possibilities. In Vitro Cell Dev Biol-Plant 35:189-199

De Paiva Neto BV, Da Mota RT, Otoni WC (2003). Direct organogenesis from hypocotyl derived explants of annatto (Bixa orellana). Plant Cell Tiss Organ Cult 75: 159-167

Deb CR, Imchen T (2010). An efficient in vitro hardening technique of tissue culture raised plants. Biotechnol 9: 79-83

Debergh PC, Maene LJ (1981). A scheme for commercial propagation of ornamental plants by tissue culture. Sci Hort 14: 335-345

208

Debnath M, Malik CP, Bisen PS (2006). Micropropagation: A tool for the production of high quality plant-based medicines. Curr Pharma Biotechnol 7: 33- 49

Debnath SC (2009). A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry. Biol Plant 45: 122-128

Deka P, Kalita MC (2005). In vitro clonal propagation and organogenesis in Spilanthes acmella (L.) Murray: a herbal pesticidal plant of North-East India. J Plant Biochem Biotechnol 14: 69-71

Deng R, Donnelly J (1993). In vitro hardening of red raspberry through CO2 enrichment and relative humidity reduction on sugar-free medium. Cannadian J Plant Sci 73: 1105-1113

Deore AC, Johnson TS (2008). High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant. Plant Biotechnol Rep 2: 10-15

Desjardins Y (1995). Photosynthesis in vitro - on the factors regulating CO2 assimilation in micropropagation systems. Acta Hort 393: 45-61

Devi PS, Arundathi A, Rao TR (2011). Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree. J Plant Biochem Biotechnol 20: 161-165

Devi YS, Mukherjee BB, Gupta S (1994). Rapid cloning of elite teak (Tectona grandis L.) by in vitro multiple shoots production. Indian J Exp Biol 32: 668-671

Dewan A, Nanda K, Gupta SC (1992). In vitro micropropagation of Acacia nilotica subsp. indica Brenan via cotyledonary nodes. Plant Cell Rep 12: 18-21

Dhabhai K, Batra A (2010). Hormonal regulation impact on regeneration of Acacia nilotica L. a nitrogen fixing tree. World App Sci J 11: 1148-1153

Dhaka N, Kothari SL (2005). Micropropagation of Eclipta alba (L.) hassk – An important medicinal plant. In Vitro Cell Dev Biol-Plant 41: 658-661

209

Dhanalakshmi S, Lakshmanan KK (1992). In Vitro somatic embryogenesis and plant regeneration in Clitoria ternatea. J Exp Bot 43: 213-219

Dhandapani M, Kim DH, Hong SB (2008). Efficient plant regeneration via somatic embryogenesis and organogenesis from the explants of Catharanthus roseus. In Vitro Cell Dev Biol-Plant 44: 18-25

Dhar U, Joshi M (2005). Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteracea): effect of explant type, age and plant growth regulators. Plant Cell Rep 24: 195-200

Dhar U, Upreti J (1999). In vitro regeneration of a mature leguminous liana (Bauhinia vahlii Wight and Arnott). Plant Cell Rep 18: 664-669

Dhawan D, Bhojwani SS (1984). Reduction in cost of tissue culture of Leucaena leucocephala (Lam) de Wit by replacing AR grade sucrose by sugarcubes. Curr Sci 53: 1159-1161

Diettrich B, Mertinat H, Luckner M (1992). Reduction of water loss during ex vitro acclimatization of micropropagated Digitalis lanata clone plants. Biochem Physiol Pflanz 188: 23-31

Dineshkumar V, Kirti PB, Sachan JKS, Chopra VL (1994). Plant regeneration via somatic embryogenesis in chickpea (Cicer arietenum L.). Plant Cell Rep 13: 468- 472

Distabanjong K, Geneve R (1997). Multiple shoot formation from cotyledonary node segments of Eastern Redbud. Plant Cell Tiss Organ Cult 47: 247-254

Dodeman VL, Ducreux G, Kreis M (1997). Zygotic embryogenesis versus somatic embryogenesis. J Exp Bot 48: 1493-1509

Dolendro SN, Sahoo L, Bhalla SN, Jaiwal PK (2003). The effect of TDZ on organogenesis and somatic embryogenesis in pigeon pea (Cajanus cajan L. Mill sp). Plant Sci 164: 341-347

Donnan A (1986). Determining and minimizing production costs. In: Zimmerman RH, Grierbach RJ, Hammerschlag FA, Lawson RH (eds). Tissue culture as a

210 plant production system for horticultural crops, Martinus Nijhoff Publishers, Boston, pp 163-173

Donnelly DJ, Skelton FE, Daubenny HA (1986). External leaf features of tissue cultured ‘Silvan’ blackberry. HortScience 21: 306-308

Donnelly DJ, Tisdall L (1993). Acclimatization strategies for micropropagated plants. In: Ahuja MR (ed). Micropropagation of woody plants, Forestry Sci 41: 153-166

Donnelly DJ, Vidaver WE (1984). Leaf anatomy of red raspberry transferred from culture to soil. J Am Soc Hort Sci 109: 172-176

Douglas GC, McNamara J (2000). Shoot regeneration from seedling explants of Acacia mangium Willd. In Vitro Cell Dev Biol-Plant 36: 412-415

Dubey NK, Kumar R, Tripathi P (2004). Global promotion of herbal medicine: India’s opportunity. Curr Sci 86: 37-41

Durham RE, Parrott WA (1992). Repetitive somatic embryogenesis from peanut cultures in liquid medium. Plant Cell Rep 11: 122-125

Durkovic J, Lux A (2010). Micropropagation with a novel pattern of adventitious rooting in American sweetgun (Liquidambar styraciflua L.). Trees 24: 491-497

Dwari M, Chand PK (1996). Evaluation of explant growth regulators and culture passage for enhanced callus induction, proliferation and plant regeneration in the tree legume Dalbergia lanceolaria. Phytomorph 46: 123-131

Eapen S, Tivarckar S, George L (1998). Thidiazuron-induced shoot regeneration in pigeonpea (Cajanus cajan L.). Plant Cell Tiss Organ Cult 53: 217-220

Earle ED, Langhans RW (1974). Propagation of Chrysanthemum in vitro. I. Multiple plantlets from shoot tips and the establishment of tissue cultures. J Am Soc Hort Sci 99: 128-132

211

Echevarrigaray S, Career RP, Andrade LB (2010). Micropropagation of Salvia guarantica Benth. through axillary shoot proliferation. Braz Arch Bio Tech 53: 883-888

Economou AS, Spanoudaki MJ (1985). In vitro propagation of Gardenia. HortScience 20: 213

El Maataoui M, Espagnac H, Jaber B, Alonso-Lopez A (1998). Regulation of in vitro callogenesis and organogenesis from Albizzia root explants by carbohydrate source modifications. J Plant Physiol 152: 494-501

Emek Y, Erdag B (2007). In vitro propagation of Gladiolus anatolicus (Boiss.) Stapf. Pak J Bot 39: 23-30

Epstein E, Müller J (1993). Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport. Physiol Plant 88: 382-389

Espino FJ, Linacero R, Rueda J, Vazquez AM (2004). Shoot regeneration in four Begonia genotypes. Biol Plant 48: 101-104

Ezura H, Nukui N, Yuhashi KI, Minamisawa K (2000). In vitro plant regeneration in Macroptilium atropurpureum, a legume with a broad symbiont range for nodulation. Plant Sci 159: 21-27

Faisal M, Ahmad N, Anis M (2005b). Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron. Plant Cell Tiss Organ Cult 80: 187-190

Faisal M, Ahmad N, Anis M (2006c). In vitro regeneration via de novo shoot organogenesis in callus culture of Ruta graveolens - a plant with medicinal and horticultural potential. Phytomorph 56: 183-187

Faisal M, Ahmad N, Anis M (2006d). In vitro plant regeneration from alginate encapsulated microcuttings of Rauvolfia tetraphylla L. Am Eur J Agric Environ Sci 1: 1-6

Faisal M, Ahmad N, Anis M (2007). An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant. Plant Biotechnol Rep 1: 155-161

212

Faisal M, Anis M (2002). Rapid in vitro propagation of Rauvolfia tetraphylla L. - an endangered medicinal plant. Physiol Mol Biol Plants 8: 295-299

Faisal M, Anis M (2003). Rapid mass propagation of Tylophora indica Merrill. via leaf callus culture. Plant Cell Tiss Organ Cult 75: 125-129

Faisal M, Anis M (2005). An efficient in vitro method for mass propagation of Tylophora indica. Biol Plant 49: 257-260

Faisal M, Anis M (2006). Thidiazuron induced high frequency axillary shoot multiplication in Psoralea corylifolia. Biol Plant 50: 437-440

Faisal M, Anis M (2007). Regeneration of plants from alginate encapsulated shoots of Tylophora indica (Burm.f.) Merrill, an endangered medicinal plant. J Hort Sci Biotechnol 82: 351-354

Faisal M, Shahzad A, Anis M (2008). Somatic embryogenesis and plant regeneration from nodal explants in Psoralea corylifolia L. Int J Plant Dev Biol 2: 111-113

Faisal M, Siddique I, Anis M (2006a). An efficient plant regeneration system for Mucuna pruriens L. (DC.) using cotyledonary node explants. In Vitro Cell Dev Biol-Plant 42: 59-64

Faisal M, Siddique I, Anis M (2006b). In vitro rapid regeneration of plantlets from nodal explants of Mucuna pruriense - a valuable medicinal plant. Ann Appl Biol 148: 1-6

Faisal M, Singh S, Anis M (2005a). In vitro regeneration and plant establishment of Tylophora indica (Burm. F.) Merrill: petiole callus culture. In Vitro Cell Dev Biol-Plant 41: 511-515

Fengyun LI, Liying H (2002). The effect of exogenous hormones on micropropagation of In vitro virus free potato plantlets. Chinese Potato J 16: 214- 216

Fett-Neto AG, Fett JP, Aquilla MEA, Ferreira AG (2000). In vitro propagation of Senna alata. Planta Med 66: 195-196

213

Feyissa T, Weiander M, Negash L (2007). Genetic stability, ex vitro rooting and gene expression studies in Hagenia abyssinica. Biol Plant 51: 15-21

Figueiredo SFL, Albarello N, Viana VRC (2001). Micropropagation of Rollina mucosa (Jacq) Baill. In Vitro Cell Dev Biol-Plant 37: 471-475

Finkelstein RR, Gibson SI (2002). ABA and sugar interactions regulating development: cross talk or voices in a crowd. Curr Opin Plant Biol 5: 26-32

Fiola JA, Hassan MA, Swartz HJ, Bors RH, McNicols R (1990). Effect of thidiazuron, light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves. Plant Cell Tiss Organ Cult 39: 371- 387

Fracaro F, Echeverrigaray S (2001). Micrpopropagtion of Cunila galioides, a popular medicinal plant of South Brazil. Plant Cell Tiss Organ Cult 64: 1-4

Franklin G, Sheeba CJ, Lakshmi Sita G (2004). Regeneration of eggplant (Solanum melongena L.) from root explants. In Vitro Cell Dev Biol-Plant 40: 188- 191

Fraternale D, Giamperi L, Ricci D, Rocchi MBL (2002). Micropropagation of Bupleurum fruticosum: The effect of triacontanol. Plant Cell Tiss Organ Cult 69: 135-140

Fuentes SI, Suarez R, Villegas T, Acero LC, Hernandez G (1993). Embryogenic response of Mexican alfalfa (Medicago sativa) varieties. Plant Cell Tiss Organ Cult 34: 299-302

Fuentes SRL, Calheiros MBP, Manetti-Filho J, Vieira LGE (2000). The effect of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell Tiss Organ Cult 60: 5-13

Gadidasu K, Umate P, Aileni M, Reddy KS, Kokkirala VR, Kasula K, Abbagani S (2011). Micropropagation of a valuable ethnomedicinal plant Streblus asper Lour. J Phytol 3: 18-23

214

Gairi A, Rashid A (2005). Direct differentiation of somatic embryos on cotyledons of Azadirachta indica. Biol Plant 49: 169-173

Gamborg OL, Miller RA, Ojima K (1968). Nutrient requirements of the suspension cultures of soybean root cells. Exp Cell Res 50: 151-158

Gamborg OL, Phillips GC (1996). Plant Cell Tissue and Organ Culture: Fundamental Methods, Narosa Publishing House, New Delhi

Ganapathi TR, Suprasanna P, Bapat VA, Rao PS (1992). Propagation of banana through encapsulated shoot tips. Plant Cell Rep 11: 571-575

Gangopadhyay G, Bandyopadhyay T, Gangopadhyay SB, Mukherjee KK (2004). Luffa sponge - a unique matrix for tissue culture of Philodendron. Curr Sci 86: 315-319

Gangopadhyay G, Das S, Mitra SK, Poddar R, Modak BK, Mukherjee KK (2002). Enhanced rate of multiplication and rooting through the use of coir in aseptic liquid culture media. Plant Cell Tiss Organ Cult 68: 301-310

Gantait S, Mandal N, Bhattacharyya S, Das PK (2010). A novel strategy for in vitro conservation of Aloe vera L. through long term shoot culture. Biotechnol 9: 326-331

Gao SL, Zhu DN, Cai ZH, Jiang Y, Xu DR (1999). Organ culture of a precious Chinese medicinal plant - Fritillaria unibracteata. Plant Cell Tiss Organ Cult 59: 197-201

Gao X, Bjork L (2000). Chemical characters, height and root weight in callus regenerated plants of Valeriana officinalis L. J Herbs Spices Med Plants 7: 31-39

García-Martín G, González-Beneto ME, Manzanera JA (2001). Quercus suber L. somatic embryo germination and plant conversion: pretreatments and germination conditions. In Vitro Cell Dev Biol-Plant 37: 190-198

Garg L, Bhandari NN, Rani V, Bhojwani SS (1996). Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica. Plant Cell Rep 15: 855-858

215

Gautam VK, Nanda N, Gupta SC (1993). Development of shoots and roots in anther derived callus of Azadirachta indica A. Juss - a medicinal tree. Plant Cell Tiss Organ Cult 34: 13-18

Gautheret RJ (1939). Sur la possibilite de realizer la culture indefinite des tissus de tubercules de carotte. CR Acad Sci Paris 208: 118-120

Gautheret RJ (1985). History of plant tissue and cell culture. In: Vasil IK (ed). Cell culture and somatic cell genetics of plants, Vol 2, Cell growth, nutrition, cytodifferentiation and cryopreservation. Academic Press, New York pp 1-59

George EF (1993). Plant propagation by tissue culture. Part 1 The Technology, second edn, Exegenetics Ltd, Edington

George EF, Hall MA, De Klerk GJ (2008a). Plant propagation by tissue culture. Vol 1, The background, third edn. Springer, Dordrecht

George EF, Sherrington PD (1984). Commercial tissue culture laboratories. In: George EF, Sherrington PD (eds). Plant propagation by tissue culture. Handbook and directory of commercial laboratories, Easter Press, Great Britain, pp 549-551

George L, Eapen S (1994). Organogenesis and embryogenesis from diverse explants in pigeon pea (C. cajan L.). Plant Cell Rep13: 417-420

George PS, Ravishankar GA, Venkataraman LV (1993). Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture. Plant Cell Rep 13: 59-62

George S, Geetha SP, Balachandran I (2008b). Micropropagation of Baliospermum montanum (Wild.) Muell. - Arg. - a red listed medicinal plant. J Plant Sci 3: 111-115

Georges D, Chenieux JC, Ochatt SJ, (1993). Plant regeneration from aged callus of the woody ornamental species, Lonicera japonica cv. ‘Halls Prolific’. Plant Cell Rep 13: 91-94

Germaná MA, Micheli M, Chiancone B, Macaluso L, Standardi A (2011). Organogenesis and encapsulation of in vitro derived propagules of Carrizo

216 citrange [Citrus sinensis (L.) Osb. X Poncirus trifoliata (L.) Raf]. Plant Cell Tiss Organ Cult 106: 299-307

Gharyal PK, Maheshwari SC (1983). In vitro differentiation of plantlets from tissue culture of Albizzia lebbeck L. Plant Cell Tiss Organ Cult 2: 49-53

Gharyal PK, Maheshwari SC (1990). Differentiation in explants from mature leguminous trees. Plant Cell Rep 8: 550-553

Gharyal PK, Rashid A, Maheshwari SC (1983). Androgenic response from cultured anthers of a leguminous tree, Cassia siamea Lam. Protoplasma 118: 91-93

Gharyal PK, Rashid A, Maheswari SC (1993). Production of haploid plantlets in anther culture of Albizzia lebbeck L. Plant Cell Rep 2: 308-309

Ghosh B, Sen S (1991). Plant regeneration through somatic embryogenesis from spear callus culture of Asparagus cooperi Baker. Plant Cell Rep 9: 667- 670

Ghosh B, Sen S (1994a). Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi Baker. Plant Cell Rep 13: 381-385

Ghosh B, Sen S (1994b). Micropropagation of Asparagus cooperi Baker. as affected by growth regulators. Plant Sci 82: 119-124

Gill R, Saxena PK (1992). Direct somatic embryogenesis and regeneration of plants from seedlings explants of peanut (Arachis hypogaea): promotive role of thidiazuron. Can J Bot 70: 1186-1192

Giridhar P, Gururaj HB, Ravishankar GA (2005). In vitro shoot multiplication through shoot tip cultures of Decalepis hamiltonii Wight and Arn., a threatened plant endemic to Southern India. In Vitro Cell Dev Biol-Plant 41: 77-80

Giridhar P, Kumar V, Ravishankar GA (2004). Somatic embryogenesis, organogenesis and regeneration from leaf callus culture of Decalepis hamiltonii Wight and Arn, an endangered shrub. In Vitro Cell Dev Biol-Plant 40: 567-571

217

Girija S, Ganapathi A, Ananthakrishnan G (2000). Somatic embryogenesis in Vigna radiata (L.) Wilczek. Indian J Exp Biol 38: 1241-1244

Girija S, Kavitha S, Deepavathi S (2006). Direct multiple shoot regeneration from shoot tip and nodal explants of Ocimum sanctum L. (Tulsi): A medicinal herb. Plant Cell Biotechnol Mol Biol 7: 23-28

Gitonga LN, Gichuki ST, Ngamau K, Muigai AWT, Kahangi EM, Wasilwa LA, Wepukhulu S, Njogu N (2010). Effect of explant type, source and genotype on in vitro shoot regeneration in Macadamia (Macadamia spp.). J Agric Biotechnol Sustain Dev 2: 129-135

Gonçalves S, Fernandes L, Romano A (2010). High frequency in vitro propagation of the endangered species Tuberaria major. Plant Cell Tiss Organ Cult 101: 359-363

Gonzalez-Benito E, Alderson PG (1990). Regeneration from Alstroemeria callus. Acta Hort 280: 135-138

González-Benito ME, García-Martín G, Manzanera JA (2002). Shoot development in Quercus suber L. somatic embryos. In Vitro Cell Dev Biol-Plant 38: 477-480

Gopi C, Natarajasekhar Y, Ponmurugan P (2006). In vitro multiplication of Ocimum gratissimum L. through direct regeneration. Afr J Biotechnol 5: 723-726

Gopi C, Vatsala TM (2006). In vitro studies on effects of plant growth regulators on suspension culture biomass yield from Gymnema sylvestre. R. Br. Afr J Biotechnol 5: 1215-1219

Gray DJ (1987). Introduction to the symposium. In: Proc Symp Synthetic seed technology for the mass cloning of crop plants: problems and prospective. HortScience 22: 796-797

Gu XF, Zhang JR (2005). An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants. Plant Cell Rep 23: 775-779

218

Guan QZ, Guo YH, Siu XL, Li W, Zhang ZX (2008). Changes in photosynthetic capacity and antioxidant enzymatic systems in micropropagated Zingiber officinale plantlets during their acclimation. Photosynthetica 46: 193-201

Gubis J, Lajchová Z, Fragó J, Jureková Z (2003). Effect of explant type on shoot regeneration in tomato (Lycopersicon esculentum Mill.) in vitro. Czech J Plant Breed 39: 9-14

Guha S, Maheshwari SC (1964). In vitro production of embryos from anthers of Datura. Nature 204: 497

Guha S, Maheshwari SC (1966). Cell division and differentiation of embryos in the pollen grains of Datura in vitro. Nature 212: 97-98

Guha-Mukherjee S (1999). The discovery of haploid production by anther culture. In Vitro Cell Dev Biol-Plant 35: 357-360

Gulati A, Jaiwal PK (1994). Plant regeneration from cotyledonary node explant of mung bean (Vigna radiata). Plant Cell Rep 13: 523-527

Gupta SK, Khanuja SPS, Kumar S (2001). In vitro micropropagation of Lippia alba. Curr Sci 81: 206-210

Gürel S, Gülsen Y (1998). The effects of different sucrose, agar and pH levels on in vitro shoot production of almond (Amygdalus communis L.). Turk J Bot 22: 363-373

Haberlandt G (1902). Kulturversuche mit isolierten Pflanzenzellen. Sitzungsber K Preuss Akad Wiss Wien, Math-Naturwiss 111: 69-92

Haccius B (1978). Question of unicellular origin of non-zygotic embryos in callus cultures. Phytomorph 28: 74-81

Hammatt N, Grant NJ (1996). Micropropagation of a mature British wild cherry. Plant Cell Tiss Organ Cult 47: 103-107

Hanning E (1904). Zur physiologie pflanzlicher Embryonen. I Uber die Culture von Cruciferen-Embryonen ausserhalb des Embryosacks. Bot Ztg 62: 45-80

219

Hariharan M, Sebastian DP, Benjamin S, Prashy P (2002). Somatic embryogenesis in Leptadenia reticulata Wight and Arn. P - a medicinal plant. Phytomorph 52: 155-160

Harikrishnan KN, Hariharan M (1996). Direct shoot regeneration from nodal explants of Plumbago rosea Linn. a medicinal plant. Phytomorph 46: 53-58

Hasan MF, Das R, Rahman MS, Rashid MH, Hossain MS, Rahman M (2008). Callus induction and plant regeneration from shoot tips of chakunda (Cassia obtusifolia L.). Int J Sust Crop Prod 3: 6-10

Hazarika BN (2003). Acclimatization of tissue cultured plants. Curr Sci 85: 1704- 1712

He WT, Hou SW, Wang CY (2006). An efficient in vitro method for mass propagation of Potentilla potaninii Wolf. In Vitro Cell Dev Biol-Plant 42: 415-417

Heide OM (1969). Non reversibility of gibberellin induced inhibition of regulation in Begonia leaves. Physiol Plant 22: 671-679

Hetherington AM (2001). Guard cell signalling. Cell 107: 711-714

Hiregoudar LV, Murthy HN, Bhat JG, Nayeem A, Hema BP, Hahn EJ, Paek KY (2006). Rapid clonal ropagation of Vitex trifolia. Biol Plant 50: 291-294

Hiren AP, Saurabh RM, Subramanian RB (2004). In vitro regeneration in Curculigo orchiodes Gaertn. an endangered medicinal herb. Phytomorph 54: 85- 95

Hisachi H, Murai Y (1996). Micropropagation of Prunus mume. Plant Cell Tiss Organ Cult 40: 159-167

Hossain M, Biswas BK, Karim MR, Rahman S, Islam R, Joarder O (1994). In vitro organogenesis of elephant apple (Feronia limonia). Plant Cell Tiss Organ Cult 39: 265-268

220

Hosseini-Nasr M, Rashid A (2003/2004). Thidiazuron-induced high-frequency shoot regeneration from root region of Robinia pseudoacacia L. seedlings. Biol Plant 47: 593-596

Hu CY, Wang PJ (1983). Meristem, shoot-tip and bud cultures. In: Evans DA, Wang WR, Ammirato PV, Yamada Y (eds). Handbook of Plant Cell Culture, Vol 1 New York, MacMillan pp 177-227

Hu Z, Hu Y, Gao HH, Guan XQ, Zhuang DH (2008). Callus production, somatic embryogenesis and plant regeneration of Lycium barbarum root explants. Biol Plant 52: 93-96

Huda KMK, Bhuiyan MSR, Zeba N, Banu SA, Mahmud F, Khatun A (2009).

Effect of FeSO4 and pH on shoot regeneration from the cotyledonary explants of Tossa Jute. Plant Omics J 2: 190-196

Huetteman CA, Preece JE (1993). Thidiazuron: a potent cytokinin for woody plant tissue culture. Plant Cell Tiss Organ Cult 33: 105-119

Hunault G, Du Manoir J (1992). Micropropagation of fennel (Foeniculum vulgare Miller). In: Bajaj YPS (ed). High-tech and micropropagation Biotechnology in agriculture and forestry, Vol 19, Berlin, Springer, Verlag pp 199-218

Hung CD, Trueman SJ (2012). Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana x C. citriodora. Acta Physiol Plant 34: 117-128

Hung FH, Al-Khayri JM, Gbur EE (1994). Micropropagation of Acacia mearnsii. In Vitro Cell Dev Biol-Plant 30: 70-74

Husain MK, Anis M (2004). In vitro proliferation of shoots of Melia azedarach L. from mature trees. In: D’Souza L, Anuradha M, Nivas S, Hegde S, Rajendra K (eds). Biotechnology for a better future. SAC, Mangalore pp 294-301

Husain MK, Anis M (2006a). Rapid in vitro propagation of Eclipta alba by shoot tip culture. J Plant Biochem Biotechnol 15: 147-149

221

Husain MK, Anis M (2006b). Rapid in vitro propagation of Eclipta alba (L.) Hassk. through high frequency axillary shoot proliferation. Acta Physiol Plant 28: 325-330

Husain MK, Anis M (2009). Rapid in vitro multiplication of Melia azedarach L. (a multipurpose woody tree). Acta Physiol Plant 31: 765-772

Husain MK, Anis M, Shahzad A (2007a). In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using Thidiazuron. In Vitro Cell Dev Biol-Plant 43: 59-64

Husain MK, Anis M, Shahzad A (2007b). Seed germination studies in Pterocarpus marsupium Roxb., an important multipurpose tree. Hamdard Med 50: 112-115

Husain MK, Anis M, Shahzad A (2008). In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol Plant 30: 353-359

Husain MK, Anis M, Shahzad A (2010). Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb. Trees 24: 781-787

Hussain TM, Thummala C, Ghanta RG (2007). High frequency shoot regeneration of Stercula urens Roxb. an endangered tree species through cotyledonary node cultures. Afr J Biotechnol 6: 1643-1649

Hussein MA, Batra A (1998). In vitro embryogenesis of cumin hypocotyl segments. Adv Plant Sci 11: 125-132

Hussey G (1980). In vitro propagation. In: Ingram DS, Helgeson JP (eds). Tissue Culture for Plant Pathologists, Oxford, Blackwell Scientific pp 51-61

Hussey G (1983). In vitro propagation of horticultural and agricultural crops. In: Mantel SH, Smith H (eds). Plant Biotechnol Cambridge, Cambridge University Press pp 111-138

222

Hussey G (1986). Vegetative propagatation of plants by tissue culture. In: Yeoman MM (ed). Plant Cell Culture Technology, Blackwell Scientific Publications, Oxford

Hutchinson MJ, Murch SJ, Saxena PK (1996). Morphoregulatory role of thidiazuron: evidence of the involvement of endogenous auxin in thidiazuron- induced somatic embryogenesis of geranium (Pelargonium x hortorum Bailey). J Plant Physiol 149: 573-579

Hutchinson MJ, Saxena PK (1996). Acetylsalicylic acid enhances and synchronizes thidiazuron induced somatic embryogenesism in geranium (Pelargonium x hortorum Bailey) tissue cultures. Plant Cell Rep 15: 512-515

Ignacimuthu S, Arockiasamy S, Antonysamy M, Ravichandran P (1999). Plant regeneration through somatic embryogenesis from mature leaf explants of Eryngium foetidum, a condiment. Plant Cell Tiss Organ Cult 56: 131-137

Ikhlaq M, Hafiz IA, Micheli M, Ahmad T, Abbasi NA, Standardi A (2010). In vitro storage of synthetic seeds: Effect of different storage conditions and intervals on their conversion ability. Afr J Biotechnol 9: 5712-5721

Inamdar JA, Nataraj M, Mohan JSS, Subramanian RB (1990). Somatic embryogenesis from callus cultures of Crataeva nurvala - Buch. Ham. Phytomorph 40: 319-322

Infante R, Magnanini E, Righetti B (1989). The role of light and CO2 in optimising the conditions for shoot proliferation of Actinidia deliciosa in vitro. Physiol Plant 77: 191-195

Irvani N, Solouki M, Omidi M, Zare AR, Shahnazi S (2010). Callus induction and plant regeneration in Dorema ammoniacum D., an endangered medicinal plant. Plant Cell Tiss Organ Cult 100: 293-299

Jablonski JR, Skoog F (1954). Cell enlargement and cell division in excised tobacco pith tissue. Physiol Plant 7: 16-24

223

Jagadishchandra KS, Rachappaji S, Gowada KRD, TharaSarswathi KJ (1999). In vitro propagatipon of Pisonia alba (Linn.) Sapanogae (Lettuce tree), a threatened species. Phytomorph 49: 43-47

Jain R, Singh N, Patni V (2009a). In vitro plantlet regeneration and isolation of quercetin from elite species of Acacia catechu (L.F.) Willd - a katha yielding plant. Plant Cell Biotechnol Mol Biol 10: 125-130

Jain R, Sinha A, Kachhwaha S, Kothari SL (2009b). Micropropagation of Withania coagulans (Stocks) Dunal: A critically endangered medicinal herb. J Plant Biochem Biotechnol 18: 249-252

Janeiro LV, Ballester A, Vieitez AM (1997). In vitro response of encapsulated somatic embryos of Camellia. Plant Cell Tiss Organ Cult 51: 119-125

Jayanand B, Sudarsanam G, Sharma KK (2003). An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro germinated seedlings. In Vitro Cell Dev Biol-Plant 39: 171-179

Jayanthi M, Mandal PK (2001). Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plantlets in Tylophora indica (Burm.f.) Merrill. In Vitro Cell Dev Biol-Plant 37: 576-580

Jeon MW, Ali MB, Hahn EJ, Paek KY (2005). Effects of photon flux density on the morphology, photosynthesis and growth of a CAM orchid, Doritaenopsis during post micropropagation acclimatization. Plant Growth Reg 45: 130-147

Jeong JH, Murthy HN, Paek KY (2001). High frequency adventitious shoot induction and plant regeneration from leaves of statice. Plant Cell Tiss Organ Cult 65: 123-128

Jethwani V, Kothari S (1996). Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis. Plant Cell Rep 15: 869-872

Jeyakumar M, Jayabalan N (2002). In vitro plant regeneration from cotyledonary node of Psoralea corylifolia L. Plant Tiss Cult 12: 125-129

224

Jha AK, Prakash S, Jain N, Nanda K, Gupta SC (2004). Micropropagation of Sesbania rostrata from the cotyledonary node. Biol Plant 48: 289-292

Jiménez VM, Guevara E, Herrera J, Bangerth F (2005). Evolution of endogenous hormone concentration in embryogenic cultures of carrot during early expression of somatic embryogenesis. Plant Cell Rep 23: 567-572

Jin S, Zhang X, Liang S, Nie Y, Guo X, Huang C (2005). Factors affecting transformation efficiency of embryogenic callus of upland cotton (Gossypium hirsutum) with Agrobacterium tumefaciens. Plant Cell Tiss Organ Cult 81: 229- 237

Johansen DA (1940). Plant Microtechnique. McGraw-Hill Book Co Inc., New York

Johnson M, Manickam VS (2003). In vitro micropropagation of Baliospermum montanum (Willb). Muell. Arg. - a medicinal plant. Indian J Exp Biol 41: 1349- 1351

Johnson TS, Narayan SB, Narayana DBA (1997). Rapid in vitro propagation of Saussurea lappa, an endangered medicinal plant, through multiple shoot cultures. In Vitro Cell Dev Biol-Plant 33: 128-130

Joseph N, Siril EA, Nair GM (2011). An efficient in vitro propagation methodology for Annatto (Bixa orellana L.). Physiol Mol Biol Plants 17: 263-270

Joseph T, Yeoh HH, Loh CS (2000). Somatic embryogenesis, plant regeneration and cryogenesis of Manihot glaziovii Muell. Agr. (Ceara rubber). Plant Cell Rep 19: 535-538

Joshi I, Bisht P, Sharma VK, Uniyal DP (2003). Studies on effect of nutrient media for clonal propagation of superior phenotypes of Dalbergia sissoo Roxb. through tissue culture. Silvae Gen 52: 143-147

Joshi M, Dhar U (2003). In vitro propagation of Saussurea obvallata (DC) Edgew.-an endangered ethnoreligious medicinal herb of Himalaya. Plant Cell Rep 21: 933-939

225

Joshi P, Dhawan V (2007a). Assessment of genetic fidelity of micropropagated Swertia chirayita plantlets by ISSR marker assay. Biol Plant 51: 22-26

Joshi P, Dhawan V (2007b). Axillary multiplication of Swertia chirayita (Roxb. Ex Fleming) H. Karst., a critically endangered herb of temperate Himalyas. In Vitro Cell Dev Biol-Plant 43: 631-638

Joshi P, Trivedi R, Purohit SD (2009). Micropropagation of Wrightia tomentosa: Effect of gelling agents, carbon source and vessel type. Indian J Biotechnol 8: 115-120

Jusaitis M (1997). Micropropagation of adult Swaisona formosa Leguminosae: Papilionoideae: Galegeae. In Vitro Cell Dev Biol-Plant 33: 213-220

Jusekutty PC, Swati S, Prathapasenan G (1993). Direct and indirect organogenesis in Coccinia indica. J Hort Sci 68: 31-35

Kadleček P, Tichá I, Capková V, Schäfer C (1998). Acclimatization of micropropagated tobacco plantlets. In: Garab G (ed). Photosynthesis: mechanism and effects, Vol 5 Kluwer Academic Publishers, Dordrecht, Boston, London pp 3853-3856

Kadleček P, Tichá I, Haisel D, Čapková V, Schäfer C (2001). Importance of in vitro pretreatment for ex vitro acclimatization and growth. Plant Sci 161: 695-701

Kaewpooa M, Te-chato S (2009). Influence of explant types and plant growth regulators on multiple shoot formation from Jatropha curcas. Sci Asia 35: 353- 357

Kala CP (2000). Status and conservation of rare and endangered medicinal plants in the Indian trans - Himalaya. Biol Conserv 93: 371-379

Kala CP (2003). Commercial exploitation and conservation status of high value medicinal plants across the borderline of India and Nepal in Pithoragarh. Indian Forester 129: 80-84

226

Kala CP (2005). Indigenous uses, population density, and conservation of threatened medicinal plants in protected areas of the Indian Himalayas. Conserv Biol 19: 368-378

Kala CP, Dhyani PP, Sajwan BS (2006). Developing the medicinal plants sector in Northern India: Challenges and opportunities. J Ethnobiol Ethnomed 2: 1-15

Kamo K, Jones B, Bolar J, Smith F (2005). Regeneration from long term embryogenic callus of the Rosa hybrida cultivar kardinal. In Vitro Cell Dev Biol- Plant 41: 32-36

Kaneda Y, Tabei Y, Nishimura S, Harada K, Akihama T, Kitamura K (1997). Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybean [Glycine max (L.) Merr]. Plant Cell Rep 17: 8-12

Kannan R, Jasrai YT (1998). Micropropagation of medicinal plants - Vitex negundo. J Med Arom Plant Sci 20: 693-696

Kapoor S, Gupta SC (1986). Rapid in vitro differentiation of Sesbania bispinosa plants a leguminous shrub. Plant Cell Tiss Organ Cult 1: 263-268

Karami O, Delijou A, Esna-Ashari M, Ostad-Ahmadi P (2006). Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.). Sci Hort 110: 340-344

Karhu ST (1997). Sugar use in relation to shoot induction by sorbitol and cytokinin in apple. J Am Soc Hort Sci 122: 47-480

Kartha KK, Gamborg OL (1978). Meristem culture techniques in the production of disease free plants and freeze preservation of germplasm of tropical tuber crops and grain legumes. In: Maraite H, Meyer JA (eds). Disease of Tropical Food Crops, Univ Catholique de Leuvain, Belgium pp 267-283

Karuppusamy S, Kalimuthu K (2010). Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis neesiana: a valuable endemic medicinal plant. Adv Biol Res 4: 211-216

227

Karuppusamy S, Kiranmai C, Aruna V, Pullaiah T (2006). Micropropagation of Vanasushava pedata - an endangered medicinal plant of South India. Plant Tiss Cult Biotechnol 16: 85-94

Karuppusamy S, Pullaiah T (2007). In vitro shoot multiplication of Bupleurum distichophyllum Wight - A native medicinal plant of Southern India. Plant Tiss Cult Biotechnol 17: 115-124

Kathiravan K, Ignacimuthu S (1999). Micropropagation of Canavalia virosa - a medicinal plant. Phytomorph 49: 61-66

Kavyashree R, Gayatri MC, Revanasiddaiah HM (2006). Propagation of mulberry variety - S54 by synseeds of axillary bud. Plant Cell Tiss Organ Cult 84: 245-249

Keng CL, Yee LS, Pin PL (2009). Micropropagation of Gynura procumbens (Lour.) Merr. an important medicinal plant. J Med Plants Res 3: 105-111

Kerns HR, Meyer MM Jr (1987). Diligence finds the chemical key to micropropagating a new maple. Am Nurseryman 165: 104, 105, 108 and 110

Ket NV, Hahn EJ, Park SY, Chakarbarty D, Paek KY (2004). Micropropagation of an endangered orchid Anoectochilus formosanus. Biol Plant 48: 339-344

Kevers C, Hausman JF, Faivre-Rampant O, Evers D, Gaspar T (1997). Hormonal control of adventitious rooting: progress and questions. Angew Bot 71: 71-79

Khalafalla MM, Daffalla HM (2008). In vitro micropropagation and micrografting of gum Arabic tree [Acacia senegal (L.) Wild]. Int J Sustain Crop Prod 3: 19-27

Khalekuzzaman M, Rahman MS, Rashid MH, Hossain MS (2008). High frequency in vitro propagation of Adhatoda vasica Nees through shoot tip and nodal explants culture. J Bio Sci 16: 35-39

Khan MI, Ahmad N, Anis M (2011). The role of cytokinins on in vitro shoot production in Salix tetrasperma Roxb.: a tree of ecological importance. Trees 25: 577-584

228

Khan MY, Aliabbas S, Kumar V, Rajkumar S (2009). Recent advances in medicinal plant biotechnology. Indian J Biotechnol 8: 9-22

Khatun MM, Hossain MS, Haque MA, Khalekuzzaman M (2010). In vito propagation of Citrullus lanatus Thumb. from nodal explants culture. J Bangladesh Agric Univ 8: 203-206

Khurana P, Bhatnagar S, Kumari S (2005). Thidiazuron and woody plant tissue culture. J Plant Biol 32: 1-12

Kidwai NR, Jain MB, Chaturvedi HC (2009). Role of thidiazuron in in vitro induction of embryogenesis in nucellar tissue of Mangifera indica L. var. Dashehari, leading to plantlets. Curr Sci 96: 1119-1124

Kim JH, LaMotte CE, Hack E (1990). Plant regeneration in vitro from primary leaf nodes of soybean (Glycine max) seedlings. J Plant Physiol 136: 664-669

Kim MK, Sommer HE, Bongarten BC, Merkle SA (1997). High frequency induction of adventitious shoots from hypocotyl segments of Liquidambar styraciflua L. by thidiazuron. Plant Cell Rep 16: 536-540

Kim YS, Lim S, Choi YE, Anbazhagan VR (2007). High frequency plant regeneration via somatic embryogenesis in Podophyllum peltatum L., an important source of anticancer drug. Curr Sci 92: 662-666

Kiran G, Kaviraj CP, Jogeswar G, Kishor KVK, Rao S (2005). Direct and high frequency somatic embryogenesis and plant regeneration from hypocotyls of chickpea (Cicer arietenum L.), a grain legume. Curr Sci 89: 1012-1018

Kiranmai C, Aruna V, Karuppusamy S, Pullaiah T (2008). Callus culture and plant regeneration from seedling explants of Pergularia daemia (forsk) chiov. J Plant Biochem Biotechnol 17: 99-101

Kishore R, Sha Valli Khan PS, Sharma GJ (2006). Hybridization and in vitro culture of an orchid hybrid Ascocenda ‘Kangla’. Sci Hort 108: 66-73

229

Kitamiya E, Suzuki S, Sano T, Nagata T (2000). Isolation of two genes that were induced upon the initiation of somatic embryogenesis on carrot hypocotyls by high concentrations of 2,4-D. Plant Cell Rep 19: 551-557

Kitto SL, Janick J (1982). Polyox as an artificial seed coat for asexual embryos. HortScience 17: 448

Kitto SL, Janick J (1985a). Production of synthetic seeds by encapsulating asexual embryos of carrot. J Am Soc Hort Sci 110: 277-282

Kitto SL, Janick J (1985b). Hardening treatments increase survival of synthetically coated asexual embryos of carrot. J Am Soc Hort Sci 110: 283-286

Knoll KA, Short KC, Curtis IS, Power JB, Davey MR (1997). Shoot regeneration from cultured root explants of spinach (Spenacia oleracea L.): a system for Agrobacterium transformation. Plant Cell Rep 17: 96-101

Knop W (1865). Quantitative Untersuchungen über den Ernährungsprozess der Pflanzen. Landwirtsch Vers Stn 7: 93-107

Kögl F, Haagen-Smit AJ, Erxleben H (1934). Über ein neues auxin (‘‘Heteroauxin’’) aus Harn. Z Physiol Chem 228: 90-103

Kohlenbach HW (1966). Die Entwicklungpotenzen explantierter und isolieter Dauerzellen. I. Das Streckungs und Teilungswachstum isolieter Mesophyllzellen von Macleaya cordata. Z Pflanzenphysiol 55: 142-157

Kohlenbach HW (1997). Basic aspects of differentiation and plant regeneration from cell and tissue culture. In: Barg W, Reinhard E, Zenk MH (ed). Plant Tissue Culture and its Biotechnological Application, Springer Verlag, New York pp 355- 368

Komalavalli N, Rao MV (2000). In vitro micropropagation of Gymnema sylvestre - a multipurpose medicinal plant. Plant Cell Tiss Organ Cult 61: 97-105

Komamine A, Murata N, Nomura K (2005). Mechanisms of somatic embryogenesis in carrot suspension cultures-morphology, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol-Plant 41: 6-10

230

Koroch A, Kapteyn J, Juliani HR, Simon JE (2003). In vitro regeneration of Echinacea pallida from leaf explants. In Vitro Cell Dev Biol-Plant 39: 415-418

Kotte W (1922). Kulturversuche mit isolierten Wurzelspitzen. Beitr Allg Bot 2: 413-443

Kozai T (1991). Micropropagation under photoautotrophic conditions. In: Debergh PC, Zimmerman RH (ed): Micropropagation. Technology and Application. Kluwer Academic Publishers, Dordrecht Boston, London pp 447-469

Kozai T, Smith MAL (1995). Environmental control in plant tissue culture general introduction and overview. In: Goto E, Kurata K, Hayashi M, Saa S (eds). Plant Production in Closed Ecosystems. Kluwer Academic Publishers, Dordrecht, Boston, London pp 153-169

Kris P, Tess A, Jerry N (2005). Tissue culture propagation of mangolian cherry (Prunus fruticosa) and nanking cherry (Prunus tomentosa). Plant Cell Tiss Organ Cult 82: 207-211

Krishnan PN, Decruse SW, Rodh PK (2011). Conservation of medicinal plants of Western Ghats, India and its sustainable utilization through in vitro technology. In Vitro Cell Dev Biol-Plant 47: 110-122

Krishnan PN, Sudha CG, Seeni S (1995). Rapid propagation through shoot tip culture of Trichopus zeylanicus Gaertn., a rare ethnomedicinal plant. Plant Cell Rep 14: 708-711

Krishnaraj S, Vasil IK (1995). Somatic embryogenesis in herbaceous monocots. In: Thorpe TA (ed). In Vitro Embryogenesis in Plants, Dordrecht, Netherland, Kluwer Academy Publishers pp 155-203

Kukreja AK, Mathur AK (1985). Tissue culture studies in Duboisia myoporoides. I. Plant regeneration and propagation by stem node cultures. Planta Med 2: 93- 96

Kukreja AK, Mathur AK, Zaim M (1990). Mass production of virus free patchouli plants [Pogostemon cablin (Blanco)] by in vitro culture. Trop Agric 67: 101-104

231

Kulkarni AA, Thangane SR, Krishnamurthy KV (2000). Direct shoot regeneration from node, internode, hypocotyl and embryo explants of Withania somnifera. Plant Cell Tiss Organ Cult 62: 203-209

Kumar A (1992). Micropropagation of a mature leguminous tree Bauhinia purpurea. Plant Cell Tiss Organ Cult 31: 257-259

Kumar A, Sood A, Palni UT, Gupta AK, Palni LMS (2001). Micropropagation of Rosa damascena Mill. from mature bushes using thidiazuron. J Hortic Sci Biotech 76: 30-34

Kumaria R, Sunnichan VG, Das DK, Gupta SK, Reddy VS, Bhatnagar RK, Leelawathi S (2003). High frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress. Plant Cell Rep 21: 635-639

Kunitake H, Mii M (1997). Somatic embryogenesis and its application for breeding and micropropagation in Asparagus (Asparagus officinalis L.). Plant Biotechnol 15: 51-63

Kuo HL, Chen JT, Chang WC (2005). Efficient plant regeneration through direct somatic embryogenesis from leaf explants of Phalaenopsis Little Steve. In Vitro Cell Dev Biol-Plant 41: 453-456

Kwada AD, Tella IO (2009). Determination of infochemicals and the phytochemical screening of the foliage and stem-bark of Senna siamea (Lam.) in Yola Adamawa State. J Med Plants Res 3: 630-634

Lakshmanan P, Lee CL, Goh CJ (1997). An efficient in vitro method for mass propagation of a woody ornamental Ixora coccinea L. Plant Cell Rep 16: 572-577

Lakshmanan Rao PV, De DN (1987). Tissue culture propagation of tree legume Albizzia lebbeck (L.) Benth. Plant Sci 51: 263-267

Lakshmi Sita G, Sreenatha KS, Sujata S (1992). Plantlet production from shoot tip cultures of red sandalwood (Pterocarpus santalinus L.). Curr Sci 62: 532-535

232

Lal N, Ahuja PS (1989). Propagation of Indian rhubarb (Rheum emodi watt) using shoot tip and leaf explant culture. Plant Cell Rep 8: 493-496

Lal N, Ahuja PS (1996). Plantlet regeneration from callus in Picrorhiza kurroa Royle ex Benth. - an endangered medicinal plant. Plant Tiss Cult 6: 127-134

Laloo RC, Kharlukhi L, Jeeva S, Mishra BP (2006). Status of medicinal plants in the disturbed and the undisturbed sacred forests of Meghalaya, North East India: population structure and regeneration efficacy of some important species. Curr Sci 90: 225-232

Lata H, Bedir E, Hosick A, Ganzera M, Khan I, Moraes RM (2002). In vitro plant regeneration from leaf - direct callus of Cimicifuga racemosa. Planta Med 68: 912-915

Lata H, Chandra S, Khan I, ElSohly MA (2009b). Thidiazuron induced high frequency direct shoot organogenesis of Cannabis sativa L. In Vitro Cell Dev Biol-Plant 45: 12-19

Lata H, Chandra S, Khan IA, ElSohly MA (2009a). Propagation through alginate encapsulation of axillary buds of Cannabis sativa L. - an important medicinal plant. Physiol Mol Biol Plants 15: 79-86

Lavanya M, Venkateshwarlu B, Devi BP (2009). Acclimatization of neem microshoots adaptable to semi - sterile conditions. Indian J Biotechnol 8: 218– 222

Lazzeri PA, Hildebrandt DF, Collins GB (1987). Soybean somatic embryogenesis: effect of hormone and culture manipulations. Plant Cell Tiss Organ Cult 10: 209-210

Lees RP, Evans EH, Nicholas JR (1991). Photosynthesis in Clematis ‘The President’ during growth in vitro and subsequent in vivo acclimatization. J Exp Bot 42: 605-610

Lemos EEP, Baker DA (1998). Shoot regeneration in response to carbon source on inter nodal explants of Annona muricata L. Plant Growth Reg 25: 105-112

233

Li ZL, Yang SY (1988). Reduction of abscisic acid and induction of sprouting in potato Solanum tuberosum L. by TDZ. J Plant Growth Reg 7: 37-44

Li ZT, Dhekeny S, Dutt M, van Anan M, Tattersll MJ, Kelley KT, Gray J (2006). Optimizing Agrobacterium - mediated transformation of grape vine. In Vitro Cell Dev Biol-Plant 42: 220-227

Linsmaier EM, Skoog F (1965). Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100-127

Lisowska K, Wysokinska H (2000). In vitro propagation of Catalpa ovata G. Don. Plant Cell Tiss Organ Cult 60: 171-176

Litvay JD, Verma DC, Johnson MA (1985). Influence of loblolly pine (Pinus taeda L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4: 325-328

Liu CZ, Murch SJ, Demerdash MEL, Saxena PK (2003). Regeneration of Egyptian medicinal plant Artemisia judaica L. Plant Cell Rep 21: 525-530

Liu Z, Li Z (2001). Micropropagation of Camptotheca acuminata Decaisne from axillary buds, shoot tips, seed embryos in a tissue culture system. In Vitro Cell Dev Biol-Plant 37: 84-88

Lloyd GB, McCown BH (1980). Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot tip culture. Proc Int Plant Prop Soc 30: 421-427

Loc NH, Duc DT, Kwon TH, Yang MS (2005). Micropropagation of zedoary (Curcuma zedoaria Roscoe) - a valuable medicinal plant. Plant Cell Tiss Organ Cult 81: 119-122

Lou H, Sako S (1995). Role of high sugar concentration in inducing somatic embryogenesis from cucumber cotyledons. Sci Hort 64: 11-20

Lu C, Zhang J (1998). Changes in photosystem II function during senescence of wheat leaves. Physiol Plant 104: 239-247

234

Lu CY (1993). The use of thidiazuron in tissue culture. In Vitro Cell Dev Biol- Plant 29: 92-96

Lulsdorf MM, Tautorus TE, Kikcio SI, Bethuna TD, Dunstan DI (1993). Germination of encapsulated embryos of interior spruce (Picea glauca engelmannii complex) and black spruce (Picea mariana Mill.). Plant Cell Rep 12: 385-389

Luo JP, Jia JF (1998). Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall. Plant Cell Rep 17: 313-317

MacKinney G (1941). Absorption of light by chlorophyll solution. J Biol Chem 140: 315-322

MacLachan S, Zalik S (1963). Plastid structure, chlorophyll concentration and free amino acid composition of chlorophyll mutant barley. Can J Bot 1053-1062

Magioli C, Rocha APM, Tarré E, Santiago-Fernandes LD, Oliveira DE, Krul WR, Mansur E (2001). Effect of morphological factors, antibiotics and Agrobacterium co-cultivation in the efficiency of somatic embryogenesis of eggplant (Solanum melongena L.). J Plant Biotechnol 3: 19-25

Maheshwari P, Kumar A (2006). In vitro high frequency of shoot regeneration in Abelmoschus moschatus. J Cell Tiss Res 6: 627-632

Maheswaran G, Williams EG (1984). Direct somatic embryoid formation on immature embryos of Trifolium repens, T. pratense, Medicago sativa and rapid clonal propagation of T. repens. Ann Botany 54: 201-211

Maity S, Ray S, Banerjee N (2005). The role of plant growth regulators on direct and indirect regeneration from various organs of Leucaena leucocephala. Acta Physiol Plant 27: 473- 480

Malik KA, Saxena PK (1992a). In vitro regeneration of plants: A novel approach. Naturwissenschaften 79: 136-137

235

Malik KA, Saxena PK (1992b). Thidiazuron induces high frequency shoot regeneration in intact seedlings of pea (Pisum culinaris Medik). Aust J Plant Physiol 19: 731-740

Malik S, Sharma S, Sharma M, Ahuja PS (2010). Direct shoot regeneration from intact leaves of Arnebia euchroma (Royle) Johnston using thidiazuron. Cell Biol Int 34: 537-542

Malik SK, Chaudhury R, Kalia RK (2005). Rapid in vitro multiplication and conservation of Garcinia indica: A tropical medicinal tree species. Sci Hort 106: 539-553

Mallikarjuna K, Rajendrudu G (2007). High frequency in vitro propagation of Holarrhena antidysenterica from nodal buds of mature tree. Biol Plant 51: 525- 529

Mamiya K, Sakamoto Y (2000). Effects of sugar concentration and strength of basal medium on conversion of somatic embryos in Asparagus officinalis L. Sci Hort 84: 15-26

Mamun AN, Matin MN, Bari MA, Siddique NA, Sultan RS, Rahman MH, Musa AS (2004). Micropropagation of woody legume Albizia lebbeck through tissue culture. Pak J Biol Sci 7: 1099-1103

Mandal AKA, Gupta SD (2001). Direct shoot organogenesis and plant regeneration in safflower. In Vitro Cell Dev Biol-Plant 37: 50-54

Mandal AKA, Gupta SD (2003). Somatic embryogenesis of safflower: influence of auxin and ontogeny of somatic embryos. Plant Cell Tiss Organ Cult 72: 27-32

Mandal J, Pattnaik S, Chand PK (2000). Alginate encapsulation of axillary buds of Ocimum americanum L. (Hoary Basil), O. basilicum L. (Sweet Basil), O. gratissimum L. (Shrubby Basil), and O. sanctum L. (Sacred Basil). In Vitro Cell Dev Biol-Plant 36: 287-292

236

Manjkhola S, Dhar U, Joshi M (2005). Organogenesis, embryogenesis and synthetic seed production in Arnebia euchorma - a critically endangered medicinal plant of the Himalaya. In Vitro Cell Dev Biol-Plant 41: 244-248

Manjula S, Job A, Nair GM (2000). Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill. Indian J Exp Biol 38: 1069-1072

Manzanera JA, Astorga R, Bueno MA (1993). Somatic embryo induction and germination in Quercus suber L. Silvae Genet 42: 90-93

Mao AH, Wetten A, Fay M, Caligari PDS (1995). In vitro propagation of Clerodendrum colebrookianum Walp: a potential natural antihypertension medicinal plant. Plant Cell Rep 14: 493-496

Marchant R, Davey MR, Lucas JA, Power JB (1996). Somatic embryogenesis and plant regeneration in Floribunda rose (Rosa hybrida L.) cvs. Trumpeter and Glad Tidings. Plant Sci 120: 95-105

Martin G, Geetha SP, Raja SS, Raghu AV, Balachandran I, Ravindran PN (2006). An efficient micropropagation system for Celastrus paniculatus Willd.: a vulnerable medicinal plant. J For Res 11: 461-465

Martin JA, Gella R, Herrero M (1988). Stomatal structure and functioning as a response to environmental changes in acclimatized micropropagated Prunus cerasus L. Ann Botany 62: 663-670

Martin KP (2002). Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis. Plant Cell Rep 21: 112-117

Martin KP (2003a). Plant regeneration through somatic embryogenesis on Holostemma ada-kodien Schult. a rare medicinal plant. Plant Cell Tiss Organ Cult 72: 79-82

Martin KP (2003b). Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoephytic woody medicinal plant. Plant Cell Rep 21: 415- 420

237

Martin KP (2004a). Benzyladenine induced somatic embryogenesis and plant regeneration of Leptadenia reticulata. Biol Plant 48: 285-288

Martin KP (2004b). Plant regeneration through somatic embryogenesis in medicinally important Centella asiatica L. In Vitro Cell Dev Biol-Plant 40: 586-591

Martin KP (2004c). Efficacy of different growth regulators at different stages of somatic embryogenesis in Eryngium foetidum L. - a rare medicinal plant. In Vitro Cell Dev Biol-Plant 40: 459-463

Martin KP, Madassery J (2005). Direct and indirect somatic embryogenesis on cotyledon explants of Quassia amara L., an antileukaemic drug plant. In Vitro Cell Dev Biol-Plant 41: 54-57

Maruyama E, Kinoshita I, Ishii K, Ohba K, Saito A (1997a). Germplasm conservation of the tropical forest trees, Cedrela odorata L., Guazuma crinita Mart., and Jacaranda mimosaefolia D Don., by shoot tip encapsulation in calcium alginate and storage at 12-25 ºC. Plant Cell Rep 16: 393-396

Maruyama E, Kinoshita I, Ishii K, Shigenaga H, Ohba K, Saito A (1997b). Alginate-encapsulation technology for the propagation of the tropical forest trees: Cedrela odorata L., Guazuma crinita Mart., and Jacaranda mimosaefolia D Don. Silvae Genet 46: 17-23

Mary JR, Jayabalan N (1997). Influence of growth regulators on somatic embryogenesis in sesame. Plant Cell Tiss Organ Cult 49: 67-70

Masmoudi R, Rival A, Nato A, Lavergne D, Drira N, Ducreux G (1999). Carbon metabolism in in vitro cultures of date palm: the role of carboxylases (PEPC and Rubisco). Plant Cell Tiss Organ Cult 57: 139-143

Mathieu M, Lelu-Walter MA, Blervacq AS, David H, Hawkins S, Neutelings G (2006). Germin-like genes are expressed during somatic embryogenesis and early development of conifers. Plant Mol Biol 61: 615-627

238

Mathur A, Mathur AK, Verma P, Yadav S, Gupta ML, Darokar MP (2008). Biological hardening and genetic fidelity testing of micro-cloned progeny of Chlorophytum borivilianum Sant. et Fernand. Afr J Biotechnol 7: 1046-1053

Mathur J, Mukunthakumar S (1992). Micropropogation of Bauhinia variegata and Parkinsonia aculeata from nodal explants of mature trees. Plant Cell Tiss Organ Cult 28: 119-121

Matsuta N, Hirabayashi T (1989). Embryogenic cell lines from somatic embryos of grape (Vitis vinifera L.) Plant Cell Rep 7: 684-687

Mauri PV, Manzanera JA (2004). Effect of abscisic acid and stratification on somatic embryo maturation and germination of holm oak (Quercus ilex L.). In Vitro Cell Dev Biol-Plant 40: 495-498

McCown BH, Sellmer JC (1987). General media and vessels suitable for woody plant cultures. In: Bonga JM, Durzan DJ (eds). Tissue culture in forestry - General principles and biotechnology, Vol 2 Martinus Nijhoff Publ, Dordrecht, Boston pp 4-6

McCoy E, O‘Connor SE (2008). Natural products from plant cell cultures. Prog Drug Res 65: 331-370

Mehrotra S, Goel MK, Kukreja AK, Mishra BN (2007). Efficiency of liquid culture systems over conventional micropropagation: A progress towards commercialization. Afr J Biotechnol 6: 1484-1492

Mercier H, Viera CCJ, Figueredo-Ribeiro RCL (1992). Tissue culture and plant propagation of Gomphrena officinalis, a Brazilian medicinal plant. Plant cell Tiss Organ Cult 28: 249-254

Merkle SA, Parrott WA, Flinn BS (1995). Morphogenic aspects of somatic embryogenesis. In: Thorpe TA (ed). In Vitro Embryogenesis in Plants, Dordrecht, The Netherlands, Kluwer Academy Publishers pp 155-203

239

Micheli M, Hafiz IA, Standardi A (2007). Encapsulation of in vitro derived explants of olive (Olea europaea L. cv. Moraiolo) II. Effects of storage on capsule and derived shoots performance. Sci Hort 113: 286-292

Miller CO, Skoog F, Von Saltza MH, Strong FM (1955). Kinetin, a cell division factor from deoxyribonucleic acid. J Am Chem Soc 77: 1392

Mishra J, Singh M, Palni LMS, Nandi SK (2011). Assessment of genetic fidelity of encapsulated microshoots of Picrorhiza kurrooa. Plant Cell Tiss Organ Cult 104: 181-186

Mishra P (2002). Direct differentiation of shoot buds from leaf explants of Cajanus cajan L. Biol Plant 45: 347-351

Missaleva N, Petri G, Szoke E (1993). Some morphological and biochemical peculiarities of Datura innoxia callus and regenerated cultures. Plant Cell Tiss Organ Cult 35: 87-92

Mithila J, Hall JC, Victor JMR, Saxena PK (2003). Thiadiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulina ionantha Wendl.). Plant Cell Rep 21: 408-414

Mohamed MAH, Alsadon AA (2010). Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets. Sci Hort 123: 295- 300

Mohamed-Yasseen Y (2002). Micropropagation of pitaya (Hylocereus undatus Britton et rose). In Vitro Cell Dev Biol-Plant 38: 427-429

Mohanraj R, Ananthan R, Bai VN (2009). Production and storage of synthetic seeds in Coelogyne breviscapa Lindl. Asian J Biotechnol 1: 124-128

Mohebodini M, Javaran MJ, Mahboudi F, Alizadeh H (2011). Effects of genotype explant age and growth regulators on callus induction and direct shoot regeneration of Lettuce (Lactuca sativa L.). Aust J Crop Sci 5: 92-95

240

Mok MC, Mok DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T (1982). Cytokinin activity of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron). Phytochem 21: 1509-1511

Morel G (1965). Clonal propagation of orchids by meristem culture. Cymbidium Soc News 20: 3-11

Morel G, Martin C (1952). Guerison de dahlias atteints d’une maladie a virus. CR Acad Sci Paris 235: 1324-1325

Morel G, Martin C (1955). Guerison de pommes de terre atteintes de maladies a virus. CR Seancer Acad Agric Fr 41: 472-475

Morton L, Browse J (1991). Facile transformation of Arabidopsis. Plant Cell Rep 10: 235-239

Mosaleeyanon K, Sha-Um S, Kirdmanadee C (2004). Enhanced growth and photosynthesis of rain tree (Samanea saman Merr.) plantlets in vitro under a

CO2-enriched condition with decreased sucrose concentrations in the medium. Sci Hort 103: 51-63

Mroginski E, Rey HY, Gonzale AM, Mroginski LA (2004). Thiadiazuron promotes in vitro plant regeneration of Arachis correntina (Leguminosae) via organogenesis. Plant Growth Reg 23: 129-134

Mullin RH, Smith SH, Frazier NW, Schlegel DE, McCall SR (1974). Meristem culture frees strawberries of mild yellow edge, pallidosis and mottle diseases. Phytopath 64: 1425-1429

Murashige T (1974). Plant propagation through tissue culture. Annu Rev Plant Physiol 25: 135-166

Murashige T (1977). Manipulation of organ culture in plant tissue culture. Botanical Bull Acad Sinica 18: 1-24

Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473-497

241

Murch SJ, Choffe KL, Victor JMR, Slimmon TY, Krishna Raj S, Saxena PK (2000). Thidiazuron-induced plant regeneration from hypocotyl cultures of St. Johns wort (Hypericum perforatum). Plant Cell Rep 19: 576-581

Murch SJ, Saxena PK (1997). Modulation of mineral and fatty acid profiles during thidiazuron mediated somatic embryogenesis in peanuts (Arachis hypogaea L.). J Plant Physiol 151: 358-361

Murch SJ, Saxena PK (2001). Somatic cell fusion: relevance to medicinal plants. In: Saxena PK (ed). Development of Plant based Medicines: Conservation, Efficacy and Safety, Kluwer Academic Publishers, Dordrecht, Netherlands pp 167-182

Murthy BNS, Murch SJ, Saxena PK (1995). Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogea): Endogenous growth regulator levels and significance of cotyledons. Physiol Plant 94: 268-276

Murthy BNS, Murch SJ, Saxena PK (1998). Thidiazuron: A potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol-Plant 34: 267-275

Murthy BNS, Saxena PK (1994). Somatic embryogenesis in peanut (Arachis hypogaea L.): stimulation of direct differentiation of somatic embryos by forchlorfenuron (CPPU). Plant Cell Rep 14: 145-150

Murthy BNS, Victor J, Singh RPS, Fletcher RA, Saxena PK (1996). In vitro regeneration of chickpea (Cicer arietinum L.) stimulation of direct organogenesis and somatic embryogenesis by thidiazuron. Plant Growth Reg 19: 233-240

Muthu C, Ayyanar M, Raja Nagappan, Ignacimuthu S (2006). Medicinal plants used by traditional healers in Kancheepuram district of Tamil Nadu, India. J Ethnobiol Ethnomed 2: 43-52

Nabi SA, Rashid MM, Al-Amin M, Rasul MG (2002). Organogenesis in teasle gourd (Momordica dioica Roxb.). Plant Tiss Cult 12: 173-180

Naik PM, Manohar SH, Praveen N, Murthy HN (2010). Effects of sucrose and pH levels on in vitro shoot regeneration from leaf explants of Bacopa monniera and

242 accumulation of abacoside A in regenerated shoots. Plant Cell Tiss Organ Cult 100: 235-239

Naik PM, Murthy HN (2010). Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.). Acta Physiol Plant 32: 75-79

Naik SK, Chand PK (2006). Nutrient alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange. Sci Hort 108: 247-252

Nair LG, Seeni S (2001). Rapid in vitro multiplication and restoration of Celastrus paniculatus (Celastraceae), a medicinal woody climber. Indian J Exp Biol 39: 697-704

Nair LG, Seeni S (2003). In vitro multiplication of Calophyllum apitalum (Clusiaceae) an endemic medicinal tree of the Western Ghats. Plant Cell Tiss Organ Cult 75: 169-174

Nakashimada Y, Uozumi N, Kobayashi T (1995). Production of plantlets for use as artificial seeds from horseradish hairy roots fragmented in a blender. J Ferment Bioeng 79: 458-464

Nalawade SM, Tsay HS (2004). In vitro propagation of some important Chinese medicinal plants and their sustainable usage. In Vitro Cell Dev Biol-Plant 40: 143-154

Nandwani D, Ramawat KG (1993). In vitro plantlets formation through juvenile and mature explants in Prosopis cineraria. Indian J Exp Biol 31: 156-160

Nassar AH (2003). Slow growth storage of encapsulated germplasm of Coffea arabica L. Int J Agric Biol 5: 517-520

Navarrete NE, Van Sambeek JW, Preece JE, Gaffney GR (1989). Improved micropropagation of white ash (Fraxinus Americana L.). Proc 7th Central Hardwood Forest Conference, Carbondale, IL pp 146-149

243

Nayak P, Behera PR, Manikkannan T (2007). High frequency plantlet regeneration from cotyledonary node cultures of Aegle marmelos (L.) Corr. In Vitro Cell Dev BIol-Plant 43: 231-236

Nef-Campa C, Chaintreuil-Dongmo C, Dreyfus BL (1996). Regeneration of the tropical legume Aeschynomene sensitiva Sw. from root explants. Plant Cell Tiss Organ Cult 4: 149-154

Negi RS, Sharma KC, Sharma M (2011). Micropropagation and anatomical comparision of in vivo and in vitro developed shoot and root in Cassia auriculata L. - a medicinally important plant. Indian J Fund App Life Sci 1: 21-29

Neuman MC, Preece JE, Sambeek JW, Gaffiney GR (1993). Somatic embryogenesis and callus production from cotyledon explants of eastern black walnut (Juglans nigra L.). Plant Cell Tiss Organ Cult 32: 9-18

Nikam TD, Bansude GM, Kumar KCA (2003). Somatic embryogenesis in sisal (Agave sisalana Perr. Ex. Engelm). Plant Cell Rep 22: 188-194

Nikam TD, Savant RS (2009). Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb. Physiol Mol Biol Plants 15: 71-78

Nikam TD, Savant RS, Parage RS (2008). Micropropagation of Ceropegia hirsuta Wt. and Arn. - a starchy tuberous asclepiad. Indian J Biotechnol 7: 129- 132

Nirmal Babu K, Sajina A, Minoo D, John CZ, Mini PM, Tushar KV, Rema J, Ravindran PN (2003). Micropropagation of camphor tree (Cinnamomum camphora). Plant Cell Tiss Organ Cult 74: 179-183

Nissen SJ, Sutter EG (1990). Stability of IAA and IBA in nutrient medium of several tissue culture procedures. HortScience 25: 800-802

Nitsch C, Nitsch JP (1967). The induction of flowering in vitro in stem segments of Plumbago indica L. the production of vegetative buds. Planta 72: 355-370

244

Nitsch JP, Nitsch C (1969). Haploid plants from pollen grains. Science 163: 85- 87

Nobécourt P (1939). Sur la pérennité et l’augmentation de volume des cultures de tissues végétaux. C R Seances Soc Biol Ses Fil 130: 1270-1271

Nolan KE, Rose RJ, Gorst JR (1989). Regeneration of Medicago trucatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants. Plant Cell Rep 8: 278-281

Nomura K, Komamine A (1995). Physiological and Biochemical aspects of somatic embryogenesis. In: Thorpe TA (ed). In vitro embryogenesis in palnts, Kluwer, Dordrecht, pp 249-266

Nonohay JS, Mariath JEA, Winge H (1999). Histological analysis of somatic embryogenesis in Brazilian cultivars of barley, Hordeum vulgare vulgare, Poaceae. Plant Cell Rep 18:929-934

Nowak J, Shulaev V (2003). Priming for transplant stress resistance in in vitro propagation. In Vitro Cell Dev Biol-Plant 39: 107-124

Nyende AB, Schittenhelm S, Wagner GM, Greef JM (2003). Production, storability, and regeneration of shoot tips of potato (Solanum tuberosum L.) encapsulated in calcium alginate hollow beads. In Vitro Cell Dev Biol-Plant 39: 540-544

Ochatt SJ, Conreux C, Jacas L (2010). In vitro production of sweet peas (Lathyrus odoratus L.) via axillary shoots. Methods Mol Biol 589: 293-301

Olmos SE, Lavia G, Di Renzo M, Mroginski I, Echenique V (2002). Genetic analysis of variation in micropropagated plants of Melia azedarach L. In Vitro Cell Dev Biol-Plant 38: 617-622

Onay A, Jefree CE, Yeoman MM (1996). Plant regeneration from encapsulated embryoids and an embryonic mass of pistachio, Pistachia vera L. Plant Cell Rep 15: 723-726

245

Oostermeijer JGB, Luijten SH, den Nijs JCM (2003). Integrating demographic and genetic approaches in plant conservation. Biol Conserv 113: 389-398

Ostrolucká MG, Libiaková G, Ondrušková E, Gajdošová A (2004). In vitro propagation of Vaccinium species. Acta Univ Latv 676: 207-212

Padmaja G, Reddy LR, Reddy GM (1995). Plant regeneration from synthetic seeds of groundnut, Arachis hypogea L. Indian J Exp Biol 33: 967-971

Palai SK, Rout GR, Das P (1997). Micropropagation of ginger (Zingiber officinale Rosc.): interaction of growth regulators and culture conditions. Proc Biotechnol Spices, Med Arom Plants Kerala, India pp 20-24

Pandey H, Chandra B, Nadeem M, Kumar A, Nandi SK, Palni LMS (2002). In vitro propagation of some alpine medicinal herbs of the Himalayan region. In: Nandi SK, Palni LMS, Kumar A (eds). Role of plant tissue culture in biodiversity conservation and economic development, Gyanodaya Prakashan, Nainital, India pp 297-323

Pandey V, Agrawal V (2009). Efficient micropropagation protocol of Spilanthes acmella L. possessing strong antimalarial activities. In Vitro Cell Dev Biol-Plant 45: 491-499

Pandeya K, Tiwari KN, Singh J, Verma JP, Dubey SD (2010). In vitro propagation of Clitoria ternatea L.: A rare medicinal plant. J Med Plants Res 4: 664-668

Park SY, Ahn JK, Lee WY, Murthy HN, Paek KY (2005). Mass production of Eleutherococcus koreanum plantlets via somatic embryogenesis from root cultures and accumulation of eleutherosides in regenerants. Plant Sci 168: 1221- 1225

Parrott WA, Bailey MA, Durham RE, Mathews HV (1992). Tissue culture and regeneration of legumes In: Moss JP (ed). In proc Int Workshop on Biotechnology and Crop Improvement in Asia, ICRISAT, India, Hyderabad pp 115-148

246

Parrott WA, Merkle SA, Williams EG (1991). Somatic embryogenesis: potential for use in propagation and gemne transfer systems. In: Murray DR (ed). Advanced Methods in Plant Breeding and Biotechnology Wallingford, CAB International pp 158-200

Parveen S, Shahzad A (2010). TDZ-induced high frequency shoot regeneration in Cassia sophera Linn. via cotyledonary node explants. Physiol Mol Biol Plant 16: 201-206

Parveen S, Shahzad A (2011). A micropropagation protocol for Cassia angustifolia Vahl. from root explants. Acta Physiol Plant 33: 789-796

Parveen S, Shahzad A (2012). In vitro propagation of medicinally important Cassia species - a review. J Funct Environ Bot 2: 1-11

Parveen S, Shahzad A, Anis M (2012). Enhanced shoot organogenesis in Cassia angustifolia Vahl. - a difficult-to-root drought resistant medicinal shrub. J Plant Biochem Biotechnol 21: 213-219

Parveen S, Shahzad A, Saema S (2010). In vitro plant regeneration system for Cassia siamea Lam., a leguminous tree of economic importance. Agroforest Syst 80: 109-116

Paterson KE, Everett NP (1985). Regeneration of Helianthus annuus inbred plants from callus. Plant Sci 42: 125-132

Patil VM (1998). Micropropagation studies in Ceropegia spp. In Vitro Cell Dev Biol-Plant 30: 240-243

Patra A, Rai B, Rout GR, Das P (1998). Successful plant regeneration from callus cultures of Centella asiatica (Linn.). Plant Growth Reg 24: 13-16

Pattnaik J, Debata BK (1996). Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture. Plant Cell Rep 15: 427-430

Pattnaik S, Chand PK (2000). Morphogenic response of the alginate encapsulated axillary buds from in vitro shoot cultures of six mulberries. Plant Cell Tiss Organ Cult 60: 177-185

247

Pattnaik SK, Chand PK (1996). In vitro propagation of the medicinal herb Ocimum americanum L. Syn. O. canumsims (Hoary Basil) and Ocimum sanctum (Holy Basil). Plant Cell Rep 15: 846-850

Pattnaik SK, Chand PK, Naik SK, Pradhan C (2000). Shoot organogenesis and plant regeneration from hypocotyl derived cell suspensions of a tree legume, Dalbergia sissoo Roxb. In Vitro Cell Dev Biol-Plant 36: 407-411

Paunescu A (2009). Biotechnology for endangered plant conservation: a critical overview. Rom Biotechnol Lett 14: 4095-4103

Peeters AJM, Gerads W, Barendse GWM, Wullems GJ (1991). In vitro flower bud formation in Tobacco: interaction of hormones. Plant Physiol 97: 402-408

Pei Shengji (2002). Ethnobotany and modernization of Traditional Chinese Medicine. In paper at a workshop on wise practices and experiential learning in the conservation and management of himalayan medicinal plants, Kathmandu, Nepal, 15-20 December 2002, supported by the Ministry of Forest and Soil Conservation, Nepal, the WWF-Nepal Program, Medicinal and Aromatic Plants Program in Asia (MAPPA), IDRC, Canada, and the WWF-UNESCO People and Plants Initiative

Perveen R, Elahi I (1987). Callus formation in Rauvolfia serpentina. Pak J Bot 10: 141-148

Perveen S, Varshney A, Anis M, Aref IM (2011). Influence of cytokinins, basal media and pH on adventitious shoot regeneration from excised root cultures of Albizia lebbeck. J For Res 22: 47-52

Pesce PG, Rugini E (2004). Influence of plant growth regulators, carbon sources and iron on the cyclic secondary somatic embryogenesis and plant regeneration of transgenic cherry rootstock ‘Colt’ (Prunus avium x P. pseudocerasus). Plant Cell Tiss Organ Cult 79: 223-232

Peshke VN, Phillips RL (1992). Genetic implications of somaclonal variation in plants. Adv Genet 30: 41-75

248

Peternal S, Gabrovsek K, Gogala N, Regvar M (2009). In vitro propagation of European aspen (Populus tremula L.) from axillary buds via organogenesis. Sci Hort 121: 109-112

Phillips GC, Collins GB (1979). In vitro tissue culture of selected legumes and plant regeneration from callus of red clover. Crop Sci 19: 59-64

Phulwaria M, Rai MK, Harish, Gupta AK, Ram K, Shekhawat NS (2012). An improved micropropagation of Terminalia bellirica from nodal explants of mature tree. Acta Physiol Plant 34: 299-305

Piatczaka E, Wielanekb M, Wysokinskaa H (2005). Liquid culture system for shoot multiplication and secoirdoid production in micropropagated plants of Centaurium erythraea Rafn. Plant Sci 168: 431-437

Piccioni E (1997). Plantlets from encapsulated micropropagated buds of M.26 apple rootstock. Plant Cell Tiss Organ Cult 47: 255-260

Piccioni E, Standardi A (1995). Encapsulation of micropropagated buds of six woody species. Plant Cell Tiss Organ Cult 42: 221-226

Pintos B, Bueno MA, Cuenca B, Manzanera JA (2008). Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring. Plant Cell Tiss Organ Cult 95: 217-225

Pospíšilová J, Solárová J, Čatský J (1992). Photosynthetic responses to stresses during in vitro cultivation. Photosynthetica 26: 3-18

Pospíšilová J, Synková H, Haisael D, Baťková P (2009). Effect of abscisic acid on photosynthetic parameters during ex vitro transfer of micropropagated tobacco plantlets. Biol Plant 53: 11-20

Pospíšilová J, Tichá I, Kadleček P, Haisel D, Plzáková S (1999). Acclimatization of micropropagated plants to ex vitro conditions. Biol Plant 42: 481-497

Pospíšilová J, Wilhelmová N, Synková H, Čatský J, Krebs D, Tichá I, Hanáčková B, Snopek J (1998). Acclimation of tobacco plantlets to ex vitro conditions as affected by application of abscisic acid. J Exp Bot 49: 863-869

249

Pradel H, Dumkelehmann U, Diettrich B, Luckner M (1997). Hairy root cultures of Digitalis lanata. Secondary metabolism and plant regeneration. J Plant Physiol 151: 209-215

Pradhan C, Kar S, Pattnaik S, Chand PK (1998a). Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes. Plant Cell Rep 18: 122-126

Pradhan C, Pattnaik SN, Chand PK, Pattnaik S, Dwari M (1998b). Efficient plant regeneration from cell suspension derived callus of East Indian rosewood (Dalbergia latifolia Roxb.). Plant Cell Rep 18: 138-142

Prakash E, Khan PSSV, Rao TJVS, Meru ES (2006). Micropropagation of red sanders (Pterocarpus santalinus L.) using mature nodal explants. J For Res 11: 329-335

Prakash MG, Gurumurthi K (2010). Effects of type of explant and age, plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in Eucalyptus camaldulensis. Plant Cell Tiss Organ Cult 100: 13-20

Prathanturarug S, Soonthornchareonnon N, Chuakul W, Phaidee Y, Saralamp P (2005). Rapid micropropagation of Curcuma longa using bud explants precultured in thiadizuron supplemented liquid medium. Plant Cell Tiss Organ Cult 80: 347-351

Preece JE, Bates S (1990). Source tree affects adventitious regeneration of white ash. Abstracts of the 23rd Intl Hort Congr. Firenze, Italy (p 4072) (Abstr)

Preece JE, Huetteman CA, Puello CH, Neuman MC (1987). The influence of thidiazuron on in vitro culture of woody plants. Hort Sci 22: 1071 (Abstr)

Preece JE, Sutter EJ (1991). Acclimatization of micropropagated plants to the greenhouse and field. In: Debergh PC, Zimmerman RH (ed): Micropropagation: Technology and Application. Kluwer Academic Publishers, Dordrecht, Boston, London pp 71-93

250

PremAnand R, Ganapathi A, Anbazhagan VR, Vengadesan G, Selvaraj N (2000). High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp. In Vitro Cell Dev Biol-Plant 36: 475-480

Prewein C, Wilhelm E (2003). Plant regeneration fom encapsulated somatic embryos of pedunculate oak (Quercus robur L.). In Vitro Cell Dev Biol-Plant 39: 613-617

Pruski KW, Lewis T, Astatkie T, Nowak J (2000). Micropropagation of Chokecherry and Pincherry cultivars. Plant Cell Tiss Organ Cult 63: 93-100

Purohit SD, Dave A (1996). Micropropagation of Sterculia urens Roxb. - an endangered tree species. Plant Cell Rep 15: 704-706

Purohit SD, Singhvi A, Nagori R (2004). In vitro shoot bud differentiation from leaf segments of Achras sapota. Biol Plant 48: 109-112

Purohit VK, Palni LMS, Nandi SK, Vyas P, Tamta S (2002). Somatic embryogenesis and regeneration in Quercus floribunda, an important central Himalyan oak. In: Nandi SK, Palni LMS, Kumar A (eds). Role of plant tissue culture in biodiversity conservation and economic development, Gyanodaya Prakashan, Nainital, India pp 41-52

Purohit VK, Palni S, Nandi SK, Rihani C (2002). In vitro regeneration of Quercus floribunda Lindl. through cotyledory nodes: an important tree of central Himalaya. Curr Sci 83: 312-316

Pyati AN, Murthy HN, Hahn EJ, Paek KY (2002). In vitro propagation of Dendrobium macrostachium Lindl, a threatened orchid. Indian J Exp Biol 40: 620-623

Quiroz-Figueroa FR, Fuentes-Cerda CFJ, Rojas-Herrera R, Loyola-Vargas VM (2002). Histological studies on the developmental stages and differentiation of two different somatic embryogenesis systems of Coffea arabica. Plant Cell Rep 20: 1141-1149

251

Quiroz-Figueroa FR, Rojas-Herrera R, Galaz-Avalos RM, Loyola-Vargas VM (2006). Embryo production through somatic embryogenesis can be used to study cell differentiation in plants. Plant Cell Tiss Organ Cult 86: 285-301

Quraishi A, Jadhav SK, Gupta S (2011). In vitro clonal propagation of Cassia tora L. (Coffee Pod): a medicinal plant. Biotechnol 10: 546-550

Raghavan V (1997). Somatic embryogenesis. In: Raghavan V (ed). Molecular Embryology of Flowering Plants, Cambridge University Press, Cambridge, New York pp 467-499

Raghu AV, Geetha SP, Martin G, Balachandran I, Ravindran PN (2006a). In vitro clonal propagation through mature nodes of Tinospora cordifolia (Willd.) Hook. F. & Thoms.: An important ayurvedic medicinal plant. In Vitro Cell Dev Biol-Plant 42: 584-588

Raghu AV, Geetha SP, Martin G, Balachandran I, Ravindran PN (2006b). Direct shoot organogenesis from leaf explants of Embelia ribes Burm. F.: a vulnerable medicinal plant. J For Res 11: 57-60

Raghu AV, Geetha SP, Martin G, Balachandran I, Ravindran PN, Mohanan KV (2007). An improved micropropagation protocol for Bael - a vulnerable medicinal tree. Res J Bot 2: 186-194

Raha S, Roy SC (2001). In vitro plant regeneration in Holarrhena antidysenterica Wall., through high frequency axillary shoot proliferation. In Vitro Cell Dev Biol- Plant 37: 232-236

Raha S, Roy SC (2003). Efficient plant regeneration in Holarrhena antidysenterica Wall. from shoot segment derived callus. In Vitro Cell Dev Biol- Plant 39: 151-155

Rahman A, Rahman MM, Sheik MMI, Rahman MM, Shadli SM, Alam MF (2008). Free radical scavenging activity and phenolic content of Cassia sophera L. Afr J Biotechnol 7: 1591-1593

252

Rai MK, Asthana P, Singh SK, Jaiswal VS, Jaiswal U (2009). The encapsulation technology in fruit plants - a review. Biotechnol Adv 27: 671-679

Rai MK, Jaiswal VS, Jaiswal U (2008). Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange. Sci Hort 118: 33-38

Rai VR, McComb J (2002). Direct somatic embryogenesis from mature embryos of sandalwood. Plant Cell Tiss Organ Cult 69: 65-70

Rajeswari V, Paliwal K (2006). In vitro propagation of Albizia odoratissima L.F. (Benth.) from cotyledonary node and leaf nodal explants. In Vitro Cell Dev Biol- Plant 42: 399-404

Rani G, Talwar D, Nagpal A, Virk GS (2006). Micropropagation of Coleus blumei from nodal segments and shoot tips. Biol Plant 50: 496-500

Rani G, Virk GS, Nagpal A (2003). Callus induction and plantlet regeneration in Withania somnifera (L.) Dunal. In Vitro Cell Dev Biol-Plant 39: 468-474

Rani S, Rana JS (2010). In Vitro propagation of Tylophora indica - influence of explanting season, growth regulator synergy, culture passage and planting substrate. J Am Sci 6: 385-392

Rao JVS, Vishnupriya KS (2002). In vitro response of embryo axis of Entada phaseoloides Merrill. Phytomorph 52: 97-102

Rao MM, Lakshmi Sita G (1996). Direct somatic embryogenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.). Plant Cell Rep 15: 355-359

Rao MS, Purohit SD (2006). In vitro shoot bud differentiation and plantlet regeneration in Celastrus paniculatus Willd. Biol Plant 50: 501-506

Rao PS, Bapat VA (1993). Micropropagation of sandalwood (Santalum album L.) and mulberry (Morus indica L.). In: Ahuja MR (ed). Micropropagation of woody plants, Kluwer Dordrecht pp 317-345

253

Rashid MH, Khalekuzzaman M, Hasan MF, Das R, Hossain MS, E Khoda SM (2009). Establishment of an efficient method for micropropagation of an important medicinal herb (Scoparia dulcis L.) from shoot tips and nodal segments. Int J Sustain Crop Prod 4: 5-9

Rashmi M, Nanda, Rout GR (2003). In vitro somatic embryogenesis and plant regeneration in Acacia Arabica. Plant Cell Tiss Organ Cult 73: 131-135

Rathore P, Suthar R, Purohit SD (2008). Micropropagation of Terminallia bellerica Roxb. from juvenile explants. Indian J Biotechnol 7: 246-249

Ravishankar GA, Grewal S (1991). Development of media for growth of Dioscorea deltoidea cells and in vitro diogenin production: influence of media constituents and nutrient stress. Biotechnol Lett 13: 125-30

Ray A, Bhattacharyaa S (2008). Storage and plant regeneration from encapsulated shoot tips of Rauvolfia serpentina - an effective way of conservation and mass propagation. S Afr J Bot 74: 776-779

Reddy PS, Gopal GR, Lakshmi Sita G (1998). In vitro multiplication of Gymnema sylvestre R. Br. - an important medicinal plant. Curr Sci 75: 843-845

Reddy PS, Rodrigues R, Rajasekharan R (2001). Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell Tiss Organ Cult 66: 183-188.

Redenbaugh K (1990). Application of artificial seed to tropical crops. HortScience 25: 251-255

Redenbaugh K (1993). Introduction In: Redenbaugh K (ed). Synthetic Seeds: Application of Synthetic Seeds to Crop Improvement. CRC Press, Boca Raton, Fal, pp 3-10

Redenbaugh K, Slade D, Viss P, Fuji J (1987). Encapsulation of somatic embryos in synthetic seed coats. HortScience 22: 803-809

Reinert J (1958). Morphogenese und ihre Kontrolle an Gewebekulturen aus Carotten. Naturwiss 45: 344-345

254

Reinert J (1959). Über die kontrolle der morphogenese und die induktion von adventivembryonen in gewebekulturen aus karotten. Planta 53: 318-333

Reisch B, Bingham ET (1980). The genetic control of bud formation from callus cultures of diploid alfalfa. Plant Sci Lett 20: 71-77

Reuther G (1991). Stimulation of the photoautotrophy of in vitro plants. Acta Hortic 300: 59-75

Ricci AP, Filho FAM, Januzzi BM, Piedade SMS (2002). Somatic embryogenesis in Citrus sinensis, C. reticulata and C. nobilis x C. deliciosa. Sci Agric 59: 41-46

Risser PG, White PR (1964). Nutritional requirement of spruce tumor cells in vitro. Physiol Plant 17: 620-635

Rival A, Beulé T, Lavergne D, Nato A, Havaux M, Puard M (1997). Development of photosynthetic characteristics in oil palm during in vitro micropropagation. J Plant Physiol 150: 520-527

Rizvi SMH, Singh RP (2000). In vitro plant regeneration from immature leaflet- derived callus cultures of Cicer arietinum L. via organogenesis. Plant Cell Biotechnol Mol Biol 1: 109-114

Robbins WJ (1922). Cultivation of excised root tips and stem tips under sterile conditions. Bot Gaz (Chicago) 73: 376-390

Robbins WJ, Maneval WE (1924). Effect of light on growth of excised root tip under sterile conditions. Bot Gaz 78: 424-432

Roja G, Benjamin BD, Heble MR, Patankar AV, Sipahimalani AT (1990). The effect of plant growth regulators and nutrient conditions on growth and alkaloid production in multiple shoot cultures of Rauwolfia serpentina. Phyto Res 4: 49-52

Romano A, Barros S, Martins-Loucáo MA (2002). Micropropagation of the Mediterranean tree Ceratonia siliqua. Plant Cell Tiss Organ Cult 68: 35-41

Romano A, Noronha C, Martins-Loucao MA (1995). Role of carbohydrate in micropropagation of Cork oak. Plant Cell Tiss Organ Cult 40: 159-167

255

Rout GR (2005a). Micropropagation of Clitoria ternatea Linn. (Fabaceae) - an important medicinal plant. In Vitro Cell Dev Biol-Plant 41: 516-519

Rout GR (2005b). Direct plant regeneration of curry leaf tree (Murraya koenigii), an aromatic plant. In Vitro Cell Dev Biol-Plant 41: 133-136

Rout GR, Mallick UC, Das P (1992). In vitro plant regeneration from leaf callus of Cephaelis ipecacuanha A. Richard. Adv Plant Sci 5: 608-613

Rout GR, Samantaray S (1995). Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu - a multipurpose leguminous tree. Plant Cell Tiss Organ Cult 42: 283-285

Rout GR, Samantaray S, Das P (2000). In vitro manipulation and propagation of medicinal plants. Biotechnol Adv 18: 91-120

Rout GR, Saxena C, Samantaray S, Das P (1999a). Rapid clonal propagation of Plumbago zeylanica Linn. Plant Growth Reg 28: 1-4

Rout GR, Saxena C, Samantaray S, Das P (1999b). Rapid plant regeneration from callus cultures of Plumbago zeylanica. Plant Cell Tiss Organ Cult 56: 47-51

Roy A, Ghosh S, Chaudhuri M, Saha PK (2008). Effect of different plant hormones on callus induction in Gymnema sylvestre R. Br. (Asclepiadaceae). Afr J Biotechnol 7: 2209-2211

Roy AT, Koutolis A, De DN (2000). Cell susupension culture and plant regeneration in the latex producing plant, Calotropis gigantea (Linn.) R. Br. Plant Cell Tiss Organ Cult 63: 15-22

Roy PK, Barat GK, Mehta SL (1992). In vitro plant regeneration from callus derived from root explants of Lathyrus sativus. Plant Cell Tiss Organ Cult 29: 135-138

Roy PK, Singh B, Mehta SL, Barat SK, Gupta N, Kirti PB, Chopra VL (1991). Plant regeneration from leaf disc of Lathyrus sativus. Indian J Exp Biol 29: 327- 330

256

Roy SK, Hossain MZ, Islam MS (1994). Mass propagration of Rauvolfia serpentina by in vitro shoot tip culture. Plant Tiss Cult 4: 69-75

Saafi H, Borthakur D (2002). In vitro plantlet regeneration from cotyledons of tree legume Leucaena leucocephala. Plant Growth Reg 38: 279-285

Saborio F, Dvorak WS, Donahue JK, Thorpe TA (1997). In vitro regeneration of plantlets from mature embryos of Pinus ayacahuite. Tree Physiol 17: 787-796

Sachs T, Thimann KV (1964). Release of lateral buds from apical dominance. Nature 201: 939-940

Sagare AP, Suhasini K, Krishnamurthy KV (1995). Histology of somatic embryo initiation and development in chickpea (Cicer arietenum L). Plant Sci 109: 87-93

Sahai A, Shahzad A (2010). In vitro clonal propogation of Coleus forskohlii via direct shoot organogenesis from selected leaf explants. J Plant Biochem Biotechnol 19: 223-228

Sahai A, Shahzad A, Anis M (2010a). High frequency plant production via shoot organogenesis and somatic embryogenesis from callus in Tylophora indica, an endangered plant species. Turk J Bot 34: 11-20

Sahai A, Shahzad A, Sharma S (2010b). Histology of organogenesis and somatic embryogenesis in excised root cultures of an endangered species Tylophora indica (Asclepiadaceae). Aust J Bot 58: 198-205

Sahoo Y, Chand PK (1998a). Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high frequency axillary shoot proliferation. Plant Cell Rep 18: 301-307

Sahoo Y, Chand PK (1998b). In vitro multiplication of a medicinal herb Tridax procumbens: Influence of explanting season, growth regulator synergy, culture passage and planting substrate. Phytomorph 48: 195-205

Sahoo Y, Pattnaik SK, Chand PK (1997). In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (Sweet basil) by axillary shoot proliferation. In Vitro Cell Dev Biol-Plant 33: 293-296

257

Sahrawat AK, Chand S (2002). Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant. In Vitro Cell Dev Biol-Plant 38: 33-38

Saiprasad GVS, Polisetty R (2003). Propagation of three orchid genera using encapsulated protocorm like bodies. In Vitro Cell Dev Biol-Plant 39: 42-48

Samake G, Folega F, Senou H, Wang H (2011). In vitro regeneration of Acacia nilotica (L.) Willd. ex Del from nodes on B5 medium. J Agric Biotechnol Sust Dev 3: 85-89

Samantaray S, Rout GR, Das P (1997). Regeneration of plant via somatic embryogenesis from leaf base and leaf tip segments of Echinochloa colona. Plant Cell Tiss Organ Cult 47: 119-125

Samy RP, Gopalakrishnakone P (2007). Current status of herbal and their future perspectives. Nature Preceedings: hdl:10101/npre.2007.1176.1: Posted 28 Sep 2007

Sanavy SAMM, Moeini JM (2003). Effects of different pH levels of medium on growth and rooting of single nodes resulted from potato meristem culture. Plant Tiss Cult 13: 151-154

Sánchez N, Manzanera JA, Pintos B, Bueno MA (2005). Agrobacterium - mediated transformation of cork oak (Quercus suber L.) somatic embryos. New For 29: 169-176

Sankhla D, Davis TD, Sankhla N (1994). Thidiazuron induced in vitro shoot formation from roots of intact seedlings of Albizzia julibrissin. Plant Growth Reg 14: 267-272

Sankhla D, Sankhla N, Davis TD (1995). Promotion of in vitro shoot formation from excised roots of silk tree Albizzia julibrissin by an oxime ether derivative and other ethylene inhibitors. Plant Cell Rep 15: 143-146

258

Sarasan V, Soniya EV, Nair GM (1994). Regeneration of Indian sarsaparillas, Hemidesmus indiculs R. Br. through organogenesis and somatic embryogenesis. Indian J Exp Biol 32: 284-287

Saravitz CH, Blazich FA, Amerson HV (1993). Histology of in vitro adventitious bud development on cotyledons and hypocotyls of Fraser fir. J Am Soc Hort Sci 118: 163-167

Sarmah DK, Borthakur M, Borua PK (2010). Artificial seed production from encapsulated PLBs regenerated from leaf base of Vanda coerulea Grifft. ex. Lindl. - an endangered orchid. Curr Sci 98: 686-690

Satheeshkumar K, Seeni S (2000). In vitro multiplication of Nothapodites foetida (Wight.) Sleumer through seedling explant culture. Indian J Exp Biol 38: 273-277

Sathyanarayana N, Kumar TNB, Vikas PB, Rajesha R (2008). In vitro clonal propagation of Mucuna pruriens var. utilis and its evaluation of genetic stability through RAPD markers. Afr J Biotechnol 7: 973-980

Saunders JW, Bingham ET (1972). Production of alfalfa plants from callus tissue. Crop Sci 12: 804-808

Savita, Vijay, Virk GS, Nagpal A (2010). Effect of explant type and different plant growth regulators on callus induction and plantlet regeneration in Citrus jambhiri Lush. Environ We Int J Sci Tech 5: 97-106

Saxena C, Palai SK, Samantaray S, Rout GR, Das P (1997). Plant regeneration from callus cultures of Psoralea corylifolia Linn. Plant Growth Reg 22: 13-17

Saxena C, Rout GR, Das P (1998). Micropropagation of Psoralea corylifolia Linn. J Med Arom Plant Sci 20: 15-18

Saxena G, Banerjee S, Rahman L, Mallavarapu GR, Sharma S, Kumar S (2000). An efficient in vitro procedure for micropropagation and generation of somaclones of rose scented Pelargonium. Plant Sci 155: 133-140

Saxena PK, Malik KS, Gill R (1992). Induction by thiadiazuron of somatic embryogenesis in intact seedlings of peanut. Planta 187: 421-424

259

Saxena S, Dhawan B (1999). Regeneration and large scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis. Plant Cell Rep 18: 438-443

Schenk RV, Hildebrandt AC (1972). Medium and techniques for induction of growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50: 166-204

Schleiden MJ (1838). Beiträge zur Phytogenesis. Arch Anat Physiol Wiss Med (J Müller) 1838: 137-176

Schmidt EDL, Guzzo F, Toonen MAJ, de Vries SC (1997). A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 124: 2049-2062

Schmülling T (2004). Cytokinin. In: LennarzW, Lane MD (eds). Encyclopedia of Biological Chemistry, Academic Press, Elsevier Science pp 1-7

Schwann T (1839). Mikroscopische Untersuchungen über die Ubereinstimmung in der Struktur und dem Wachstum des Tiere und Pflanzen. W Engelmann: Leipzig No 176

Seetharam YN, Rajanna LN, Jyothishwaran G, Aravind B, Sharanabasappa G, Mallikharjun PB (2007). In vitro shoot regeneration from leaf and nodal explants of Vernonia ceneria L. Less. Indian J Biotechnol 6: 418-420

Sehgal CB, Abbas NS (1994). Somatic embryogenesis and plant regeneration from hypocotyl tissue of Trachyspermum ammi (L.) Sprague. Phytomorph 44: 265-271

Sen J, Sharma AK (1991). Microprpagation of Withania somnifera from germinating seeds and shoot tips. Plant Cell Tiss Organ Cult 26: 71-73

Senaratna T, Saxena PK, Rao MV, John A (1995). Significance of the zygotic seed coat on quiscensce and desiccation tolerance of Medicago sativa L. somatic embryos. Plant Cell Rep 14: 375-379

260

Shahzad A, Ahmad N, Anis M (2006). An improved method of organogenesis from cotyledon callus of Acacia sinuata (Lour.) Merr. using thidiazuron. J Plant Biotechnol 8: 15-19

Shahzad A, Ahmad N, Rather MA, Husain MK, Anis M (2009). Improved regeneration system through leaf derived callus and nodule culture of Sansevieria cylindrica. Biol Plant 53: 745-749

Shahzad A, Faisal M, Ahmad N, Anis M, Alatar A, Hend AA (2012). An efficient system for in vitro multiplication of Ocimum basilicum through node culture. Afr J Biotechnol 11: 6055-6059

Shahzad A, Faisal M, Anis M (2007). Micropropagation through excised root culture of Clitoria ternatea L. and comparison between in vitro regenerated plants and seedling. Ann App Biol 150: 341-349

Shahzad A, Hasan H, Siddiqui SA (1999). Callus induction and regeneration in Solanum nigrum L. in vitro. Phytomorph 49: 215-220

Shahzad A, Parveen S, Fatema M (2011). Development of a regeneration system via nodal segment culture in Veronica anagallis-aquatica L. - an amphibious medicinal plant. J Plant Interact 6: 61-68

Shahzad A, Siddiqui SA (2000). In vitro organogenesis in Ocimum sanctum, a multipurpose herb. Phytomorph 50: 27-35

Shaik NM, Arha M, Nookaraju A, Gupta SK, Srivastava S, Yadav AK, Kulkarni PS, Abhilash OU, Vishwakarma RK, Singh S, Tatkare R, Chinnathambi K, Rawal SK, Khan BM (2009). Improved method of in vitro regeneration in Leucaena leucocephala - a leguminous pulpwood tree species. Physiol Mol Biol Plants 15: 311-318

Sharma A, Meena MC, Patni V, Arora DK (2007). In vitro multilplication of Acalypha wilkesiana dwarf through nodal segment culture. J Plant Sci Res 23: 79-82

261

Sharma K, Yeung EC, Thorpe TA (1993). Histology of shoot bud ontogeny from seedling root segments of Brassica napus L. Ann Botany 71: 461-466

Sharma N, Chandel KPS (1992). Effect of ascorbic acid on axillary shoot induction in Tylophora indica (Burmf.). Merrill. Plant Cell Tiss Organ Cult 29: 109-113

Sharma RK, Wakhlu AK (2003). Regeneration of Heracleum candicans Wall. plants from callus cultures through organogenesis. J Plant Biochem Biotechnol 12: 71-72

Sharma S, Rathi N, Kamal B, Pundir D, Kaur B, Arya S (2010). Conservation of biodiversity of highly important medicinal plants of India through tissue culture technology - a review. Agric Biol J N Am 1: 827-833

Sharma S, Shahzad A (2011). High frequency clonal multiplication of Stevia rebaundiana Bertoni, sweetener of the future. J Funct Environ Bot 1: 70-76

Sharma S, Shahzad A (2012). Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn. Plant Cell Tiss Organ Cult DOI 10.1007/s11240-012-0183-0

Sharma S, Shahzad A, Jan N, Sahai A (2009). In vitro studies on shoot regeneration through various explants and alginate-encapsulated nodal segments of Spilanthes mauritiana DC., an endangered medicinal herb. Int J Plant Dev Biol 3: 56-61

Sharma SK, Millam S (2004). Somatic embryogenesis in Solanum tuberosum L.: a histological examination of key developmental stages. Plant Cell Rep 23: 115- 119

Sharp WR, Sondahl MR, Caldas LS, Maraffa SB (1980). The physiology of in vitro asexual embryogenesis. Hortic Rev 2: 268-310

Shasany AK, Khanuja SPS, Dhawan S, Yadav U, Sharma S, Kumar S (1998). High regenerative nature of Mentha arvensis internodes. J Biosci 23: 641-646

262

Sheen J (1990). Metabolic repression of transcription in higher plants. Plant Cell 2: 1027-1038

Shekhawat MS, Shekhawat NS (2011). Micropropagation of Arnebia hispidissima (Lehm) DC. and production of alkannin from callus and cell suspension culture. Acta Physiol Plant 33: 1445-1450

Shekhawat NS, Rathore TS, Singh RP, Deora NS, Rao SR (1993). Factors affecting in vitro clonal propagation of Prosopis cineraria. Plant Growth Reg 12: 273-280

Shepley S, Chen C, Chang JLL (1972). Does gibberellic acid stimulate seed germination via amylase synthesis. Plant Physiol 49: 441-442

Shibli RA, Abu-Ein AM, Ajlouni MM (2001). In vitro and in vivo multiplication of virus-free ‘Spunta’ potato. Pak J Bot 33: 35-41

Shibli RA, Smith MAL (1996). Direct shoot regeneration from Vaccinium pahalae (ohelo) and Vaccinium myrtillus (bilberry) leaf explants. HortScience 31: 1225- 1228

Shirin F, Kumar S, Mishra Y (2000). In vitro plantlet production system for Kaempferia galanga, a rare Indian medicinal herb. Plant Cell Tiss Organ Cult 63: 193-197

Shirin F, Rana PK, Mandal AK (2005). In vitro clonal propagation of Tectona grandis through axillary bud proliferation. J For Res 10: 465-469

Short KC, Roberts AV (1991). Rosa spp. (Roses). In: Bajaj YPS, (ed). In vitro culture, micropropagation and production of secondary products. Biotechnology in Agriculture and Forestry, Vol 15 Medicinal and Aromatic Plants III. Berlin, Springer, Verlag pp 376-397

Short KC, Wardle K, Grout BW, Simpkins I (1984). In vitro physiology and acclimatization of asseptically cultured plantlets. In: Novák FJ, Havel L, Doležel J (ed). Plant Tissue and Cell Culture Application to Crop Improvement, Inst Exp Bot Czechoslov Acad Sci, Prague pp 475-486

263

Shrivastava N, Patel T, Srivastava A (2006). Biosynthetic potential of in vitro grown callus of Cassia senna L. var-senna. Curr Sci 90: 1472-1473

Shrivastava N, Rajani M (1999). Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell. Plant Cell Rep 18: 919-923

Shrivastava V, Khan SA, Banerjee S (2009). An evaluation of genetic fidelity of encapsulated microshoots of the medicinal plant: Cineraria maritime following 6 months of storage. Plant Cell Tiss Organ Cult 29: 623-629

Shyamkumar C, Anjaneyulu, Giri CC (2003/4). Multiple shoot induction from cotyledonary node explants of Terminalia chebula. Biol Plant 47: 585-588

Siddique I, Anis M (2006). Thidiazuron induced high frequency shoot formation and plant regeneration from cotyledonary node explants of Capsicum annuum L. Indian J Biotechnol 5: 303-308

Siddique I, Anis M (2007a). In vitro shoot multiplication and plantlet regeneration from nodal explants of Cassia angustifolia (Vahl.): a medicinal plant. Acta Physiol Plant 29: 233-238

Siddique I, Anis M (2007b). High frequency multiple shoot regeneration and plantlet formation in Cassia angustifolia (Vahl.) using thidiazuron. Med Arom Plant Sci Biotechnol 2: 282-284

Siddique I, Anis M, Aref IM (2010). In vitro adventitious shoot regeneration via indirect organogenesis from petiole explants of Cassia angustifolia Vahl. - a potential medicinal plant. Appl Biochem Biotechnol 162: 2067-2074

Simões C, Albarello N, Callado CH, de Castro TC, Mansur E (2010). Somatic embryogenesis and plant regeneration from callus cultures of Cleome rosea Vahl. Braz Arch Biol Tech 53: 679-686

Singh AK, Chand S (2003). Somatic embryogenesis and plant regeneration from cotyledon explants of a timber yielding leguminous tree, Dalbergia sissoo Roxb. J Plant Physiol 160: 415-421

264

Singh AK, Sharma M, Varshney R, Agarwal SS, Bansal KC (2006a). Plant regeneration from alginate encapsulated shoot tips of Phyllanthus amarus Schum and Thonn, a medicinally important plant species. In Vitro Cell Dev Biol- Plant 42: 109-113

Singh AK, Varshney R, Sharma M, Agarwal SS, Bansal KC (2006b). Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species. J Plant Physiol 163: 220-223

Singh B, Sharma S, Rani G, Virk GS, Zaidi AA, Nagpal A (2007). In vitro response of encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora). Plant Biotechnol Rep 1: 101-107

Singh HP, Singh RPS, Saxena KP, Singh RK (1993). In vitro bud break in axillary nodal segments of mature trees of Acacia nilotica. Indian J Plant Physiol XXXVI: 21-24

Singh J, Tiwari KN (2010). High frequency in vitro multiplication system for commercial propagation of pharmaceutically important Clitoria ternatea L. - a valuable medicinal plant. Indust Crops Prod 32: 534-538

Singh K, Sudarshana MS, Singh K (2003). In vitro micrpropagation of Baliospermum axillare Blume. Indian J Plant Physiol 8: 125-128

Singh SK, Rai MK, Asthana P, Pandey S, Jaiswal VS, Jaiswal U (2009). Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species. Acta Physiol Plant 31: 649-653

Singh SK, Rai MK, Asthana P, Sahoo L (2010). Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.). Acta Physiol Plant 32: 607-610

Sinha PK, Mallick R (1991). Plantlets from somatic tissue of woody legume Sesbania bispinosa (Jacq.) W.F. Wight. Plant Cell Rep 10: 247-250

265

Sinha RK, Majumdar K, Sinha S (2000). In vitro differentiation and plant regeneration of Albizia chinensis (OBS.) Merr. In Vitro Cell Dev Biol-Plant 36: 370-373

Sivanesan I, Jeong BR (2007). Micropropagation and in vitro flowering in Pentanema indicum Ling. Plant Biotechnol 24: 527-532

Sivanesan I, Jeong BR (2009). Micropropagation of Plumbago zeylanica L. Afr J Biotechnol 8: 3761-3768

Sivaprakash N, Pental D, Sarin NB (1994). Regeneration of pigeon pea from cotyledonary nodes via multiple shoot formation. Plant Cell Rep 13: 623-627

Skoog F, Miller CO (1957). Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11: 118-131

Skoog F, Tsui C (1951). Growth substances and the formation of buds in plant tissues. In: Skoog F (ed). Plant Growth Substances. University of Wisconsin Press, Madison, pp 263-285

Slavtcheva T, Dimitrova V (2001). Gas exchange of in vitro and ex vitro grown grapevine plants. Photosynthetica 39: 29-33

Smith EF, Roberts AV, Mottley J (1990a). The preparation in vitro of Chrysanthemum for transplantation to soil. 2. Improved resistance to desiccation conferred by paclobutrazol. Plant Cell Tiss Organ Cult 21: 133-140

Smith EF, Roberts AV, Mottley J (1990b). The preparation in vitro of Chrysanthemum for transplantation to soil. 3. Improved resistance to desiccation conferred by reduced humidity. Plant Cell Tiss Organ Cult 21: 141-145

Smith EF, Roberts AV, Mottley J, Denness S (1991). The preparation in vitro of Chrysanthemum for transplantation to soil. 4. The effect of eleven growth retardants on wilting. Plant Cell Tiss Organ Cult 27: 309-313

Soneji JR, Rao PS, Mhatre M (2002). Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.). Plant Cell Rep 20: 891-894

266

Sonia, Jaiwal PK, Gulati A, Dahiya S (1998). Direct organogenesis in hypocotyls cultures of Tamarindus indica. Biol Plant 41: 331-337

Soniya EV, Das MR (2002). In vitro micropropagation of Piper longum - an important medicinal plant. Plant Cell Tiss Organ Cult 70: 325-327

Sood H, Chauhan RS (2009). High frequency callus induction and plantlet regeneration from different explants of Picrorhiza kurroa - a medicinal herb of Himalayas. Afr J Biotechnol 8: 1965-1972

Sopher CR, Krol M, Huner NPA, Moore AE, Fletcher RA (1999). Chloroplastic changes associated with paclobutrazol induced stress protection in maize seedlings. Can J Bot 7: 279-290

Sreekumar S, Seeni S, Pushpangadan P (2000). Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy-4-methyl benzaldehyde. Plant Cell Tiss Organ Cult 62: 211-218

Sridhar TM, Naidu CV (2011). Effect of different carbon sources on in vitro shoot regeneration of Solanum nigrum (Linn.) - an important antiulcer medicinal plant. J Phytol 3: 78-82

Srivastava N, Rajani M (1999). Multiple shoot regeneration and tissue culture studies on Bacopa monneiri L. Pennell. Plant Cell Rep 18: 919-923

Srivastava R (2000). Studying the information needs of medicinal plant stakeholders in Europe. TRAFFIC Dispatch 15: 5

Standardi A, Piccioni E (1998). Recent perspectives on synthetic seed technology using non-embryogenic in vitro derived explants. Int J Plant Sci 159: 968-978

Stapfer RE, Heuser CW, Deneke CF (1985). Rapid multiplication of Veronica Red Fox propagated in vitro. HortScience 20: 866-867

Stephan R, Jayabalan N (2001). Propagation of Coriandrum sativum L. through somatic embryogenesis. Indian J Exp Biol 39: 387-389

267

Steward FC, Mapbs MO, Mears K (1958). Growth and organized development of cultured cells. II. Organization in cultures grown from freely suspended cells. Am J Bot 45: 705-708

Steward FC, Mapes MO, Kent AE, Holsten RD (1964). Growth and development of cultured plant cells. Science 143: 20-27

Subrat N (2002). Ayurvedic and herbal products industry: an overview. In. Paper at a workshop on wise practices and experiential learning in the conservation and management of himalayan medicinal plants, Kathmandu, Nepal, 15-20 December 2002, supported by the Ministry of Forest and Soil Conservation, Nepal, the WWF Nepal Program, Medicinal and Aromatic Plants Program in Asia (MAPPA), IDRC, Canada, and the WWF-UNESCO People and Plants Initiative pp 10

Sudarsono, Goldy RG (1991). Growth regulator and axillary bud position effects on in vitro establishment of Vitis rotundifolia. HortScience 26: 304-307

Sudha CG, Seeni S (1994). In vitro multiplication and field establishment of Adhatoda beddomei CB Clarke, a rare medicinal plant. Plant Cell Rep 13: 203- 207

Sudha CG, Seeni S (2006). Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F. - a rare medicinal plant. In Vitro Cell Dev Biol-Plant 42: 119-123

Sudhersan C, Abo El-Nil M (2002). Somatic embryogenesis of Sturt’s Desert Pea. In: Program and Abstracts of the 7th meeting of the Intl Ass Plant Tiss Cult Biotechnol (Australian Branch) pp 63

Sujana P, Naidu CV (2011). High frequency rapid plant regeneration from shoot tip and nodal explants of Mentha piperita (L.) - an important multipurpose medicinal plant. J Phytol 3: 9-13

Sujatha G, Kumari BDR (2008). Micropropagation, encapsulation and growth of Artemisia vulgaris node explants for germplasm preservation. S Afr J Bot 74: 93- 100

268

Sunnichan VG, Shivanna KR, Mohan Ram HY (1998). Micropropagation of gum karaya (Sterculia urens) by adventitious shoot formation and somatic embryogenesis. Plant Cell Rep 17: 951-956

Sutheer RK, Habibi N, Purohit SD (2011). Influence of agar concentration and liquid medium on in vitro propagation of Boswellia serrata Roxb. Indian J Biotechnol 10: 224-227

Swamy BVR, Himabindi K, Sita LG (1992). In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb). Plant Cell Rep 11: 126-131

Swamy SL, Ganguli JL, Puri S (2004). Regeneration and multiplication of Albizia procera Benth. through organogenesis. Agroforest Syst 60: 113-121

Synková H (1997). Sucrose affects the photosynthetic apparatus and the acclimation of transgenic tobacco to ex vitro culture. Photosynthetica 33: 403- 412

Tabassum B, Nasir IA, Farooq AM, Rehman Z, Latif Z, Husnain T (2010). Viability assessment of in vitro produced synthetic seeds of cucumber. Afr J Biotechnol 9: 7026-7032

Tawfik AA, Noga G (2002). Cumin regeneration from seedling derived embryogenic callus in response to amended kinetin. Plant Cell Tiss Organ Cult 69: 35-40

Taylor JLS, van Staden J (2001). The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets. Plant Growth Reg 34: 49-56

Teixetra SL, Da Silva LL (1990). In vitro propagation of adults Eucalyptus grandis Hill Ex. Maiden from epicormic shoots. VII Intl Cong on Plant Tiss and Cell Cult (IAPTC), Amsterdam, pp 218

269

Tetu T, Sangwan RS, Sangwan-Norreel BS (1990). Direct somatic embryogenesis and organogenesis in immature zygotic embryos of Pisum sativum L. J Plant Physiol 137: 102-109

Thakur R, Rao PS, Bapat VA (1998). In vitro plant regeneration in Melia azedarach L. Plant Cell Rep 18: 127-131

Thangavel P, Britto SJ, Senthilkumar SR (2011). Adventitious shoot regeneration from leaf explants of the valuable medicinal herb Plectranthus barbatus Andrews. Afr J Biotechnol 10: 8562-8569

Thengane SR, Kulkarni DK, Shrikhande VA, Krishnamurthy KV (2001). Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida. In Vitro Cell Dev Biol-Plant 37: 206-210

Thimann KV (1935). On the plant hormone produced by Rhizopus suinus. J Biol Chem 109: 279-291

Thirumalai S, Thamburaj S (1996). Plantlet regeneration in sweet orange (Citrus sinensis) var. sathgudi and acid lime (Citrus aurantifolia) var. PKM 1. S Indian Hortic 44: 62-64

Thom TA, Maretzki Komer E, Sakai WS (1981). Nutrient uptake and accumulation by sugarcane tissue culture in relation to growth cycle. Plant Cell Tiss Organ Cult 1: 3-14

Thomas TD (2003). Thidiazuron induced multiple shoot induction and plant regeneration from cotyledonay explants of mulberry. Biol Plant 46: 529-533

Thomas TD, Jacob A (2004). Direct somatic embryogenesis of Curculigo orchioides Gaertn., an endangered medicinal herb. J Plant Biotechnol 6: 193- 197

Thomas TD, Maseena EA (2006). Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant. Sci Hort 108: 332-336

270

Thomas TD, Philip B (2005). Thidiazuron-induced high frequency shoot organogenesis from leaf derived callus of a medicinal climber Tylophora indica (Burm. F.) Merrill. In Vitro Cell Dev Biol-Plant 41: 124-128

Thomas TD, Puthur JT (2004). Thidiazuron induced high frequency shoot organogenesis in callus from Kigelia pinnata L. Bot Bull Acad Sin 45: 307-313

Thomas TD, Yoichiro H (2010). In vitro propagation for the conservation of a rare medicinal plant Justicia gendarussa Burm. F. by nodal explants and shoot regeneration from callus. Acta Physiol Plant 32: 943-950

Thorpe T, Stasolla C, Yeung EC, De Klerk GJ, Roberts A, George EF (2008). The components of plant tissue culture media II: Organic additions, osmotic and pH effects and support systems. In: George EF et al (eds). Plant Propagation by Tissue Culture, 3rd edition, Vol 1 The Background, Springer, Dordrecht pp 115- 175

Thorpe TA (1980). Organogenesis in in vitro: structural, physiological and biochemical aspects. In: Vasil IK (ed). Perspectives in plant cell and tissue culture, Academic, New York, pp 71-111

Thorpe TA (2007). History of plant tissue culture. Mol Biotechnol 37: 169-180

Thorpe TA, Harry IS, Kumar PP (1991). Application of micropropagation to forestry. In: Debergh PC, Zimmerman RH (eds). Micropropagation: Technology and application. Kluwer Academic publishers, Dordrecht pp 311-336

Thorpe TA, Murashige T (1970). Some histochemical changes underlying shoot initiation in tobacco callus cultures. Can J Bot 48: 277-285

Tiwari KN, Sharma NC, Tiwari V, Singh BD (2000). Micropropagation of Centella asiatica (L.), a valuable medicinal herb. Plant Cell Tiss Organ Cult 63: 179-185

Tiwari S, Shah P, Singh K (2004). In vitro propagation of Pterocarpus marsupium Roxb.: an endangered medicinal tree. Indian J Biotechnol 3: 422-425

Tiwari SK, Kashyap MK, Ujjaini MM, Agrawal AP (2002). In vitro propagation of Lagerstromia parviflora Roxb. from adult tree. Indian J Exp Biol 40: 212-215

271

Tiwari V, Tiwari KN, Singh BD (2001). Comparative studies of cytokinins on in vitro propagation of Bacopa monniera. Plant Cell Tiss Organ Cult 66: 9-16

Toth KF, Lacy ML (1992). Micropropagation of celery (Apium graveolens var dulce). In: Bajaj YPS (ed). High-tech and micropropagation III. Biotechnology in agriculture and forestry. Vol 19 Berlin, Springer, Verlag pp 217-229

Trejgell A, Dąbrowska G, Tretyn A (2009). In vitro regeneration of Carlina acaulis subsp. simplex from seedling explants. Acta Physiol Plant 31: 445-453

Trejgell A, Michalska M, Tretyn A (2010). Micropropagation of Senecio macrophyllus M. Bieb. Acta Biol Craco Series Botanica 52: 67-72

Tremblay L, Tremblay FM (1991). Carbohydrates requirement for the development of a black spruce [Picea mariana (Mill). B.S.P.] and red spruce (P. rubens Sarg) somatic embryo. Plant Cell Tiss Organ Cult 27: 95-99

Trigiano RN, Geneve RL, Merkle SA, Preece JE (1992). Tissue and cell cultures of woody legumes. Hort Rev 14: 265-332

Trillas MI, Serret MD, Jorba J, Araus JL (1995). Leaf chlorophyll fluorescence changes during acclimatization of the rootstock GF677 (peach × almond) and propagation of Gardenia jasminoides E. In: Carre F, Chagvardieff P (ed). Ecophysiology and Photosynthetic In Vitro Cultures. CEA, Centre ďÉtudes de Cadarache, Saint-Paul-lez-Durance pp 161-168

Tripathi L, Tripathi JN (2003). Role of biotechnology in medicinal plants. Tropical J Pharma Res 2: 243-253

Tripathi M, Kumari N (2010). Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis. Acta Physiol Plant 32: 1011-1015

Tsay HS, Gau TG, Chen CC (1989). Rapid clonal propagation of Pinellia ternata by tissue culture. Plant Cell Rep 8: 450-454

Tsay HS, Lee CY, Agrawal DC, Basker SG (2006). Influence of ventilation closure, gelling agent and explants type on shoot bud proliferation and

272 hyperhydricity in Scrophularia yoshimurae - a medicinal plant. In Vitro Cell Dev Biol-Plant 42: 445-449

Tsvetkov I, Hausman JF (2005). In vitro regeneration from alginate encapsulated microcuttings of Quercus sp. Sci Hort 103: 503-507

Turker AU, Camper ND, Gurel E (2001). In vitro culture of common mullein (Verbascum thapsus L.). In Vitro Cell Dev Biol-Plant 37: 40-43

Turker AU, Yucesan B, Gurel E (2010). Adventitious shoot regeneration from stem internode explants of Verbena officinalis L., a medicinal plant. Turk J Biol 34: 297-304

Tuteja N (2007). Abscisic acid and abiotic stress signaling. Plant Signal Behav 2: 135-138

Tyagi RK, Agrawal A, Mahalaxmi C, Hussain Z, Tyagi H (2007). Low cost media for in vitro conservation of turmeric (Curcuma longa L.) and genetic stability assessment using RAPD markers. In Vitro Cell Dev Biol-Plant 43: 51-58

Ugraiah A, Karuppusamy S, Pullaiah T (2010). Micropropagation of Marsdenia brunoniana Wight & Arn. ‐ a rare antidiabetic plant. Plant Tiss Cult Biotechnol 20: 7-12

Uozumi N, Nakashimada Y, Kato Y, Kobayashi T (1992). Production of artificial seed from horseradish hairy root. J Ferment Bioeng 74: 21-26

Upadhyay N, Makoveychuk AYU, Nikolaeva LA, Batygina TB (1992b). Organogenesis and somatic embryogenesis in leaf callus culture of Rauvolfia caffra Sond. J Plant Physiol 140: 218-222

Upadhyay R, Arumugam N, Bhojwani SS (1989). In vitro propagation of Picrorhiza kurroa Royle Ex. Benth.: an endangered species of medicinal importance. Phytomorph 39: 235-242

Upadhyay A, Davis TD, Sankhla D, Sankhla N (1992a). Micropropagation of Lupinus texensis from cotyledonary node explants. HortScience 27: 1222-1223

273

Uranbey S (2005). Thidiazuron induced adventitious shoot regeneration in Hyoscyamus niger. Biol Plant 49: 427-430

Vadodaria HK, Samantaray S, Maiti S (2007). Micropropagation of Glycyrrhiza glabra Linn. an important medicinal plant. J Cell Tiss Res 7: 921-926

Van der Krieken WM, Breteler H, Visser MHW, MAvridou D (1993). The role of conversion of IBA into IAA on root regeneration in apple: introduction of a test system. Plant Cell Rep 12: 203-206

Van Huylebroeck JM, Piquerasm A, Deberghm PC (2000). The evolution of photosynthetic capacity and the antioxidant enzymatic system during acclimatization of micropropagated Calathea plants. Plant Sci 155: 59-66

Van Huylenbroeck JM, Debergh PC (1996). Impact of sugar concentration in vitro on photosynthesis and carbon metabolism during ex vitro acclimatization of Spathiphyllum plantlets. Physiol Plant 96: 298-304

Van Huylenbroeck JM, Piqueras A, Debergh PC (1998). Photosynthesis and carbon metabolism in leaves formed prior and during ex vitro acclimatization of micropropagated plants. Plant Sci 134: 21-30

Van Huylenbroeck JM, Piqueras A, Debergh PC (2000). The evolution of photosynthetic capacity and the antioxidant enzymatic system during acclimatization of micropropagated Calathea plants. Plant Sci 155: 59-66

Van Nieuwkerk JP, Zimmerman RH, Fordham I (1986). Thidiazuron stimulation of apple shoot proliferation in vitro. HortScience 21: 316-518

Van Overbeek J, Conklin ME, Blakeslee AF (1941). Factors in coconut milk essential for growth and development of very young Datura embryos. Science 94: 350-351

Varshney A, Shahzad A, Anis M (2009). High frequency induction of somatic embryos and plantlet regeneration from nodal explants of Hygrophila spinosa T. Anders. Afr J Biotechnol 8: 6141-6145

274

Vasil IK (1959). Nucleic acids and the survival of excised anthers in vitro. Science 129: 1487-1488

Vasil IK (1987). Developing cell and tissue culture systems for improvement of cereal and grass crops. J Plant Physiol 128: 193-218

Vasil IK, Vasil V (1972). Totipotency and embryogenesis in plant cell and tissue cultures. In Vitro 8: 117-127

Vasil V, Hildebrandt AC (1965). Differentiation of tobacco plants from single, isolated cells in microcultures. Science 150: 889-892

Vasil V, Hildebrandt AC (1967). Further studies on the growth and differentiation of single, isolated cells of tobacco in vitro. Planta 75: 139-151

Vengadesan G, Ganapathi A, Amutha S, Selveraj N (2003a). High frequency plant regeneration from cotyledon callus of Acacia sinuata (Lour.) Merr. In Vitro Cell Dev Biol-Plant 39: 28-33

Vengadesan G, Ganapathi A, Anand RP, Anbazhagan VR (2000). In vitro organogenesis and plant formation in Acacia sinuata. Plant Cell Tiss Organ Cult 61: 23-28

Vengadesan G, Ganapathi A, Anand RP, Anbazhagan VR (2002). In vitro propagation of Acacia sinuata (Lour.) Merr. via cotyledonary nodes. Agroforest Syst 55: 9-15

Vengadesan G, Ganapathi A, Preamanand R, Selvaraj N (2003b). In vitro propagation of Acacia sinuata (Lour.) Merr. from nodal segments of a 10-year- old tree. In Vitro Cell Dev Biol-Plant 39: 409-414

Venkatachalam P, Geetha N, Khandelwal A, Shaila MS, Sita GL (1999a). Induction of direct somatic embryogenesis and plant regeneration from mature cotyledons explants of Arachis hypogea L. Curr Sci 77: 269-273

Venkatachalam P, Kavi Kishore PB, Geetha N, Thankavelu M, Jayabalan NA (1999b). Rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogea L.). In Vitro Cell Dev Biol-Plant 35: 409-412

275

Vibha JB, Choudhary K, Singh M, Rathore MS, Shekhawat NS (2009). An efficient somatic embryogenesis system for velvet bean [Mucuna pruriens (L.) DC.]: a source of anti Parkinson’s drug. Plant Cell Tiss Organ Cult 99: 319-325

Victor JMR, Murthy BNS, Murch SJ, Krishnaraj S, Saxena PK (1999). Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.). Plant Growth Reg 28: 41-47

Vijaya Chitra DS, Padmaja G (2001). Seasonal influence on axillary bud sprouting and micropropagation of elite cultivars of Mulbery. Sci Hort 92: 55-68

Vincent KA, Mathew KM, Hariharan M (1992). Micropropagation of Kaempferia galanga L. - a medicinal plant. Plant Cell Tiss Organ Cult 28: 229-230

Vinocur B, Carmi T, Altman A, Ziv M (2000). Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media. Plant Cell Rep 19: 1146-1154

Virchow R (1858). Die Cellullarpathologie im ihrer Begrüngungund physiologische und pathologische Gewebelehre. A Hirschwald, Berlin

Visser C, Qureshi JA, Gill R, Saxena PK (1992). Morphoregulatory role of thidiazuron: substitution of auxin-cytokinin requirements of somatic embryogenesis in hypocotyl cultures of geranium. Plant Physiol 99: 170-177

Vöchting H (1878). Über Oganbildung im Pflanzenreich. Max Cohen, Bonn

Von Arnold S, Eriksson T (1981). In vitro studies of adventitious shoot formation in Pinus contorta. Can J Bot 59: 870-874

Wala BB, Jasrai YT (2003). Micropropagation of an endangered medicinal plant: Curculigo orchioides Gaertn. Plant Tiss Cult 13: 13-19

Wang H, Liu H, Wang W, Zu Y (2008). Effects of thidiazuron, basal medium and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba x P. berolinensis. J For Res 19: 257-259

276

Wang H, Zu Y, Dong F, Zhao X (2005). Assessment of factors affecting in vitro shoot regeneration from axillary bud explant of Camptotheca acuminata. J For Res 16: 52-54

Wang HC, Chen JT, Wu SP, Lin MC, Chang WC (2003). Plant regeneration through shoot formation from callus of Areca catechu L. Plant Cell Tiss Organ Cult 75: 95-98

Wang SY, Steffens GL, Faust M (1986). Breaking bud dormancy in apple with a plant bioregulator, thidiazuron. Phytochem 25: 311-317

Warakagoda PS, Subasinghe S (2009). In vitro culture establishment and shoot proliferation of Jatropha curcas L. Trop Agric Res Exten 12: 77-80

Weekley CW, Race T (2001). The breeding system of Ziziphus celata Judd and D.W. Hall (Rhamnaceae), a rare endemic plant of the Lake Wales Ridge, Florida, USA: Implications for recovery. Biol Conserv 100: 207-213

Wei X, Gou X, Yuan T, Russell SD (2006). A highly efficient in vitro plant regeneration system and Agrobacterium mediated transformation in Plumbago zeylanica. Plant Cell Rep 25: 513-521

Went FW (1928). Wuchstoff und Wachstum. Rec Trav Bot Neerl 25: 1-116

West TP, Ravindra MB, Preece JE (2006). Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments. Plant Cell Tiss Organ Cult 87: 223-231

Wetmore R, Wardlaw CW (1951). Experimental morphogenesis in vascular plants. Annu Rev Plant Physiol 2: 269-292

White PR (1934). Potentially unlimited growth of excised tomato root tips in a liquid medium. Plant Physiol 9: 585-600

White PR (1939). Potentially unlimited growth of excised plant callus in an artificial nutrient. Am J Bot 26: 59-64

277

White PR (1963). The cultivation of animal and plant cells. The Ronald Press, New York pp 228

White PR, Braun AC (1941). Crown-gall production by bacteria-free tumour tissues. Science 94: 239-241

Wickson M, Thimann KV (1958). The antagonism of auxin and kinetin in apical dominance. Physiol Plant 11: 62-74

Williams RR, Taji AM, Winney KA (1990). The effect of Ptilotus plant tissue on pH of in vitro media. Plant Cell Tiss Organ Cult 22: 153-158

Wojcik A, Podstolski A (2007). Leaf explant in in vitro culture of St. John’s Wort (Hypericum perforatum L.). Acta Physiol Plant 29: 151-156

Wondyifraw T, Surawit W (2004). Micropropagation of krawan (Amomum krervanh Pierre ex Gagnep). Sci Asia 30: 149-150

Xie D, Hong Y (2001b). In vitro regeneration of Acacia mangium via organogenesis. Plant Cell Tiss Organ Cult 66: 167-173

Xie DY, Hong Y (2001a). Regeneration of Acacia mangium through somatic embryogenesis. Plant Cell Rep 20: 34-40

Yadav R, Arora P, Kumar D, Katyal D, Dilbaghi N, Chaudhury A (2009). High frequency direct plant regeneration from leaf, internode, and root segments of Eastern Cottonwood (Populus deltoides). Plant Biotechnol Rep 3: 175-182

Yadav SK, Sreenu P, Maheswari M, Venkateswarlu B (2010). Efficient shoot regeneration from double cotyledonary node explants of green gram [Vigna radiata (L.) Wilczek]. Indian J Biotechnol 9: 403-407

Yan H, Liang C, Yang L, Li Y (2010b). In vitro and ex vitro rooting of Siratia grosvenorii, a traditional medicinal plant. Acta Physiol Plant 32: 115-120

Yan SF, Zhang Q, Wang JE, Sun YQ, Daud MK, Zhu SJ (2010a). Somatic embryogenesis and plant regeneration in two wild cotton species belong to G genome. In Vitro Cell Dev Biol-Plant 46: 298-305

278

Yang X, Zhang X (2010). Regulation of somatic embryogenesis in higher plants. Critical Rev Plant Sci 29: 36-57

Yemets AI, Klimkina LAA, Tarassenko LV, Blume YB (2003). Efficient callus formation and plant regeneration of goosegrass (Eleusine indica). Plant Cell Rep 21: 503-510

Yeung EC (1999). The use of histology in the study of plant tissue culture systems - some practical comments. In Vitro Cell Dev Biol-Plant 35: 137-143

Yip WK, Yang SF (1986). Effect of thidiazuron, a cytokinin active urea derivative, in cytokinin dependent ethylene production system. Plant Physiol 80: 515-519

Yoshimatsu K, Shimomura K (1994). Plant regeneration on cultured root segments of Cephalis ipecacuanha A Richard. Plant Cell Rep 14: 98-101

You XL, Han JY, Choi YE (2007). Plant regeneration via direct somatic embryogenesis in Panax japonicas. Plant Biotechnol Rep 1: 5-9

Young AJ (1991). The protective role of carotenoids in higher plants. Physiol Plant 83: 702-708

Yucesan B, Turker AU, Gurel E (2007). TDZ induced high frequency plant regeneration through multiple shoot formation in witloof chicory (Cichorium intybus L.) Plant Cell Tiss Organ Cult 91: 243-250

Yusnita S, Geneve RL, Kester ST (1990). Micropropagation of white flowering eastern red bud Cercisc anadensis var. alba. J Environ Hortic 8: 177-179

Zambre M, Chowdhury B, Kuo YH, Montagu MV, Angenon G, Lambein F (2002). Prolific regeneration of fertile plants from green nodular callus induced from meristematic tissues in Lathyrus sativus L. (grass pea). Plant Sci 163: 1107- 1112

Zanol GC, Fortes GR, de L, Campos AD, Centellas AQ, da Silva JB (1998). In vitro rooting and peroxidase activity of apple root stock cv, Marubakadlo treated with indole butyric acid and phloroglucinol. Rev Brasil Fisiol Veg 10: 65-68

279

Zhang T, Wang XY, Cao ZY (2005). Plant regeneration in vitro directly from cotyledon and hypocotyl explants of Perilla frutescens and their morphological aspects. Biol Plant 49: 423-426

Zhang ZM, Davis FT Jr (1986). In vitro culture of Crape myrtle. HortScience 21: 1044-1045

Zhou X, Han Y, Yang W, Xi T (1992). Somatic embryogenesis and analysis of peroxidase in cultured lettuce (Lactuca sativa L.) cotyledons. Ann Botany 69: 97- 100

Ziv M, Schwartz A, Fleminger D (1987). Malfunctioning stomata in vitreous leaves of carnation (Dianthus caryophyllus) plants propagated in vitro, implications for hardening. Plant Sci 52: 127-134

280