In vitro studies on growth and morphogenesis of some potential medicinal plants through tissue culture

DISSERTATION

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

iRas^ter of ^fjilosiopbp 31n ^ iBotanp ^ /•#

L By

SHEIKH ALTAF HUSSAIN

DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH -202002 (INDIA) 2014 2 4 NOV 201i

DS4373 m

f^>^a(pra6le &ra4ferUA

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Certificate

Certified that the dissertation entitled "In vitro studies on growth and morphogenesis

of some potential medicinal plants through tissue culture" submitted to the Aligarh

Muslim University, Aligarh, in fulfillment of the requirement for the award of Master of

Philosophy, embodies the original research work carried out by Mr. Sheikh Altaf Hussain

under my guidance and supervision. The work has not been submitted elsewhere in part

or full for the award of any other degree of this or any other university.

(ProfessorJAoham^^FAms) ^ - "^^^--"^ap^fvfsor ^ X ) 4

Tel. Off: 0571-27020]6: 2700920, 21, 22 (Ext. 3308). Home: 07. Rainbow Roofs-I. Anoopshahar Road. Aligarh -202 122. UP (India) CONTENTS

Acknowledgements

Abbreviations CHAPTER 1: INTRODUCTION 1-10 1.1. Micropropagation 1.2. Micropropagation ofR. tetraphylla and R.serpentina 1.3. Classification of ^. tetraphylla 1.3.1 Distribution 1.3.2 Botanical description 1.3.3 Characteristics and constituents 1.3.4 Medicinal importance 1.4. Classification of/?, serpentina 1.4.1 Distribution 1.4.2 Botanical description 1.4.3 Medicinal importance 1.5 Objectives

CHAPTER 2: REVIEW OF LITERATURE 11-18 2.1 Historical background 2.2 Micropropagation 2.3 Explant type 2.4 Plant growth regulators 2.5 Subculturing 2.6 Rooting 2.7 Hardening and Acclimatization

CHAPTER 3: MATERIALS AND METHODS 19-25 3.1 Plant material and explant sources 3.2 Surface sterilization of explants 3.3 Establishment and Preparation of aseptic explants 3.4 Culture media 3.5 Preparation of stock solution 3.6 Growth regulator and carbon source 3.7 Adjustment of pH and gelling of medium 3.8 Sterilization of glasswares and instruments 3.9 Sterilization of laminar airflow hood 3.10 Inoculation 3.11 Incubation 3.12 Subculturing 3.13 Rooting 3.14 Hardening and Acclimatization 3.15 Chemical and glasswares 3.16 Statistical analysis

CHAPTER 4: RESULTS (with Tables & Figures) 26-36 4.1 Direct shoot regeneration of/?, tetraphylla 4.1.1. Effect of Cytokinins 4.1.2. Combined effect of Cytokinins and auxin 4.1.3. Effect of thidiazuron (TDZ) 4.1.4. Combined effect of TDZ and BA 4.1.5. Effect of metatopolin 4.1.6. Rooting of regenerated shoots 4.1.7. Acclimatization 4.2 Direct shoot regeneration in R. serpentina 4.2.1. Combined effect of cytokinin and auxin 4.2.2. Rooting 4.2.3. Acclimatization

CHAPTERS: DISCUSSION 37-42 5.1. Direct shoot regeneration in R. tetraphylla 5.2. In vitro Rooting in regenerated shoots of R. tetraphylla 5.3. Direct shoot regeneration in R. serpentina. 5.4 In vitro Rooting in regenerated shoots of R. serpentina 5.5 Hardening and Acclimatization CHAPTER 6: SUMMARY AND CONCLUSIONS 43-44

REFERENCES 45-60 (praise 6e to JiClali, tfie cHerisHer ancf tHe sustainer of the worCd, 'Who Bestowed on me the divine guidance, enough courage and patience to successfuCCy compete thu worH^

It 6rings me coCossaC pCeasure while placing on record my sincere gratitude and reverence to my esteemed supervisor (prof. ((Dr.) 'Mohammad J/lnis for his unparaCleC and sincere guidance, ^en interest, incessant encouragement, strong motivation, constructive criticism and idea oriented discussion, which has enabled me to accomplish this wor^

It is my proud privilege to express my sense of than^ulness to (prof. Tiroz Mohammad, Chairman (Department of (Botany, JUMV for providing necessary infrastructure and technical facilities during my wor^

I deem it a privilege to ey^ress my sincere gratitude to (Dr. Jlnwar Shazad(Jlssistant (professor) and (Dr. ^aseem Jlhmad ('Young scientist 0)31) for all time support, encouragement, motivation and suggestions all through my wor^

I am highly appreciative of the support, encouragement and fruitful suggestions expended all through the course 6y my seniors and laB-mates especially (Dr. J^n({ita varsheny (Young scientist (DST), ^r. S^igar Tatima, Ms. jAfshan Jiaaz, Ms. (Rjuphi 'Xaz, Saad (Bin Javid, Md (Rafique Jlhmed, Ms. Jifsheen Shaid, J^aushad jAlam, Jlness Jlhmad and Ms. Jltjumund Shaeen. JL to^n of appreciation will always remain for (Dr. JVaseem JLhmadfor his support cooperation in creating a congenial, amicadle and friendly atmosphere in the la6.

M this inexplicahk moment of joy, I deem it a privilege to thanl^ all my friends especially Manzoor (Raiees %han, Jiddil(Rashid, Imran Mustafa Mali^ Jl6. Latif "Wani, Sheilih Mohammad jAsif Kjianday Tanveer, Mir (Rayees, (Pervaiz J4hmad, SheiUJi (Rameez Jlli and(Bilaljlhmadfor their incessant motivation, encouragement and support throughout the wor^ I extend my special than^ to (Dr Manzoor J^hmadfor his sincere help, advice and support during the wor^ I feeC -pCeasure to pay su6Cime oSeisance ancCgratitude to my reverecf ancC Bethvecf parents, ivdose e^cjuisite Benedictions, unconditionaC Cove and affection, unfCincHing care and support, prayers and 6(essings right from tHe cradCe ignited my confidence to exceC academicaCCy andfufiCC my wisfies. (Dedicating this wor^ to my deCoved parents, I apohgize to them for Searing with aCC the faiCures in commitment on my part, arising out of Susy worli^schedufe during my studies. 94.y heartfeCt devotion is due to my sisters who extended uCtimate cooperation and every possiSCe support aCC through my studies. I may 6e ungratefuC without than^ng aCCmy relatives for their affection, support and encouragement throughout my studies.

Last 5ut not the [east, I wish to offer my than^ to aCCthose names which couhdnot 6e incCudedhut wiCCaCways hefondCy rememhered.

(Sheikh ACtaf^Hussain)

11 ABBREVIATIONS

2-iP 2-isopentyl adenine 2, 4-D 2, 4-dichlorophenyoxy acetic acid BA 6-benzyladenine CH Casein hydrolysate

cm Centimeter

DDW Double distilled water

EDTA Ethylene diamine tetra acetic acid

g Gram

GA3 Gibberellic acid

CM Coconut milk

h Hour

HCl Hydrochloric acid

lAA Indole-3-acetic acid

IBA Indole-3-butyric acid

Kin Kinetin

mT metatopolin

|iM Micromolar

mg Milligram mg 1 • Milligram per liter min Minute ml Milliliter mM Millimolar

MS Murashige and Skoog's medium

N Normality

NAA a-napthalene acetic acid NaOH Sodium hydroxide

PABA Para- amino benzoic acid

PGRs Plant growth regulators

TDZ Thidiazuron

UV Ultraviolet v/v Volume by volume w/v Weight by volume

% Percentage

C Degree celsius ^/udftet^ - /

1^ Introuction

Introduction

Biodiversity is the store house of species richness and serves as the cushion against the potentially dangerous environmental changes and economic reforms (Yadav et ui. 2012). It encompasses ail the biological entities that occurs due to their dynamic interactions among different levels of integration within the living world, in an ecosystem and in their habitat. Biodiversity of plants is collectively known as plant genetic resources, and serves as the major biological basis of the world food securit> and in all means they support the livelihoods of every life form on earth (Alexander ei ai. 2011). India with wide spectrum of phytogeographic and agro-climatic zones is blessed with rich diversity of plants. Plants form the key element in maintaining the biodiversity on this planet Earth. Plants are endowed with the unique capacity to convert solar energy into chemical energy and synthesize thousands of chemical substances which are very important to our lives.

India has very rich plant diversity and is one of the 12 mega biodiversity hot spot centers of the world with 4"^ rank in Asia. India represents about 12% of the world s recorded flora with 49,000 plant species, among them 20,000 species belongs to higher plants, one third of it being endemic and 1000 species being medicinally important (Krishnan et al., 2011; Singh et ai, 2003). At least 40% of the world economy and 80% of the needs of the poor people are derived from biological resources, in addition, the richer the diversity of life, greater the opportunity for medicinal discoveries, economic development and adaptive responses to such new challenges as climate change.

Since the beginning of human civilization, medicinal plants have been used virtual!) in all social cultures for its therapeutic value. Nature has been a source of medicinal assets from prehistoric times and an impressive number of modem and valuable drugs have been isolated from these natural sources. Many of these isolations were based on the uses of agents in herbal medicine. The plant-based traditional medicine system continues to play an essential role in health care, with about 80% of the world s inhabitants relying mainly on traditional, largely on herbal medicine for their primar) health care. The ancient texts like Rig veda, and Atharva Veda mention the use of several plants as medicine. The books on ayurvedic medicine such as Charaka Samhita and Susruta Samhita refer to the use of more than 700 herbs (Jain, 1968). Introtiction

According to WHO, 80% of world population and 65% of Indian rural population uses traditional form of medicine to meet their primary health care needs (Jain and Singh. 2010), because they are easily available, non narcotic and also having no side effects.

India is called the botanical garden of the world because it houses the largest number of the medicinal plants and acts as the gold mine, serves as the well recorded and most practiced knowledge of herbal medicine. India officially recognizes about 3000 plants for their medicinal value. It is generally estimated that over 6000 plants in India are in use. in traditional, folk and herbal medicine representing about 75% of the medicinal needs of the third world. Medicinal herbs are moving from fringe to mainstream use, with a greater seeking remedies and health approaches free from side effects as caused by synthetic chemicals.

Out of the 17.500 flowering species found in India, 1600 are used in traditional medicine system, and more than 1000 plants are used in indigenous system of medicine like Ayurveda, Sidha and Unani. thus gaining wide currency and acceptability for the purpose of financial gain. Market demand has lead to an increased pressure on these natural resources. The demand of the majority of the people in developing countries for medicinal plants have been met by indiscriminate harvesting of spontaneous flora include those in forests, over exploitation of these wild sources had led many species being extinct, threatened, or endangered.

The big concern is that these medicinal plants are collected from their natural habitat and involves the destructive harvesting when they are collected. Sometimes leafs, flowers, seeds, stems, bark, roots, wood or whole plant are used (Anis et ai, 2009). Due to their multipurpose use they are highly exploited for their market value. The increase in demand and ruthless collection of these plants are the key factors for their extinction. Other challenging factors for their existence are population explosion, rapid industrialization, urbanization, agricultural encroachment; forest degradation, unscientific harvesting etc. have lead to extinction of more than 150 plants.

It is therefore necessary that systematic cultivation, maintance and assignment for future use of these medicinal plants be introduced in order to conserve biodiversity and politic threatened species. Conservation of these medicinal plants is possible either through in situ or ex situ or by both. The major problems and challenges in in Introuction situ conservation of these plants include slow regeneration rates, over exploitation and destruction of their natural habitat. Plant improvement for agriculture, forestry and ecological conservation by traditional methods has been a slow process, limited b> slow growth and life cycles.

Plant biotechnology has provided a large number of tools and techniques which arc more precise and highly reproducible for rapid multiplication and in generating no\ el genetic variability, enhancement and transformation. Biotechnology has revolutionized the fields like basic biology, agriculture, horticulture and forestry. Biotechnology revolution is the best option for solving the problems of food securitv. poverty reduction, ecological and environmental conservation in developing countries (Serageldin 6^/a/., 1999).

Plant tissue culture is one of the important techniques of biotechnology. It involves the in vitro aseptic culture of cells, tissues, organs and their components in an artificially prepared medium under defined physical and chemical conditions. It is an important tool in both basic and applied studies as well as in commercial application. It was the availability of these techniques that led to the application of tissue culture to five broad areas i.e. cell behavior, plant modification and improvement, pathogen free plants and germplasm storage, clonal propagation and product formation. Plant tissue culture involves the following types of aseptic cultures.

Embryo culture Seed culture Meristem culture

Cell culture ••- Plant Tissue Culture •Protoplast culture

Callus culture Bud culture Organ culture.

Types of cultures Introuction

Tissue culture propagation also known as micropropagation is a way of dividing cells and reproducing clones of the mother plant. It also helps to study the mechanism by which cell differentiates, thus providing an experimental approach to link the genotype with the phenotype. It is clear that without tissue culture, plant biotechnology is immature and totally incomplete because it cements together its various aspects; to a large extent revolution in tissue culture has occurred because of the needs of the new biotechnology (Varshney and Anis, 2014).

The main advantage of the clonal propagation is the production of large number of plants that are clones to each other. Desirable stock plants can be chosen and thousands of high quality identical clones can be produced. Also helps in germplasm conservation, providing viable methods of regenerating genetically modified cells and to offer customer - tailored improved material. Tissue culture often promotes genetic disturbances which resulted in somaclonal variation, thus greatly extending the range of useful variation available to the breeder (Rout et al, 2000, Kukreja and Dawan 2000 and Sevon and Oksman caldentry 2002).

1.1 Micropropagation

Plant tissue culture techniques are a part of large group of strategies and technologies, ranging through molecular genetics, recombinant DNA studies, genomic characterization, gene transfer techniques, aseptic growth of cells, tissues, organs and in vitro regeneration of plants etc.

In vilro techniques are used mainly for the regeneration of organs or somatic embryos for propagation, disease elimination and pathogen free plant multiplication, transformation and regeneration of transgenic plants for improvement in their traits. It is possible to achieve rapid propagation of selected and elite genotypes and important varieties in a short time. Micropropagation is the first and major commercial application of tissue culture techniques. It is currently used in a large number of medicinaly important plants ranging from a herb to a big tree, both in angiosperms and gymnosperms. through enhanced axillary bud formation, organogenesis or somaticembryogenesis. Micropropagation of elite, selected or recalcitrant genotypes generally involve four distinct stages: explants establishment, regeneration and proliferation, rooting and acclimatization and finally transplanting ex vitro. The micropropagation technology has a vast potential to produce plants of superior Introuction quality, isolation of useful variants in well adapted high yielding genotypes with better disease and stress tolerance capacities.

In the present investigation, two important medicinal plants, Ravuolfia tetraphyllu and R. serpentina has been selected for their large scale propagation and conservation purposes through tissue culture techniques.

1.2 Micropropagation of Rauvolfia tetraphylla and Rauvolfia serpentina

Earlier reports on micropropagation of R. tetraphylla and R. serpentina using different explants have been depicted in Table 1 and 2.

Table. 1. Current status oiRauvolfia tetraphylla in tissue culture:

Explant Response PGR used References

Nodal Multiple shoot, plant BAP and Kin Patil and Janyanti regeneration (1997) Nodal Multiple shoot, plant BAP, Kin and Sarma et al., regeneration NAA (1999) Nodal Multiple shoot. plant BA and NAA Faisal and Anis regeneration (2002) Nodal Multiple shoot and plant TDZ Faisal et at., regeneration (2005b) Shoot and nodal in vitro flowering BAP and GA3 Anitha and explants Raj itha (2006) Leaf Effect of NaCI on Callus lAA, NAA, IBA Anitha and Raj itha induction and its growth and 2,4-D (2006)

Nodal Syn. seed. plant BA, NAA and Faisal et al.. regeneration IBA (2006a)

Nodal Clonal propagation KIN, BA and Harisaranraj. NAA (2009) Nodal Clonal multiplication and BA and NAA/ Faisal et al.. evaluation of genetic RAPD-DNA (2012) stability by using DNA marker markers

Nodal Syn. seed and evaluation BA and NAA Altar et al, of genetic fidelity by using (2012) RAPD/ISSR Markers Introiiction

Table. 2. Current status of Rauvol/ia serpentina in tissue culture. Explant Response PGR used Refrences Leaf and stem Induction and growth 2,4-D, NAA, Parveen and Ilahi segments of callus IBA, Kin, (1978) CM and CH Auxiliary Clonal propagation BA and NAA Roja and Heble meristem (1996) Nodal Multiple shoot BA and NAA Sarker et ah, regeneration (1996) Shoot tips and Multiple shoot BAP and IBA Jain et al., Nodal segments regeneration (2003) Nodal Callus morphogenesis NAA and Panday et al., and shoot proliferation BAP (2007) Nodal Callus and multiple 2,4-D, NAA, Bhandra et al, shoot formation lAA, BAP (2008) and Kin Shoot tips, leaves Callus induction and 2,4-D, BAP Bhatt et al, and Nodal direct shoot regeneration and IBA (2008) segments Nodal Callus and multiple NAA and Pant et al., shoot regeneration BAP (2008) Leaf Callus culture and multiple BAP and lAA Singh et al., shoot formation (2009) Leaf Direct root regeneration PABA, NAA Pandey et al., and IBA (2010) Leaf and stem Callus induction and 2,4-D, BAP Pan war et al., multiple shoot and NAA (2011) regeneration Leaf and stem Callus induction and 2,4-D, BAP Mallick et al, multiple shoot and NAA (2012) regeneration Shoot tips and Multiple shoot BAP and Ranjusha et al., Nodal segments regeneration NAA (2012) Nodal Multiple shoot BAP, Kin Singh and Dash regeneration and GA3 (2012) Nodal Multiple shoot BA, Kin Alatar et al, regeneration and 2ip (2012) Nodal Multiple shoot BAP and Pan et al., regeneration NAA (2013) Shoot tip Multiple shoot BA and NAA Susila et al., regeneration (2013) Mature and Induction of callus and 2,4-D and Shetty et al, immature seeds hairy root formation BAP (2014) Introiiction

1.3 Rauvolfia tetraphylla L.

Classification

Kingdom : Plantae Division : Angiosperms Class : Rudicots Series: Asterids Order: Gentianales Family: Apocynaceae Genus: Rauvolfia Species : tetraphylla Binomial Name: Rauvolfia tetraphylla

Synonyms: Rauvolfia canescens L., R. heterophylla Roem & Schult., R. hirsuta Taeq., R.latifolia var.minor Mull.Arg., R. odontophora van Heurck & Mull. Arg.. R. suhpuhescens L, R. tomentosa Jacq.

Common Names:

English : Devil pepper. Be-still, Milk bush,etc.

Hindi : Barachadrika.

1.3.1. Distribution It is native to Tropical America and also distributed in Tropical Asia, and Africa. In India it is found as an escape from cultivation in various states like Uttar Pradesh. Bihar. Orissa, Madhya Pradesh, Andhra Pradesh, West Bengal and Kerala (Anonymous, 2003). It is abundantly found in moist and warm regions of West Bengal, particularly in 24 Parganas and Howrah and Kerala (Jyothi et al., 2012). It is also grown as ornamental plant in house gardens, despite of the fact that it is medicinally very important.

1.3.2. Botanical description

It is an evergreen woody, perennial shrub, growing upto the height of 1.5 meter. Leaves are found in whorls, with four unequal leaves at each node and hence the name Rauvolfia tetraphylla. Leaves are dark green above and pale green below. Roots are very bitter and odourless. Bark is pale brown, corky, irregular and having longitudinal fissures. Flowers are creamy white and fruits are drupes (Anonymous, 2003). Introiiction

1.3.3 Characteristics and constituents

It is rich source of phytochemical alkaloids and about 30 indole alkaloids have been reported from it and among them, reserpine is the major and most potent alkaloid which is pharmacologically most important. The other main alkaloids present in this plant are ajmaline, alistonine, canescine, corynathene, desperdine, isoreserpiline, isoreserpine. serpagine, serpentine, tetraphyllicine, yohimbine and pseudoyohimbine (Anonymous. 2003. Anitha and Rajitha 2004, Jyothi et ai, 2012).

1.3.4. Medicinal importance

In India, it is used as a medicine since from the prehistoric times to cure and relief the diseases like central nervous system disorders. Reserpine is the potent alkaloid and pharmacologically most important that depresses the central nervous system; produces sedation and lowers blood pressure. The root extract of this plant is used to cure the diseases like diarrhea, dysentery and other intestinal infections, also used as anthelmithic (for the expulsion of intestinal worms). The plant is known in folk medicine as a treatment of snake poisoning and for external application for skin aliments. The roots of this plant are also used to stimulate uterine contraction and are recommended in difficult child birth cases (Faisal et al., 2006)

Rauvolfia serpentina (L).

1.4, Classification

Kingdom: Plantae Division: Angiosperms Class: Rudicots Series: Asterids Order: Gentianales Family: Apocynaceae Genus: Rauvolfia Species: serpentina Binomial Name: Rauvolfia serpentina L.

Synonyms: Rauvolfia serpentina (L.) Ex Kurz., Rauvolfia trifoliata (Gaertn.) Baill..Iava devil pepper (Engl.). Ophioxylon serpentimm L. Rauwolfia (Engl.)

8 Introuction

Common names:

English : Serpentine wood, Snakeroot. Hindi : Sarpagandha, Chota cliand etc. 1.4.1. Distribution

It is found in Bangladesh, Bhutan, China, Indonesia, India, Malayasia, Mynamar, Nepal. Pakistan, Srilanka and Vietnam. In India it is reported in the Himalayas, Assam, Java, and the Malaya peninsula.

1.4.2. Botanical description

It is an evergreen small, woody, perennial and erect shrub grows upto the height of 60cm. Roots are tuberous with pale brown cork. Leaves are in whorls of three, elliptic to lanceolate or obovate, bright green above and pale green below. Petioles are long. Flowers are white, clustered in axillary cymes, with slender corolla tube and ^ spreading lobes. Fruit is a small drupe (Anonymous, 2003).

1.4.3. Medicinal importance

Rauvolfia serpentina (L.) Benth, is an important medicinal shrub of faniil\ Apocynaceae. Its roots contain about 50 indole alkaloids, including the therapeutically important reserpine, ajmalicine, rescinnamine and yohimbine. Phc herbal plant is used as medicine for high blood pressure, insomnia, anxiety and other disorders of the central nervous system and epilepsy (Ghani 1998). This species has been listed as an endangered in the red-data book because of its fast disappearance and indiscriminate collection from the wild and depletion from the natural habitat due to poor seed germination.

Micropropagation is specifically used for the species whose clonal propagation is needed. In the present study attempt has been made to propagate the plant by multiple shoot regeneration and ultimately production of complete plantlets using different cvtokinins and auxins alone or in combination of different ratio. Introuction

1.5. Objectives

The present experimental work was undertaken with an aim of following objectives:

1. To establish and proliferate axenic cultures from juvenile and mature nodal explants.

2. To formulate culture conditions for regeneration, multiplication in somatic tissues and regenerant differentiation.

3. To evaluate the optimal medium for direct regeneration.

4. To optimize the media for in vitro rooting.

5. To standardize the hardening and acclimatization procedure for in vitro raised plants

^Rfvieiv of Literature

REVIEW OF LITERATURE

Historical background

In the history of tissue culture, the pioneer step was made by Henri-Lous Duhumel du Monceau in 1756, who during his pioneering studies on wound-healing in plants, observed callus formation (Gautheret, 1985). The science of the tissue culture cemented its roots from the path breaking research in botany like the discovery of the cell theory. In 1839, Schleiden and Schwann proposed that cell is the basic, structural and fundamental unit of an organism. They visualized that plant cell is capable of autonomy and therefore capable to generate the whole plant from the single plant cell (totipotentcy). This idea was tested from time to time by various researchers, but the work of Vochting (1878) and on the limits of divisibility of plant segments was perhaps the most important. He showed that the upper part of the stem segment produced buds and the lower end produced callus or roots. Thus he suggested the polar development is the key feature that guides the development of the plant fragments.

In 1902. a German Botanist, Gottlieb Haberlandt was the first to try to obtain experimental evidence of totipotency by culturing single cells of Lamium purpureiim and Eicchornia crassipes on Knop's salt solution with sucrose and observed ob\ ious growth in palisade cells. Although, Haberlandt remained unsuccessful in getting the desired results but he clearly established the concept of totipotency by predicting that one could successfully cultivate the artificial embryos from vegetative cells and the technique of cultivating isolated cells in nutrient solution permits the investigation of important problems from a new experimental approach. Due to this endeavor. Gottlieb Haberlandt is regarded as the father of tissue culture.

From 1902 to 1930 several attempts were made for organ culture. In 1904, Hanning isolated embryos of some crucifers like Raphanus caudatus, R. landra, R. stavis etc. and successfully cultured on the mineral salt and sugar solution under aseptic conditions, thus laid the foundation of embryo culture. In 1908, Simon regenerated callus, buds and roots from Poplar stem segments and established the basis for callus culture. Using a different approach, Kotte (1922), a student of G. Haberlandt and Robbins (1922) succeeded in culturing of isolated root and shoot tips in vitro. White (1934) had established successfully indefinite culture of tomato root fips, using 1(fview of Literature

excised explants with merismatic cells, on the excellent defined medium containing ingredients of sucrose, mineral salts and yeast extract. At the same time, in 1934 Roger Gautheret grew callus from cambial tissues isolated from woody plants including Willow sycamore. These were the first sustained dividing callus cultures. Soon after Gautheret, Noubecourt 1939 reported the successful growth of plant callus cultures, proving the one of the assumptions of G. Haberlandt true. Van Overbeeck and his coworkers in 1941 demonstrated for the first time, the stimulatory effect of coconut milk on embryo development and callus formation in Datura innoxia.

In 1950, there was an immense advancement in the field of plant tissue culture, after the discovery of effect of different PGRs on plant development. Skoog and Tsui in 1957 demonstrated the induction of cell division by the addition of adenine in the tobacco plant. Steward and Reinert (1958) reported the regeneration in callus culture of carrot. The tremendous increase in the in vitro cultivation of valuable number of plant species was made by the discovery of different cytokinins.

Skoog and Miller (1957) putforth the concept of hormonal control of organ formation after the discovery of cytokinin. Since then, tissue culture techniques have revolutionized the improvement in plant species and conservation. The dream of Haberlandt for the cultivation of somatic embryos became true when the first report appeared on somatic embryogenesis in carrot tissues by Reinert (1958, 1959) and steward et al.. (1958). In early 1960s, the most significant breakthrough in the field of tissue culture was the development of well defined culture medium which originally devised for the rapid growth and bioassay for the tobacco callus (Murashige and Skoog 1962). This medium contains all the desired salt composifion which is widely used to induce organogenesis and regeneration of various plant parts in cultured tissues. Even today this medium is recognized most effective and is commonly used medium in plant tissue culture.

Maheshwari and his coworkers became actively engaged in in vitro techniques in 1960 and laid the foundation of new mile stone by raising haploids, through anther culture of Datura innoxia (Guha and Maheshwari, 1964, 1966). In 1971, Takebe et al.. first demonstrated the totipotency of protoplast and regenerated the tobacco plants from mesophyll protoplasts. Thrope, in (1980), reported de novo organogenesis by interchanging auxin and cytokinin ratio in the medium. In addition, explants such as cotyledonary node, hypocotyls, callus and thin superficial cell layers have been used

12 ^Rfview of Literature in traditional morphogenetic studies as well as to produce de novo organs and plantlets in various plant species (Murashige 1974, 1979).

2.2. Micropropagation

In vivo clonal propagation of plants is tedious, expensive and frequently unsuccessful. Micropropagation is an alternative method of vegetative plant propagation and produces plants in a rapid way not only generating clonal planting stocks, but also effectively captures genetic variation while by passing long breeding cycles (Giri ei ill.. 2004). Further, micropropagation is a process that involves exposing plant tissue to a specific regime of nutrients and hormones under sterile or in artificial conditions outside the mother plant to produce many new plants.

In vitro clonal propagation through tissue culture is referred as micropropagation. Use of tissue culture technique for micropropagation was first started by Morel (1960) for propagation of orchids, and is now applied to several plants. The main aim of micropropagation is to provide a sufficient number of plantlets within a short duration and in a limited space. Micropropagation offers a viable tool for mass propagation and germplasm preservation of many medicinal and aromatic plants. As a result. laboratory-scale micropropagation protocols are available for a wide range of species and at present micropropagation is the widest use of plant tissue culture technology.

There are three methods by which micropropagation can be achieved. These are enhancing axillary bud breaking, production of adventitious buds directly or indirectly via callus and somatic embryogenesis directly or indirectly on explants (Murashige. 1974). Tissue culture techniques are being used in all types of plants like temperate, tropical, forest and ornamental, gymnosperms or angiosperms.

Micropropagation techniques are preferred over the conventional propagation methods because a small amount of fissue is required to produce millions of clonal plants in a short period.

Micropropagation generally involves four distinct stages (Murashige 1974).

i) Inifiation of asepfic culture - from explants ii) Shoot multiplication of the explants iii) Rooting of the in vitro regenerated shoots. iv) Transplantation - Transfer of the acclimafized plants to green house or field conditions.

13 1(fview of Literature

Debergh and Maene (1981) introduced the stage 0 (preparation of explants) making micropropagation a five stage process. George Morel (1960) was the pioneer in applying shoot tip culture as a clonal multiplication tool. After his success in cloning the orchid, Cymbidium, the clonal muUiplication gained momentum in the 1970s. Furthermore, in vitro growth and development of a plant is determined by a number of complex factors.

i) The genetic makeup of the plant.

ii) Nutrients; water, macro and micro elements and sugars.

iii) Physical growth factors, light, temperature, pH, O2 and CO2 concentrations.

iv) Some organic substances; regulators and vitamins etc.

2.3, Explant type

The regeneration power of the explants depends upon the size, nature of plant woody/ herbaceous, season of collection and healthiness of the plant. Any piece of the plant tissue can be used as an explant such as shoot apices, hypocotyls, epicotyls, mesocotyl, cotyledon, cotyledonary node, leaf and stem segments which have been known to possess the potential for shoot induction in aseptic culture (Bajaj and Gosal, 1981). A popular explant widely utilized for multiple shoot production is nodal explants with axillary bud of field grown medicinal plants including Raulvofia tetraphylla (Faisal et al, 2005), Psoralen corylifolia (Faisal and Anis 2006), Capssicum annum (Ahmad et al., 2006), Tylophora indica (Faisal and Anis, 2007), Cassia angiistifolia (Siddique and Anis 2007), Vitex negundo (Ahmad and Anis, 2010), Nyctanthes arbor tristis (Jahan et al., 2010a and b), Salix tetrasperma (Khan et al. 2011), Withania somnifera (Fatima and Anis, 2011^ Euphorbia continifolia (Perveen et al. 2013), Abrusprecatorius (Perveen et al, 2013), Vitex trifolia (Ahmad et al. 2013) and Cassia alata (Ahmed et al, 2013).

2.4. Plant Growth Regulators

Plant growth regulators are the synthetic organic substances which in minute amount act like the natural hormones, other than the vitamins and micro elements to promote, inhibit, or otherwise modify the physiological process of plants. These are the main components of media in determining the growth and developmental pathway of in vitro plant cell totipotency (Davies, 1995). Auxins, cytokinins and their different

14 ([(fvie-w of Literature

combinations and interactions are considered most important PGR sclieme for regulating growth, development and morphogenesis in vitro regeneration of plants The influence of PGRs and their interaction on in vitro regeneration of different plant species have been reported by Skirvin et al., (1990), Rout and Das (1997). Komalavalli and Rao (2000), Ahmad and Anis (2007), Siddique and Anis (2008). Faisal and Anis ( 2006 and 2009). Khan et ai, (2010), Perveen and Anis (2011). Fatima et al. (2011), Naz and Anis (2012), Seemab et ai, (2012), and Ahmad et ciL. (2013).

Cytokinins like 6-benzyl adenine (BA), Kinetin (Kin), and Metatopolin (niT) are adenine derivatives, promote cell division, shoot proliferation and shoot morphogenesis (Skoog and Miller, 1953 and Miller, 1961). Cytokinins are the most important additives of the plant tissue culture media which regulate cell division, stimulate axillary and adventious shoot bud proliferation and differentiation (Sugiyama, 1999). Tissue culture is all about the modifications of the media thus the optimization of the culture media is affected by the change in concentrations of the applied cytokinins because their uptake, transport and metabolism differ between varieties and during the interaction with the endogenous physiology of explants (Staden et al, 2008).

BA is considered as the most widely used cytokinin for shoot induction, proliferation and multiplication in wide variety of plant species. The edge of BA over other cytokinins is well documented in many plants including Fragaria Xananassu. (Kang el al.. 1994), Eucalyptus impensa (Bunn 2005), Psoralea corylifolia (Jeyakumar and Jayabalan 2002), Bupleurum kaoi (Chin et a/., 2006) and Rauvolfia tetraphyllu (Faisal el al.. 2006). Also in Cassia siamea (Sreelatha et al, 2007), highest shoot multiplication was observed on MS medium containing BA. Further in Rauvolfia tetraphyllu (Harisararaj et al, 2009) reported good shoot proliferation on BA than Kn.

The successful node cultures were developed in Paraserianthes falcutaria (Sasmitamihardja et al, 2005) on the medium supplemented with BA. SimilarU nodal segment of various leguminous tree species showed good axillary bud proliferation on MS medium supplemented with BA including Albizia chinensis ( Sinha et ai. 2000). Pterocarpous marsupium (Chand and Singh 2004) and Albizia lehbek (Per\een et ai. 2012). Maximum shoot multiplication in Terminalia bellerica (Metha et ai. 2012).

15 ^Rfview of Literature was observed when nodal segments were cultured on MS medium supplemented with BA.

Thidiazuran (TDZ) (A'- phenyl-A^-1, 2, 3-thidiazol-5-yl urea) is a plant bio regulator that induces axillary shoots bud (Binzel et ai, 1996 and Pardhan et al, 1998). TDZ was reported to be more effective in lower concentration for shoot bud induction and at higher concentration produces callus in Hagenia abyssinica (Feyissa et al, 2005). TDZ was also tested best for shoot induction in Cardiospermum halicacabum (Jahan and Anis 2009) on nodal segments. Similar positive effects were also reported in Cannabis sativa (Lata et al, 2009) when the nodal segments were cultured on MS medium supplemented with TDZ. A good effect of TDZ was investigated on in vitro shoot proliferation from nodal segments of Rauvolfia tetraphylla (Faisal et al, 2005).

Metatopolin (niT), is used in the plant tissue culture either alone or in combination with the BA. mT has the direct effect on shoot length enhancement and increase the secondary metabolite production. In Juglans nigra (Pijut et al, 2007) cultured nodal segments on MS basal medium supplemented with mT (4.1 |iM), a significant increase in shoot number, elongation and number of nodes per explants was observed. mT enhanced the shoot proliferation rate in Uniola paniculata (Carmen valego et al, 2009). In Aloe arhorescens, (Amoo et al. 2012), studied the evalvated roles of different aromatic cytokinins types (mT, mTR, MemT and BAR) on direct organogenesis in vitro and anti oxidant activity of regenerated shoots of secondary metabolite production, improvement in every aspect was reported and mT (5.0|iM) gave the largest number of transplantable shoots.

In addition, a range of auxins and cytokinins in combination played a significant role in the multiplication of many plant species. Among the different tested combinations of different cytokinins (BA and Kin) with auxins (NAA, lAA and IBA), BA with NAA has proven to be optimal for regeneration of shoots from nodal segments as is reported in Alhizia lebbeck (Perveen and Anis, 2012), Withania somnifera (Fatima and Anis 2012), Acacia ehrenbergiania (Javed et al, 2013) and Euphorbia cotinifolia (Perveeent'/a/., 2013).

2.5. Subculturing

The rapid rate of shoot regeneration, proliferation and multiplication depends upon the subculturing of the shoot cultures because to avoid toxic effect of closed vessels

16

In Picrorhiza kurro (Upadhyay et al., 1989) shoot number increased as the number of subcultures were increased. 2200 plantlets were obtained from single shoot explants in Thymus vulgaris (Bajaj et al., 1998), grown in vitro for four months followed b> four subculturings. In Kaempferia galanga (Shirin et al., 2001 ). observed 13 fold increase in the plantlet production after every four weeks of subculturing, cultured on MS medium containing BA (12.0 \xM) and NAA (3.0 \xM). Mass multiplication of shoots was reported in Cunila galiodes (Fracaro and Echeverrigaray 2001), without any decline in shoot number up to eight months and were subcultured after every four weeks. Significant shoot improvement was observed in Leptadenia reticulata (Arya ei al., 2003) through repeated transfer of explants on the same fresh medium after ever\ four weeks. Effect of sub culture passage on shoot multiplication was also observed by Siddique and Anis 2007 in Cassia angustifolia where shoot number increased up to fourth passage, became stable during the fifth passage, beyond which a gradual decrease in multiplication rate was observed.

2.6. Rooting

Rooting is the main step of the micropropagation and considerable work has been done to achieve rooting in various plant species like Anemopaegma arvense (Pereira et al.. 2003), Pongamiapinnata (Sugla et al., 2007), Ocimum hasilicum (Siddique and Anis, 2009) and Cassia angustifolia (Siddique et al, 2010). In many plant species rooting greatly depends upon the strength of the MS medium with or without plant growth regulator. For root induction, optimization of basal medium, strength and hormone concentration is very effective. MS medium was found satisfactory for root induction in number of plant species e.g., Vitex negundo (Ahmad and Anis, 2011). Ruta graveolens (Ahmad et al, 2012). Also, good results for root induction were reported on half strength MS medium in Albizia lebheck (Perveen et al, 2011). Bauhinia tomentosa (Naz et al, 2011), Salix tetrasperma (Khan et al, 2011), Cassia angustifolia (Parveen et al. 2012), Cuphea procumbens (Fatima et al, 2012). and Withania somnifera (Fatima and Anis 2012) whereas in Cardiospermum halicacabum (.lahan and Anis, 2009) one-third concentration of MS salts was effective for root formation.

17 ^ffview of Literature

Addition of different auxin (lAA, NAA and IBA) have been proven to be more effective for rhizogenesis in number of woody plants for instance in Liquidambar styraiflua (Pareira et al, 2003), Pterocarpous marsurpium (Anis et al, 2005), Garcinia indica (Malik et al, 2005) and in Rauvolfia tetraphylla (Faisal et al, 2005) and in Mucuna pruriens (Faisal et al. 2006). 100% rooting was achieved in Withania sommferu (Fatima and, Anis 2011), in vitro raised shoots when transferred to half strength MS medium supplemented with 0.5 \iM NAA.

2.7. Hardening and Acclimatization

Aclimatization of the obtained micropropagated plantlets is a critical moment for establishment of good protocol for mass production. Adaptation of plants in greenhouse, field or in the nature is another delicate and difficuh stage. Easy acclimatization depends primarily on the formation and establishment of autotrophic growth. Successful results can be achieved by careful environmental control during acclimatization. Plantlets developed in the in vitro environment faces different conditions like aseptic environment, low diffused light, high relative humidity, low temperature and the chemically well defined medium, rich in sugar and nutrients which is responsible for their heterotrophic growth. These plantlets being very fragile face a number of problems when they are transferred to ex vitro environment due to high rates of water loss when taken out from the culture vessels. Even if the water potential of the substrate is higher than the water potential of the media and due to the poor development of cuticle and stomata, the plantlet may wilt quickly.

In addition, water supply may be limiting because of low hydraulic conductivity of roots and root stem connections (Fila et al, 1998 and Pospisilova et al, 2007). The heterotrophic mode of nutrition and poor mechanism of water control loss make these plantlets vulnerable to transplantational shocks, when directly placed in the green house or field. Many plants die during this transitional period. Therefore, prior to ex vitro transplantation, plantlets needs 2-4 weeks of acclimatization with gradual lowering of humidity and high irradiance to check photo inhibition, caused due to the under developed physiological system. Acclimatizafion period of micropropagated plantlets is thus necessary and very crucial for the better survival in the ex vitro conditions before transferring them in to the garden, field or green house condhions.

Materials and Methods

Materials and Methods

3.1. Plant material and explants sources:

Nodal explants were collected in the month of June 2013 and April 2014 from a 5 year old Rauvolfia tetraphylla and 1 year old R. serpentina respectively, from the field grown plants, maintained in the Botanical Garden of the Aligarh Muslim University, Aligarh.

3.2. Surface sterilization of explants

The young nodal explants of size 1.5cm were collected in glass beaker, washed carefully under the running tap water for 30 minutes, treated with 5% (v/v) savlon (a liquid disinfectant) for 3 minutes, followed by soaking in 5% (v/v) labolenc (Qualigens, Mumbai, India) for 8 min., followed by thorough washing under tap water. The explants were surface sterilized with freshly prepared 0.1% (w/v) mercuric chloride (HgCb) for 3 min. followed by repeated rinsing with sterilized single distilled water 5 times till all the traces of HgCb were removed. The sterilized explants were inoculated onto the freshly prepared culture medium for shoot bud induction, proliferation and multiplication.

3.3. Establishment and preparation of aseptic explants

The aseptic or disinfected Nodal segment explants were inoculated on MS (Murashige & Skoog, 1962) basal media alone or supplemented with different concentrations t)f 6-bezyladenine (BA) 1.0, 2.5, 5.0, 7.5 and 10|iM, kinetin (Kin) 1.0, 2.5, 5.0, 7.5 and lO^M. thidiazuron (TDZ) 0.2, 0.4, 0.6, 0.8 andl.O^M and metatopolin (mT) 5.0, 7.5.

10. 12.5 and 15|J.M for shoot regeneration. The explants used were of 1.5 cm in length.

3.4. Culture media In vitro growth, development and morphogenesis of explants is mainly governed by the composition and modification of the culture media. In the present work MS (Murashige & Skoog, 1962) basal medium was used throughout the experimental work. The chemical composition of various constituents of the MS basal media along with their concentration used, are listed in table 2. 3.5. Preparation of stock solution

MS stock solution was prepared in a series of four stocks- stock I- Macro nutrients (20X). stock II - Micronutrients (200X), stock III Iron salt (lOOX) and stock IV MateriaCs amfMetfiocCs Vitamins (lOOX) except sucrose. All the stocks were prepared separately by dissolving the solute in required amount of double distilled water. MS III stock solution being very photosensitive was immediately wrapped with black paper or aluminum foil to avoid photo oxidation. To prepare one liter of medium, 50 ml of stock 1, 5 ml of stock II, 10 ml each of stock III and IV are required (composition is given in table 3). The stock solutions were stored in a refrigerator at A^C and were regularly checked for any kind of visible or bacterial contamination.

For each plant growth regulator, separate stock solutions were prepared by dissolving them in a few drops of appropriate solvent (IN NaOH for cytokinins and IN absolute alcohol for auxins) and then required amount of DDW was added to make the overall solution of Ijj-M concentration. In the present study, different concentrations of plant growth regulators were prepared from the stock solutions by using the formula:

SiVrS.V.

Here

Si = Strength of stock solution

Vi= Volume of the stock solution required

S2= Strength of the desired solution

Vi = Volume of the desired solution

20 MateriaCs andMetHocfs

Table.3. Nutritional components of MS basal media

Components Amount (mg/1) Macronutrients MgS04.7H20 370 KH2PO4 170 KNO3 1900 NH4NO3 1650 CaCl2.2H20 440 Micron utrients H3BO3 6.2 MnS04.7H20 22.3 ZnS04.7H20 8.6 Na2Mo04.2H20 0.25 CUSO4.5H2O 0.025 C0CI2.6H2O 0.025 KI 0.83 FeS04.7H20 27.8 Na2EDTA.2H20 37.3 Organic supplements (Vitamins) Thiamine HCl 0.5 Pyridoxine HCl 0.5 Nicotinic acid 0.5 Myo-inositol 100 Others Glycine 2.0 Sucrose (g) 30

21 ^Materials atufMetfiods Table. 4. Stock solutions for MS basal medium.

Constituents Amount (mg/1)

Stock solution I Macronutrients (20X) MgS04.7H20 7400 KH2PO4 3400 KNO3 38000 NH4NO3 33000 CaCl2.2H20 8800 Stock solution II Micronutrients(200X) H3BO3 1240 MnS04.7H20 4460 ZnS04.7H20 1720 Na2MoO4.2H20 50 CUSO4.5H2O 05 C0CI2.6H2O 05 KI 166 Stock solution III Iron Salt (lOOX) FeS04.7H20 2780 Na2EDTA.2H20 3730 Stock solution IV Vitamins(lOOX) Thiamine HCL 50 Pyridoxine HCl 50 Nicotinic acid 50 Myo-inositol 10000 Glycene 200

22 MateriaCs andMetHods 3.6. Growth regulators and carbon source

Depending upon the need of experimental plan, MS medium was differently supplemented with plant growth regulators like 6-benzyladenine (BA), Kinetin (kin). Thidiazuron (TDZ) and Metatopolin (mT) either singly or in combination with different levels (O.I, 05, 1.0, 1.5 and 2.0) (iM of auxins a-naphthalene acetic acid (NAA) and indole-3-butaric acid (IBA), as mentioned in the results. Sucrose (3"o) was used as the key carbon source in all the experiments.

3.7. Adjustment of pH and gelling of medium pH 5.8 was maintained by using IN NaOH or IN HCl with the aid of pH meter (Toshcon Industries Pvt. Ltd. Hardwar India) of all the media before adding the gelling agent. The medium was solidified by adding 0.8% (w/v) agar (Thermo Fisher Scientific Qualigens Pvt Ltd., Mumbai India), dissolved in the microwave oven until the clear gel was formed. The medium was then poured in 25 x 150 mm culture tubes and 100 ml Erlenmeyer culture flasks (Borosil, India), each containing 20 ml and 50 ml medium respectively. The medium was also dispensed in the jam bottles with plastic caps. The culture tubes and culture flasks were closed with the cotton plugs. wrapped with butter paper and followed by the sterilization of medium in an autoclave at 1.06 kgcm''^ or 15 psi pressure at a temperature of 121 *^C for 18 minutes.

3.8. Sterilization of glasswares and instruments

All the glassware and instruments (wrapped with the aluminum foil) and distilled water were sterilized by autoclaving at 1.06 kgcm" for 20 minutes. The forceps, scalpel etc made of stainless steel was re-sterilized by dipping them in 70% ethanol followed by flame sterilization and cooling before each inoculation made.

3.9. Sterilization of laminar air flow hood

Laminar air flow hood (NSW, New Delhi) was sterilized by switching on the ultraviolet (UV-B) lamp for 20 minutes prior to the inoculation and then wiping the working surface area with 70% alcohol (ethanol) before the initiation of ever\ operation inside the cabinet.

3.10. Inoculation

All the inoculations were carried out under the aseptic conditions of laminar air flow hood. Every time before starting the inoculation process, the hands were rinsed with

23 Materials and Methods 70% (v/v) ethanol. The instruments used for inoculation were resterlized time to time by dipping them in rectified spirit followed by flaming and cooling. Expalnts were transferred to sterilized petridishes and then inoculated into the culture vials with the aid of sterilized forceps followed by immediate plugging infront of the spirit lamp to avoid any kind of physical or biological contamination.

3.11. Incubation

All the cultures were labeled and placed inside the culture room at 24 ± 2°C, having the relative humidity of 60 - 65 %, photoperiod of 16 h, photosynthetic photon flux density (PPFD) of 20 (im/m^/sec provided by cool white fluorescent tube lights (40W, Phillips, India).

3.12. Subculturing

For further shoot multiplication and proliferation, the inoculated explants were transferred onto the same fresh medium after every three weeks.

3.13. Rooting

The proliferated microshoots of 4-6 cm length were transferred to the freshly prepared MS liquid media, supplemented with the root inducing hormones NAA and IBA with different concentrations. Individual shoots were employed for rooting by two step culture procedure with the aid of filter paper bridge (Whatman No.l). After 5 weeks rooted plantlets were transferred to plastic pots containing sterilized soilrite, and then covered by polybags to retain the proper moisture for ex-vitro conditions. After 2 weeks of exposure to soilrite these plantlets were transferred to the pots containing normal garden soil and manure in the ratio of 3:1. Data on percentage of rooting mean number of roots and root length per shoot were recorded after 4 weeks of inoculation i.e. before their transplantation to the sterilized soilrite.

3. 14. Hardening and acclimatization

Plantlets with well developed shoots and roots were transferred from the culture medium with the help of forcep, washed carefully under the running tapwater and then transferred to the plastic cups containing sterilized soilrite ( Keltech Energies Ltd., Banagalore) under diffused light conditions (16/8h photoperiod). Potted plantlets were covered with the transparent polythene bags to ensure and retain high humidity inside the polybag and were watered with half strength MS salt solution without the vitamins,

24 Materials and Methods for 2 weeks on respective alternate days. The poly-bags were removed after 2 weeks, in order to acclimatize the plantlets to ex-vitro conditions and after 5 weeks, the plantlets were potted in garden soil and were maintained in the green house.

3.15. Chemicals and Glasswares

Plant growth regulators like BA, Kin. TDZ, NAA, IBA and metatopolin were obtained from Sigma- Aldrich (St. Louis. USA). Macro, micro salts, agar, sucrose, and mercuric chloride were purchased from Qualigens, Merck; India. Glasswares, such as culture tubes (25 x 150mm), petridishes (17 x 100 mm), and wide mouth Erlenmeyer flasks (100 ml and 250 ml) were from Borosil India.

3.16. Statistical analysis

In all the experiments, ten replicates per treatment were used and all the experiments were repeated thrice. The effect of different treatments was quantified and data was analyzed statistically using SPSS Ver-10 (SPSS Inc, Chicago,USA). The significance of differences among means were carried out using Duncans Multiple Range Test at P=0.05. The results are expressed as the means ± SE.

25

^suCts

RESULTS

Rauvolfia teiraphylla L. 4.1. Direct shoot regeneration 4.1.1. Effect of Cytokinins The nodal explants were obtained from the plants maintained at Botanical garden o^i the AMU, Aligarh. The explants cultured on hormone free MS media did not show any growth and eventually necrosed. However, when cultured on MS medium supplemented with BA (1.0, 2.5, 5.0, 7.5 or lO^M) and Kin (1.0. 2.5, 5.0. 7.5 or lOfiM). a differential response to shoot induction and proliferation was observed. Multiple shoot buds were observed after two weeks of incubation. BA was found more effective than Kin, for shoot bud induction, multiplication and subsequent proliferation. The maximum regeneration frequency (73%) was observed on MS medium supplemented with BA 5.0 |iM, which also induced the highest mean number (21.6 ± 0.66) of shoots, with the maximum shoot length of (5.5 ± 0.81 cm) after 8 weeks of inoculation. Among all the tested concentrations of Kin, highest shoot regeneration frequency (66%), highest shoot number (7.6 ± 0.33) and maximum shoot length (5.5 ± 0.81cm) were observed on MS medium supplemented with 2.5 i^M Kin. Regeneration frequencies and number of shoots per explant first increased up to the optimal level (BA 5.0|aM) and in case of Kin (2.5^M). On increasing the concentration above the optimal one, the regeneration frequencies and number of shoots got decreased as mentioned in the table 5.

4.1.2. Combined effect of Cytokinin and Auxin

The synergetic effect of the optimal concentration of cytokinins viz. BA (5.0|iM) and Kin (2.5|aM) with different concentrations of NAA was also evaluated. Nodal explants cultured on MS medium supplemented with BA (5.0 |iM) + NAA (0.1, 0.5, 1.0. 1.5 or 2.0 |uM) and Kin (2.5 [M) + NAA (0.1, 0.5, 1.0, 1.5 or 2.0 ^M) separately, showed emergence of multiple shoots after two weeks of inoculation. Presence of high cytokinin and low auxin positively influenced the regeneration frequencies of shoot number and multiplication. The combination of BA with NAA gave significantly more number of shoots than Kin with NAA. Among the various tested combinations BA5.0)aM + NAAO.SjiM, yielded highest regeneration frequency (85%), maximum shoot number (26.3 ± 0.66) with maximum shoots length (6.1 ±

26 (RfsuCts

0.33cm). Among all the tested combinations of Kin 2.5 ^M with NAA (0.1. 0.5, 1.0, 1.5 or 2.0 (JM), highest shoot number (11.0 ± 1.00) with maximum height (5.7 ± 0.39cm) was observed at Kin (2.5|aM) + NAA (0.5|aM) Table 6. The explants cultured on MS medium supplemented with (BA + NAA) were having large and condensed nodes as compared to the (Kin + NAA), which were having larger internodes.

4.1.3. Effect of TDZ

Nodal explants when cultured on the growth regulator free MS medium failed to respond morphologically but when cultured on MS medium supplemented with different concentrations (0.2. 0.4. 0.6. 0.8 or 1.0 |iM) of TDZ; multiple shoot induction was observed after three weeks of inoculation. The explants were transferred onto the same fresh medium after every three weeks. Positive effect of TDZ on shoot induction, multiplication and proliferation was observed upto the four weeks of inoculation. The highest shoot regeneration frequencies (65%) with maximum shoot number (7.6 ± 0.33) and shoot length (4.6 ± 0.19cm) were observed on MS media supplemented with TDZ 0.4 |iM as compared to the other tested concentrations table.7. Shoot number and shoot proliferation increased upto the four weeks. After four weeks the regenerated shoots became hyperhydric, desiccated and distorted when cultured on the same fresh medium.

4.1.4. Combined effect of TDZ and BA

To overcome ill effects of prolonged TDZ exposure, a combined effect of TDZ (0.2, 0.4. 0.6. 0.8 or 1.0 jiM) with BA (l.OjiM) was evaluated. Among all the tested concentrations of TDZ (0.2, 0.4, 0.6. 0.8 and 1.0 \iM) with BA (1.0 |aM), TDZ (0.4 |iiM) + BA (1.0 |iM) was most effective and produced highest shoot number (8.6 ± 0.33) and maximum shoot length (5.2 ± 0.25cm). There was normal shoot development and proliferation after four weeks of inoculation. Shoot number and proliferation first increased up to optimal concentration (0.4jiM TDZ + 1.0 jaM BA) and then decreased on increasing the concentrations of TDZ (0.6, 0.8 and 1.0 ^iM) with BA 1.0 |iM as is depicted well in the table 8.

4.1.5. Effect of metatopolin

Nodal explants failed to grow and did not responded morphologically to the growth regulator free MS media. But when the nodal explants were inoculated on the MS

27 ^suCts medium supplemented with different concentrations (5.0. 7.5, 10, 12.5 or 15|.iM) of mT, emergence of multiple shoot buds was observed after two weeks of inoculation. The number and frequency of shoot bud regeneration and proliferation with regard to the concentration used also varied. On moving from lower (5.0 |j.M) to higher concentration (12.5|aM), an increase in shoot number (4.6 ± 0.17 to 15.6 ± 0.33) and proliferation was observed, however, beyond optimal concentration (12.5|iM m\). shoot number and proliferation decreased as depicted in table (9). Among all the tested concentrations (5.0, 7.5, 10, 12.5 or 15|iM) of mT, maximum regeneration frequency (80%), and highest shoot number (15.6 ± 0.33) with maximum height (5.7 ± 0.25cm) per explant was observed at mT (12.5|iM) after eight weeks.

4.1.6. Rooting of regenerated shoots

For rooting, the in vitro regenerated shoots (4-5cm) were excised and transferred to the MS liquid medium, supplemented with different concentrations of IBA and NAA (0.1. 0.3, 0.5, 0.8 or 1.0 \xM) separately. The basal cut portion of the shoots supported by the Whatman filter paper number 1, resulted in the root formation after two weeks of culture and no rooting was observed on growth regulator free MS liquid medium. The maximum frequency of root formation 90%, number of roots (9.3 ± 1.45) and maximum root length (3.3 ± 0.33 cm) per shootlet was achieved on medium containing IBA (0.3|j,M) after four weeks of incubation. MS medium supplemented with NAA also induced roots on varying degree and gave the optimal rooting at NAA (0.8|iM). Of the two auxins used, IBA was found to be more effective than NAA for root induction as depicted well in the table 10.

4.1.7. Acclimatization

Plantlets having 4-6 fully expanded leaves with well developed roots were successfully hardened off inside the culture room in Soilrite^'^ for 4 weeks and eventually established in garden soil. The percentage survival of the in vitro raised plants was (80%) in Soilrite and about 80% plantlets survived well when transferred to the mixture of garden soil and manure in the ratio of 3:1 (fig. C). The micropropagated plants appeared morphologically similar with normal leaf, shape and growth patterns, without any detectable variation.

28 (l^suCts

Table. 5. Effect of cytokinins (BA and Kin), on shoot regeneration from nodal segments of R. tetraphylla in MS medium after eight weeks of culture.

Growth] regulators |iiM) o//o Mean number Mean shoot BA Kin Response of shoots/ explant length (cm)

0.0 0.0 00 0.0 ± 0.00' 0.0 ± 0.00*

1.0 0.0 10 9.6 ± 1.45''^ 3.6 ± 0.50"'

2.5 0.0 40 12.6± 3.71' 5.5 ± 0.73'

5.0 0.0 90 21.6 ± 0.66' 5.5 ± 0.81"

7.5 0.0 55 16.6 ± 0.66" 4.6 ± 0.24'"

10 0.0 49 13.3 ± 0.33"' 3.9 ± 0.32"'

0.0 1.0 30 5.0 ± 0.57' 4.6 ± 0.16'"

0.0 2.5 76 7.6 ± 0.33"' 4.9 ± 0.86'"

0.0 5.0 53 6.3 ± 0.88"' 4.6 ± 0.28'"

0.0 7.5 49 3.6 ± 0.33" 3.7 ± 0.35"'

0.0 10 38 4.0 ± 0.00' 3.1 ± 0.33'

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.

29 (I(fsuCts

Table.6. Combined effect of cytokinins (BA and Kin) with NAA on in vitro shoot regeneration in R. tetraphylla after eight weeks of culture.

Growth regulators;(HM ) % Mean number Mean Shoot BA Kin NAA Response of shoots/ explant Lengt h (cm). 5.0 0.0 0.1 40 16.0 ± 1.52^ 5.0 ± 0.35'''

5.0 0.0 0.5 85 26.3 ± 0.66^* 6.1 ± 0.33"

5.0 0.0 1.0 72 20.0 ± 1.00^ 4.9 ± 0.31'"

5.0 0.0 1.5 66 16.3 ± 0.33'^ 4.8 ± 0.24*^'

5.0 0.0 2.0 55 14.3 ± 0.88' 4.6 ± 0.14'''

0.0 2.5 0.1 30 6.6 ± 0.88' 4.9 ± 0.45"'

0.0 2.5 0.5 80 11.0 ± l.OO" 5.7 ± 0.39'''

0.0 2.5 1.0 72 9.6 ± 0.88"' 5.3 ± 0.28"^'

0.0 2.5 1.5 65 8.0 ± 0.57' 4.5 ± 0.38'

0.0 2.5 2.0 48 6.6 ± 1.20' 4.6 ±0.41*'^

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.

30

Table. 7. Effect of TDZ on shoot regeneration in R. tetraphylla after four weeks of culture.

TDZ(^M) % Mean number Mean Shoot Response of Shoots/ explants Length (cm) 0.0 0 0.0 ± 0.00" 0.0 ±0.00' 0.02 30 4.6 ± 0.33'' 4.1 ±0.26"'' 0.04 65 7.6 ± 0.33' 4.6 ±0.19' 0.06 60 5.3 ± 0.33" 3.9 ±0.28'" 0.08 40 5.0 ± 0.57'' 3.7 ±0.37'' 1.0 10 2.6 ± 0.33'^ 3.6 ± 0.29" Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.

Table. 8. Combined effect of TDZ and BA on shoot regeneration in R. tetraphylla after four weeks of culture.

Growth regulators o//o Mean number Mean shoot ofshoots/ explant length (cm) TDZ BA Rsponse 0.2 1.0 50 5.3 ±0.33' 4.5 ± 0.25'" 0.4 1.0 76 8.6 ±0.33' 5.2 ±0.25' 0.6 1.0 63 6.6 ±0.33" 4.5 ±0.31'" 0.8 1.0 44 5.6 ± 0.66"' 4.0 ±0.35" 1.0 1.0 25 4.6 ±0.33' 4.0 ±0.35"

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test. 1(fsu[ts

Table. 9. Effect of mT on shoot regeneration in R. tetraphylla after eight weeks of culture. metatopolin (^M) % Mean number Mean Shoot Response of Shoots/explants Length(cni) _ _ 00 0.0 ± 0.00' 0.0 ± 0.00' 5.0 16 5.0 ± 0.57" 4.6 ±0.17" 7.5 33 8.3 ±0.33' 4.7 ± 0.09" 10.0 61 11.3 ±0.33" 5.0 ±0.15" 12.5 75 15.6 ±0.33' 5.7 ±0.25' 15.0 55 12.0 ± l.OO" 4.8 ±0.35"

Values represent means ± SE. Means followed by the same letter are not significantl\ different using Duncan's multiple range test.

32 (RfsuCts

Table. 10. Effect of auxins on root induction from in vitro raised microshoots of R. tetraphylla after four weeks of culture. Auxins (^M) % Mean number Mean root IBA NAA Rooting of roots / shoot length(cm) "(To oo (xo 0.0 ± 0.0^' ooToW

0.1 0.0 30 6.6 ± 0.88"''^ 1.4 ±0.19'^

0.3 0.0 80 9.3 ±1.45' 3.3 ±0.33'

0.5 0.0 68 8.0 ±1.15'"= 1.6 ± 0.0.32*''

0.8 0.0 42 6.3 ±0.88''"' 1.7 ±0.23*''

1.0 0.0 39 4.3 ±0.88"*^ 1.3 ±0.25'

0.0 0.1 15 3.0 ±0.57' 1.2 ±0.24'

0.0 0.3 35 4.6±0.66''' 1.8 ±0.38"'

0.0 0.5 60 5.6 ±0.33'" 2.0 ±0.51"'

0.0 0.8 42 8.6 ±0.88'" 2.6 ±0.38'"

0.0 1.0 36 5.3 ±0.33''' 1.6 ±0.39"'

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.

33 Explanation of figure 1 (A-D)

Multiple shoot induction in R. tetraphylla from mature nodal explants on MS + BA (5.0 ^M)

A. after two weeks of culture.

B. after four weeks of culture.

Inset in figure A- mature nodal explant.

Multiple shoot induction from mature nodal explants of R. tetraphylla on MS + Kin (2.5 \M)

C. after two weeks of culture. D. after four weeks of culture.

Explanation of figure 2 (A-D)

Multiple shoot induction and proliferation in R. tetraphylla from nodal explants on MS + BA (5.0 ^M) + NAA (0.5 |aM)

A. after four weeks of culture. B. after eight weeks of culture.

Multiple shoot induction and proliferation from nodal explants on MS + Kin (2.5 |iM) + NAA (0.5 \iU)

C. after four weeks of culture. D. after eight weeks of culture.

Explanation of figure 3 (A-D)

Multiple shoot induction from mature nodal explants of i?. tetraphylla on MS + TDZ (0.4 (iM),

A. after two weeks of culture B. after four weeks of culture.

Multiple shoot induction and proliferation from nodal explants of R. tetraphylla on MS + TDZ (0.4 |iM) + BA (1.0 ^M)

C. after four weeks of culture. D. after six weeks of culture.

Explanation of figure 4 (A-B)

Multiple shoot induction, multiplication and proliferation from nodal explants on MS+ mT(12.5^M).

A. after four weeks of culture. B. after eight weeks of culture.

Explanation of figure 5 (A-C)

A. In vitro rooting of R. tetraphylla on MS + IBA (0.3 \xM) employing filter paper bridge technique after four weeks of incubation. B. Plantlets with well developed roots and shoots before transplantation in soilrite. C. Hardened and acclimatized plantlets of R. tetraphylla, after four weeks of transplantation to soilrite.

^suCts

Rauvolfia serpentina L.

4.2. Direct shoot regeneration of Rauvolfia serpentina.

4.2.1. Combined effect of cytokinin and auxin.

Nodal explants from the field grown one year old plants of R. serpentina were cultured on MS medium supplemented with various concentrations of BA and NAA in combination (table 11). Initiation of multiple shoot regeneration and elongation of shoot primodia started after two weeks of culture. The best and rapid multiple shoot production was observed on MS medium supplemented with BA (8.0|iM) + NAA (2.0|^M), in which highest shoot percentage (85%) with (7.6 ± 0.70) multipleshoots per culture, within 4 weeks was recorded. An average of 20 cultures showed 35 shoots per flask when subcultured on the same fresh medium every eight weeks.

4.2.2. Rooting

For root induction, individual regenerated (5-6cm) healthy shoots were excised and transferred to half strength MS medium supplemented with (2.0, 4.0 or 8.0|aM) IBA or NAA). Good rooting was attained in a medium containing NAA (2.0)aM) and sucrose (30g/l) within 10-15 days (table 12).

4.2.3 Acclimatization

The plantlets with well developed roots and shoots were transfersd into plastic pots containing a mixture of soil and sand (2:1) and then acclimatized gradually to the natural sunlight. Almost 75% of the regenerated plants survived and showed a vigorous growth without any morphological variation.

34 (RfsuCts

Table. 11. Combined effect of cytokinin (BA) and auxin NAA on multiple shoot formation in Rauvolfia serpentina after four weeks of incubation.

Growth reguIators(|iiM) % Mean number Mean shoot Response of shoots/explant Length (cm) BA NAA To ZO 55 2.2 ± 0.07" 1.6^=0.05"

4.0 2.0 60 3.2 ±1.00' 1.5 ±0.05"

6.0 2.0 75 4.6 ±0.70" 2.6 ±0.07"

8.0 2.0 85 7.6 ±0.70' 4.2 ±0.09'

10.0 2.0 50 2.2 ±0.09" 1.6 ±0.05'

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test. ^suCts

Table.l2. Effect of different auxins in half strength MS media on rooting of miroshoots of Rauvolfia serpentina, after four weeks of incubation.

Auxins itiM) % Mean number of Mean root Rooting roots/shoot length(cm) NAA IBA

0.0 0.0 00 0.0 ± O.OO'' 0.0 ± O-O'

2.0 0.0 95 3.6 ±0.05' 3.0 ±0.07'

4.0 0.0 70 2.7 ±0.05" 2.2 ±0.37'"

8.0 0.0 55 1.7±0.01' 2.0 ± 0.30'"

10.0 0.0 50 1.5 ±0.01' 1.1 ± 0.09"'

0.0 2.0 80 2.8 ± 0.05" 2.4 ±0.41'

0.0 4.0 62 1.9 ±0.10" 2.2 ± 0.30'

0.0 8.0 48 1.7 ±0.05" 1.8 ±0.30'"

0.0 10.0 40 1.2 ±0.10' 1.2±0.10"

Values represent means ± SE. Means followed by the same letter are not significantly different using Duncan's multiple range test.

36 Explanation of figure 6 (A-C)

Induction of multiple shoots and from mature nodal explants in R. serpentina on MS + BA (8.0 |JM) + NAA (2.0^M)

A. after two weeks of culture. B. after four weeks of culture. C. Shoot elongation and proliferation in R. serpentina after eight weeks of culture.

Explanation of figure 7 (A-C)

A. In vitro rooting of R. serpentina on MS + NAA (2.0 i^M) employing filter paper bridge technique after four weeks of incubation.

B. Plantlet with well developed roots and shoots before transplantation in soilrite.

C. Hardened and acclimatized plantlets of R. serpentina, after four weeks of transplantation to soilrite. p m

^ki^ter^ -S m.^i6euA6ian/ (Discussion

DISCUSSION

Increasing world population and decreasing aerable lands have made it a challenge to produce food, medicine and energy that would suffice. One of the most neglected biological entities are the medicinal plants growing in the wild and are diminishing quickly around the globe because of ruthless overexploitation and even more b> the environmental degradation. The need for herbal medicine in recent years has highlighted the importance for conservation and propagation of many prized medicinal plants. An efficient and most suited alternative solution to the problems faced by the phytopharmaceutical industry is the development of in vitro regeneration system for important medicinal species. Plant tissue culture serves as a savior for many prized medicinal plants and the process can be exploited for continuous supply of plant materials to various pharmaceutical companies as well as for generating large number of cloned plants for their rehabilitation in natural habitat, conservation and sustainable utilization.

During the last 10 years, a number of woody plants have been successfully propagated in vitro using juvenile and mature nodal explants for direct shoot regeneration Ahmad et al, (2006), Faisal and Anis (2007), Khan et al, (2011), and Parveen et al, (2013). Micropropagation offers means for rapid and mass multiplication of the existing stock of germplasm for biomass production and conservation of the rare and threatened species (Hussain and Anis 2004, Anis et al, 2005 and Parveen et al, 2010) and Ahmad et al., 2013). Keeping in mind the need for micropropagation, the results obtained during the study in R. tetraphylla and R. serpentina have been discussed in the light of existing literature on the subject.

5.1. Direct shoot regeneration in R. tetraphylla.

Microproagation of R.tetraphylla has been achieved through rapid proliferation of nodal segments on MS medium, supplemented with different cytokinins like BA, Kin, TDZ and mT singly or in combination with auxin (NAA). The nodal segments failed to respond morphogenetically when cultured on growth regulator free MS media. All the cytokinins used in the present study facilitated axillary bud induction but the maximum shoot bud induction and proliferation was recorded on MS medium supplemented with 5.0^M BA. The edge of BA over other cytokinins has also been reported in several medicinal and aromatic plants viz. Tylophora indica (Faisal and

37 (Discussion

Anis, 2003), Pterocarpous marsupium (Anis et al, 2005), Mucuna pruriens (Faisal et a/., 2006). Cassia siamea (Sreelatha et al, 2007), Rauvolfia tetraphylla (Harisararaj et al. 2009), Terminalia bellehca (Metha et al. 2012) and Albizia lebbeck ( Perveen et al, 2013).

Variation in shoot growth parameters in terms of shoot number and shoot length was found when minor differences were made in the respective concentrations. Increase in concentration of BA beyond the optimal level had a negative effect and exhibited reduction in regeneration frequencies and shoot number from each explant. Similar investigations have been reported in many plant species Albizia chinensis (Sinha et al, 2000) and Pterocarpous marsupium (Anis et al, 2005). The combined effect of auxins and cytokinins on shoot induction and multiplication of various medicinal plants have been reported (Jahan et al, 2011, Perveen et al, 2011 and Varshney and Anis, 2012).

Cytokinin is required, in optimal concentration for shoot induction and proliferation in many genotypes however, inclusion of auxin along with cytokinin enhanced the shoot regeneration (Rout et al, 1999, Ahmad et al, 2013). Highest shoot number and maximum shoot length was obtained when optimal cytokinin BA (5.0^M) was combined with lower concentration of an auxin NAA (0.5|iM). The synergistic effect of BA and NAA has been reported in many medicinal plant species such as Vitex nigundo (Ahmad et al, 2013), Rauvolfia tetraphylla (Faisal et al, 2012), thus, individual and interactive effect of BA and NAA could ensure better in vitro regeneration and their synergism in proper concentration was extremely favourable for shoot multiplication. In accordance with these reports, the present study also demonstrated the positive modification of shoot bud induction efficacy, obtained by employing low concentration of auxin in addition with cytokinins.

Among the different tested combinations of cytokinins with auxin, BA with NAA was found optimal for regeneration of shoots from nodal segments. Similar observations have been reported in various plant species like Albizia lebbeck (Perveen and Anis 2012), Withania somnifera (Fatima and Anis 2012), Acacia ehrenbergiania (Javed et al, 2013) and Euphorbia cotinifolia (Perveeen et al, 2013). These results are further supported, by the earlier findings of several workers, who reported the addition of low level auxin with cytokinin promoted shoot induction and proliferation as in Wrightia tinctoria (Purohit and Kukda, 1994, Acacia catechu (Kaur et al, 1998), Psoralea

38 (Discussion corylifolia (Anis and Faisal 2005), Rauvolfia tetraphylla (Faisal et al, 2005). Terminalia arjuna (Pandey et a/., 2006)., R. serpentina (Baksha et al., 2007) and R. serpentina (Panwar et al., 2011).

Thiazuron (N-phenyl-N-1, 2, 3-thidiazol-5-yl urea) has cytokinin like activity and has been effective in low concentrations to stimulate shoot formation (Sankhla et al, 1996. Binzel et al, 1996, Murthy et al, 1998). Use of TDZ for woody plant regeneration has been excellently reviewed by Huetteman and Preece (1993). fhis potent cytokinin has been used for most of the plant species including Psoralea corylifolia (Faisal and Anis 2006), Pterocarpous marsupium (Hussain et al, 2007) and Vitex negundo (Ahmad and Anis 2007).

In addition to the other cytokinins nodal explants were also cultured on MS medium supplemented with TDZ. Among all the tested concentrations (0.2, 0.4, 0.6, 0.8 or 1.0 \iM) of TDZ, 0.4^M was more effective for maximum number of shoot and shoot length after four weeks of culture. The shoots became hyperhyric, distorted and fasciated when subcultured on the same fresh medium after four weeks. Prolonged exposure to TDZ had also caused hyperhydric, stunted, distorted and fasciated shoots as reported in Rhododendron (Preece and Imel 1991), Dalbergia sisso (Pradhan et al. 1998), Rauvolfia tetraphylla (Faisal et al, 2005) and Cassia angustifolia (Siddique and Anis 2007). To overcome these ill effects, the nodal explants were cultured on the MS medium supplemented with BA and TDZ. Among all the tested combinations of (TDZ and BA), TDZ (0.4 f^M) + BA (1.0 |aM) proved to be optimal in regenerating normal shoots.

Metatopolin (mT), is used in the plant tissue culture either alone or in combination with the BA and has a direct effect on shoot length enhancement and increase in secondary metabolite production (Randall et al, 2011). In sea oats Uniola paniculala L. genotypes. Carmen valego et al, 2009 reported the positive effect of mT over BA for shoot multiplication. In Aloe arborescens, Amoo et al, (2012) evaluated the role of different aromatic cytokinin types and concentrations on direct organogenesis and anti-oxidant activity of regenerated shoots, and secondary metabolite production and reported improvement in every aspect. In the present study, nodal segments of R. tetraphylla were cultured on MS medium supplemented with different concentrations of mT and 12.5|LIM mT was found to be optimal for maximum shoot regeneration and proliferation. Also, Steven et al, (2007) reported that, when nodal segments of

39 (Discussion

Juglam nigra were cultured on MS medium supplemented with mT, significant increase in shoot number, elongation and number of nodes per micro shoot was observed.

5,2. In vitro rooting in regenerated shoots oiR. tetraphylla

Rooting of in vitro regenerated microshoots and transplantation of the plantlets to the field is the most important, crucial and essential step, but a difficult task in tissue culture of woody plants. The microshoots failed to develop roots on growth regulator free MS medium. The regenerated well developed shoots (4-5 cm) were isolated from the proliferated cultures and were transferred to full strength MS medium supplemented with different concentrafions (0.1, 0.3, 0.5, 0.8 and 1.0 fxM ) of NAA and IBA separately. Among all the tested concentrations of auxins NAA and IBA, 0.3|iM IBA was most effective and proved to be optimal for in vitro rooting of R. tetraphylla, on which 80% of the regenerated shoots developed roots with an average root number (9.3 ± 1.45) and length (3.3 ± 0.33cm) per shoot after four weeks of incubation. Out of the two auxins tested, IBA was found more effective than NAA.

Effectiveness of IBA for in vitro rooting has been reported for several medicinal plants including Gymnema sylvestre (Komalavalli and Rao 2002), Tylophora indica (Faisal and Anis 2003), Pesudarthria viscida (Baskar et al., 2006), Clitoria ternatea (Shazad et al., 2007) and Abrus precatorius (Perveen at al, 2013). The edge of IBA over NAA in root development may be due to several factors such as its preferential uptake, transport and stability over other auxins and subsequent gene activation (Ludwig -Mller, 2000).

5.3. Direct shoot regeneration in R. serpentina

Micropropagation of R. serpentina has been achieved through rapid proliferation of nodal segments on MS medium supplemented with different concentratios (2.0 - 10|iM) of BA with NAA (2.0|iM). All the combined concentrations of cytokinin (BA) with auxin (NAA) facilitated axillary bud induction and shoot proliferation but BA (8.0 laM) along with NAA (2.0^M) proved to be optimal for the highest shoot regeneration frequency (85%) with maximum shoot number (7.6 ± 0.70) and shoot length (4.2 ± 0.09)cm, after four weeks of culture. The synergisfic effect of higher concentration of cytokinin (BA) with low concentration of auxin (NAA) has proved to be optimal for best shoot regeneration, proliferafion and mulfiplication in many

40 (Discussion

medicinal plants {Mucuna pruriens Faisal et al., 2005, Rauvolfia tetraphylla Faisal et ai. 2012, Vitex trifolia Ahmad et a/.. 2013 and Acacia ehrenbergiana Javed et al. 2013).

5.4, In vitro rooting in regenerated stioots of R. serpentina

The regenerated well developed microshoots (4-5cm) were isolated from the proliferated cultures and were transferred to the half strength MS medium supplemented with different concentrations (2.0-10.0 \iM) of auxin NAA and IBA separately. Among all the tested concentration half strength MS medium supplemented

with NAA (2.0|JM) proved to be optimal for the highest rooting regeneration frequency (95%) with maximum root number (3.6 ± 0.05) and root length (3.0 ± 0.07 cm). Similar findings have been reported in Vitex trifolia (Ahmed and Anis 2012) and Withania somnifera (Fatima and Anis 2012). Further work is in progress.

Hardening and Acclimatization

Hardening and acclimatization are the most important and crucial steps for achieving success in micropropagation program. Micropropagated plantlets when transferred from in vitro to ex vitro conditions like green house or field show low survival or reduced growth rate due to sudden changes in the environment (Eliasson et al.. 1994 and Pospisilova et al, 1999). The special conditions during in vitro culture results in the formation of plantlets of abnormal morphology, anatomy and physiology, when such in vitro raised plantlets are transferred to a relatively less humid external environment they undergo desiccation, water loss and finally death (Selvapandiyan et a/., 1988). Thus, an acclimatization period of 2-3 weeks to overcome these abnormalities is required.

In the present study, the rooted plantlets of R. tetraphylla and R. serpentina were successfully hardened off inside the growth room in a selected planting substrate (soilrite). Type of potting mixture used to acclimatize plants under ex vitro conditions is one of the important factors in determining the survival percentage of the plants during acclimatization process.

Similarly various hardening substrates like garden soil, vermiculite, soilrite etc, were used by several workers, Faisal et al., (2006), Tiwari et al., (2001) and Varshney and Anis (2012). Soilrite being more porous substrate holds more water than vermiculite and garden soil and thus promote better growth of tender roots of tissue culture raised

41 (Discussion plants during hardening. The plantlets were covered with transparent polythene bags to maintain high humidity and also to prevent desiccation with the control temperature, photoperiod and irradiance. These were then transferred to the garden soil after four weeks.

42

Summary and ConcCusion

SUMMARY AND CONCLUSION

Plant tissue culture technique provides a viable alternative for managing the valuable assets (phytodiversity) in a sustainable manner. Micropropagation provides an efficient method for ex situ conservation of plant biodiversity and mass multiplication of various plant species from a very small piece of available plant material.

Plant species and cultivars are genetically specific to their nutrient requirements hence no universal medium has been devised for in vitro cultures. An understanding of optimal nutrient concentration could lead to increased growth and evoke morphogenesis in vitro more efficiently. Axillary shoot bud proliferation is known to be a reliable method for clonal propagation of many economic plants.

Micropropagation is the only aspect of plant tissue culture that has been convincingly documented with regard to its feasibility for mass scale propagation commercially. Success of in vitro regeneration depends on the control of morphogenesis, which is influenced by several factors namely genetic back ground, kinds of tissue or explants, nutritional components, growth regulators and culture environment.

The findings of my experimental investigation lead to the following conclusions;

1. Direct multiple shoot regeneration has been obtained from nodal explants using different cytokinins (BA, Kin, mT) either singly or in combination with auxins (IBA and NAA) in R. tetraphylla. 2. Among different cytokinins tested, MS medium supplemented with BA (5.0 ^M) was found suitable for multiple shoot regeneration in R. tetraphylla. 3. Maximum shoot numbers 26.3 ± 0.33 with 5.0 ± 0.33 cm shoot length was achieved on MS medium containing BA (5.0|iM) + NAA (0.5|iM) in nodal explants of 7?. tetraphylla, after 8 weeks of culture. 4. Of the different concentrations of mT tested, 12.5 |aM was found best for the maximum shoot regeneration where 15.6 ± 0.33 shoots whh 5.7 ± 0.25 cm shoot length was obtained after eight weeks in R. tetraphylla.

43 Summary and ConcCusion

5. A combination of TDZ (0.4|iM) and BA (1.0 |aM) was found optimal for (76%) regeneration, 8.6 ± 0.33 shoots and 5.2 ± 0.25 cm shoot length after four weeks of culture in R. tetraphylla. 6. MS medium supplemented with 0.3 |aM IB A gave maximum (80%) rooting after four weeks of culture in R. tetraphylla. 7. A combination of BA and NAA proved effective for the direct shoot regeneration in R. serpentina. MS medium supplemented with BA (8.0|iM) + NAA (2.0|am) proved to be optimal for maximum shoot regeneration. 8. Vi MS medium supplemented with NAA (2.0|iM) showed best Rooting in R. serpentina, after four weeks of incubation. 9. The in vitro raised plantlets were successfully hardened off in Soilrite followed by their transfer to garden soil.

44

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