1

Chapter I

In vitro regeneration studies on Dioscorea prazeri

Global view

1.1Objectives of the study

1.2Introduction1.2.1 Micropropagation1.2.2 History of the plant Dioscorea prazeri1.2.3 Taxonomy of the plant Dioscorea prazeri1.2.4 Fingerprinting analysis for Dioscorea prazeri1.2.5 Need for in vitro propagation studies

1.3Materials and Method1.3.1 Media Preparation1.3.2 Plant Material1.3.3 In vitro establishment of explants1.3.4 Acclimatisation of Plantlets1.3.5 Morphological, biochemical and genetic evaluation1.3.6 Statistical analysis

1.4Results1.4.1 Authentification of D. prazeri with pharmacological aspects1.4.2 Micropropagation1.4.3 Genetic stability assessment1.4.4 Statistical data analysis

1.5Discussion1.5.1 Micropropagation1.5.2 Genetic fidelity assessment

1.6References I

2

Global view

With a growing focus on the importance of medicinal plants and traditional health

systems the international trade of medically significant plants has shown a phenomenal

growth. Interest in natural materials by the dominant economic powers enhanced with

emergence of new possibilities in biotechnology and drug synthesis. It is only during the

last decade that the real significance of the medicinal plants sector has begun to be

realized. By the mid-1980s, there was a renewed interest in natural products and

approaches to health care, with recognition that technology alone could not solve the

pressing health care needs of the world’s population (Tempesta and King, 1994). The

failure of modern medicine to provide effective treatment for chronic diseases and

emergence of multi-drug resistant bacteria and parasites has resulted in an increase in the

use of traditional medicine in developed countries. Alternative treatments are now

increasingly favoured with the adverse effects of chemical drugs becoming known. The

approaches and assumptions of allopathic medicine have been met with apprehensions

and their increasing costs and greater public access to information on traditional medicine

has evoked greater interest for the latter, especially in rural localities. This is evident from

studies that reveal that there are more traditional medicine providers than the allopathic

providers in the rural areas (WHO, 2002). More than 30% of the entire plant species are

used for medicinal purposes and over three-quarters of the world population trust mainly

on plants and plant extracts for health care. According to the World Health Organization,

over 80% of the world’s population, or 4.3 billion people, rely upon such traditional

plant-based systems of medicine to provide them with primary health care (Bannerman et

al., 1983).

A strong revival of the Indian systems of medicine like Ayurveda and Siddha and

a thoughtful, high level investment in developing the medicinal plant base and manpower

is needed to translate our traditional skills and resources to a functional, modern system.

India’s diversity is unmatched due to the presence of 16 different agro-climatic zones, 10

vegetation zones, 25 biotic provinces and 426 biomes (habitats of specific species). Of

these, about 15000-20000 plants have good medicinal value. However, traditional

communities in India use only 7000-7500 species for their medicinal values. Drugs of

herbal origin have been used in traditional systems of medicines such as Unani and

3

Ayurveda since ancient times. The Ayurveda system of medicine uses about 700 species,

Unani 700, Siddha 600, Amchi 600 and modern medicine around 30 species. The drugs

are derived either from the whole plant or from different organs, like leaves, stem, bark,

root, flower, seed, etc. Some drugs are prepared from excretory plant product such as

gum, resins and latex. Even the Allopathic system of medicine has adopted a number of

plant-derived drugs, which form an important segment of the modern pharmacopoeia.

Some important chemical intermediates needed for manufacturing the modern drugs are

also obtained from plants (Diosgenin, solasodine and β-ionone). Not only does plant-

derived drug offer a stable market worldwide, but also plants continue to be an important

source for new drugs.

According to the International Trade Centre, as far back as 1967, the total value of

imports of starting materials of plant origin for the pharmaceutical and cosmetics industry

was of the order of USD 52.9 million. From this amount, the total values grew to USD

71.2 million in 1971, and then showed a steady annual growth rate of approximately 5-

7% through to the mid-1980s. It is estimated that world market for plant derived drugs

may account for about Rs.2, 00,000 crores. Presently, Indian contribution is less than

Rs.2000 crores. Indian export of raw drugs has steadily grown at 26% to Rs.165 crores in

1994-’95 from Rs.130 crores in 1991-’92. The annual production of medicinal and

aromatic plant’s raw material is worth about Rs.200 crores. This is likely to touch US

$1150 by the year 2000 and US $5 trillion by 2050. Green plants synthesize and preserve

a variety of biochemical products, many of which are extractable and used as chemical

feed stocks or as raw material for various scientific investigations. Many secondary

metabolites of plant are commercially important and find use in a number of

pharmaceutical compounds. However, a sustained supply of the source material often

becomes difficult due to the factors like environmental changes, cultural practices,

diverse geographical distribution, labour cost and selection of the superior plant stock and

over exploitation by pharmaceutical industry. Over–exploitation has even led to the

detriment of natural habitats and indigenous populations.

The scientific study of traditional medicines, derivation of drugs through bio-

prospecting and systematic conservation of the concerned medicinal plants are thus of

great importance (Joy et al., 2001). This new drive for natural and plant-based medicines

4

was observed in the market from the mid-1980s onwards. Growth in the market in

various regions is now on average 3 to 4 times the average growth rates of the national

economies in the same regions. Some of these phenomenal rates, in some cases nearly

20%, imply that the market is now doubling in size every 4-5 years. Dioscorea is one

among the major medicinal plants that is indigenous to India and have established

demand for their raw materials (Kumar et al., 1997). D. prazeri, which belongs to the

dioscorea genus, is an endangered plant having immense pharmaceutical value.

Hence, this research on conservation and in vitro propagation of the endangered

medicinal plant, Dioscorea prazeri is highly advantageous and necessary for rapid

multiplication. The enhancement of active components in plant system and the extraction and

characterization of these active components, using the advances in plant tissue culture and the

biotechnological tools enable sustainable use of medicinal plants for present and future

generations.

1.1 Objectives of the study

1. In vitro regeneration studies on Dioscorea prazeri.

2. Development of Germplasm conservation technique for Dioscorea prazer.

3. Diosgenin extraction and characterisation in Dioscorea prazeri.

4. (I)Agrobacterium mediated genetic transformation of Dioscorea prazeri.

(II)Morphological, Biochemical, Molecular analysis and Cell assays.

5

Sterile Explants

Somatic Embryo

MicropropagationTechniques

Obtaining Endangered MedicinalPlants

Explants

Adventitious ShootCultures In vitro Shoot cultures

Diosgenin Pathway AnalysisGermplasm Conservation

Techniques

Establishment ofplants

Genetic Transformationstudies

Product Formation

Extraction andAnalysis

Morphological, Molecular and Biochemical analysis

Establishment ofplants

Objective – Overview

6

1.2 Introduction

Medicinal plants play an undisputed role in today’s health care needs. There are over

1500 species of significant medicinal plants reported thus far from India. The demand for

medicinal plants is ever increasing and some medicinal plants are in great demand for

pharmaceutical industries. Medicinal plants provide an important therapeutic aid,

particularly in the third world countries to meet their health care needs. It is of utmost

importance that these plants be propagated as it will reduce the pressure on natural

resources, share the raw materials to the pharmaceuticals and other concern industries

and help in conservation of medicinal plants that are being pushed to the danger of

extinction.

Yams are monocots belonging to the family Dioscoreaceae (Ayensu E.S. and

Coursey D.J, 1972). Yam tubers are staple food for millions in many tropical and sub-

tropical countries (Onwueme, 1978). Dioscorea, one of the important genera of yams,

comprises of about 600 species that have medicinal and edible importance. The large-

scale cultivation is, however, restricted to three main areas: West Africa, Southeast Asia

including adjacent parts of China, Japan, and Oceania, and the Caribbean. It can be

cultivated only in specific climatic zones like Northwestern and Northeastern Himalayas.

Dioscorea species were investigated from Ghana such as, D. bulbifera, D. burkilliana, D.

hirtiflora, D. munutiflora, D. praehensilis, D. togoensis and D. zingibarensis (Quigley,

1978). The significant species of the genus are Dioscorea prazeri and D. deltoidia found

at high altitudes in India, D. composita, D. floribunda and D. mexicana in Mexico, D.

elephantipes (L.) and D. sylvatica in South Africa. D. composita, D. floribunda and D.

mexicana in Mexico, D. elephantipes (L.) and D. sylvatica in South Africa. While some

species i.e., Mexican D. floribunda appears to adapt throughout north Indian plains and

south India, all other species are difficult to collect and grow. Dioscorea prazeri was

distributed in the moist regions of the Himalayas in North Bihar, Nepal, West Bengal,

Sikkim, Bhutan and Abor hills of Arunachal Pradesh. The plant has also been detected at

an altitude of 1,220 m in the Himalayas, or perhaps even higher (Asolkar, 1979). Over-

harvesting has threatened the natural populations of D. prazeri and efforts at cultivation

have met with limited success (Coursey, 1976). The long-term availability of many

7

Himalayan herbs has become uncertain due to indiscriminate harvesting resulting in

possible threat of extinction (Badoni et al., 2010).

Most of the Diosgenin in India was from the indigenous Dioscorea and this

species has been extensively exploited for collection and processing by various drug

companies because of high Diosgenin content in its tubers. D. prazeri is one of the best

sources of Diosgenin, found at high altitudes in India. Due to indiscriminate harvesting

of this species from natural habitat, the population of the species has shrunken in such a

way that it has been listed amongst the threatened genera in the Red Data Book (Global

environment facility and UNDP, 2002; Batugal et. al., 2004). However, this species still

remains remarkably important because of its pharmaceutical benefits and the tuber and

Diosgenin are sold at high price. Furthermore, reliable proliferation of shoots and

subsequent plant regeneration are important for massive plant propagation studies on D.

prazeri for utilization of its therapeutic properties and commercial applicability.

Tissue culture offers the means for rapid and mass multiplication of existing

stock of germplasm and also for conservation of important, elite endangered plants

(Razdan, 2003). Among the different approaches, in vitro culture method provides new

means of conserving and rapidly propagating valuable, rare and endangered medicinal

plants from extinction (Nalawade et al., 2003; Thomas and Shankar, 2009; Rahman et

al., 2009). Micropropagation of the D. prazeri will be the perfect tool for re-establishing

the plant in the natural habitat. Advantages of this technique would further include

improvement in growth conditions of the plants, space usage and and better facilitation

of international germplasm exchange (Alderete et al., 2006). Propagation of endangered

species is most appropriate for the species that have strongly reduced population and

species with low germinative activity of seeds

In vitro production of the related species of D. floribunda (Sengupta et al., 1984),

D. alata (Jasik and Mantell, 2000) D. composita (Alizadeh, 1998) have been reported.

Micropropagation of the endangered species of Dioscorea prazeri, an important

medicinal yam of India has not been carried out. Since the micropropagation protocols

are mostly clonal specific in nature, development of these procedures for D. prazeri is

imperative.

8

The tissue culture technique reported herein demonstrates consistent production

and proliferation of shoots and subsequent plant regeneration, important for massive

plant propagation studies on D. prazeri for utilization of its therapeutic properties and

commercial applicability. In vitro propagation is a possible means of avoiding loss of

embryogenic potential and maintaining genetic stability of highly significant medicinal

plant like Dioscorea prazeri with the optimised hormonal treatment and

micropropagation strategies. Occurrence of somaclonal variability can be successfully

eliminated by suitable methods of micropropagation (Mikulik, 1999). The study on

morphological, genetic and biochemical stability of the plant under the standardised in

vitro growth conditions reaffirms the applicability of the method for large-scale

propagation of this indigenous, endangered medicinal plant.

1.2.1 Micropropagation

Micropropagtion exploits the aspects of cellular totipotency and can be defined as the

propagation of a whole plantlet from isolated cells or a small piece of tissue and organs

under asceptic conditions. This multidimensional science has proved to be highly

significant in a variety of ways including plant propagation, in vitro raising and

maintenance of high health status plants, germplasn storage and a valuable technique in

crop improvement by genetic engineering.

Plant regeneration from a cultured tissue can be achieved by culturing tissue

sections lacking preformed meristems, which is adventitious in origin or from callus and

cell cultres. Axillary buds are preformed meristems while adventitious regeneration

occurs at unusual sites such as internode, leaf blade, cotyledon or root elongation zone.

The different regeneration approaches may yield different propagation rates. Axillary bud

propagation and culture of individual nodes are techniques most widely used in

commercial micropropagation and which shows the least variation among the propagated

plants (Chu, 1992).

1.2.2 History of the plant Dioscorea prazeri

The genus Dioscorea is accredited to a renowned Greek Physician Dioscorides Pedanius

and the specific epithet prazeri after the name of J.G. Prazer, a collector of Sir George

9

King, who was the Director of Royal Botanic garden, Calcutta. D. prazeri (Prain and

Burkill, 1936) plant belongs to a conserved family Dioscoreaceae, which was primarily

described by Robert Brown on 27th march 1810 and was based on the type genus

Dioscorea. Several species of Dioscorea collectively known as yams have significant

economic value. Their tubers contain a large content of starch and have been used as a

famine food. It is an excellent source of raw material for the manufacture of steroids like

cortisones. The highly vital compound obtained from tubers on hydrolysis gives ‘steroidal

sapogenins’. Chemically saponins are glycosides. There are various classes of sapogenins

found in different species like Diosgenin, Yamogenin, Botogenin, Krytogenin etc.

Various steroid drugs have been synthesized from Diosgenin like corticosteroids, sex

hormones, anabolic steroids and oral contraceptives. Dr. R.N Chakravarty (School of

tropical medicine), Calcutta discovered that D. deltoidea and D. prazeri contained

appreciable amount of Diosgenin in rhizome. In 18th and 19 centuries, herbalist used wild

yam to treat menstrual cramps and problems related to childbirth. The subsequent

discovery of the steroidal sapogenin, Diosgenin in tubers of wild yam, revolutionized

pharmaceutical industry. Diosgein involved in the production of the hormone

progesterone. Diosgenin has saved a key role for the production of hormones and for the

development of birth control pills, considered as two of the major advances in the plant

drug medicine. Wild yams continue to be used for treating menstrual cramps, nausea and

morning sickness, inflammation, osteoporosis, menopausal symptoms and at present

chiefly as anti-cancerous and other health conditions.

1.2.3 Taxonomy of the plant Dioscorea prazeri

Dioscorea prazeri is a rhizomatous plant. The rhizomes are branched, stout, gray

brownish or black with fibrous roots scattered all over the surface. The stem is 2.0mm in

diameter, glabrous, unarmed, smooth, green and twining to the left. The leaves are

alternate, rarely opposite at the base of the stem, cordate, gradually acuminate or with a

short acumen at the apex; lobes at he base of the leaves are rounded. Male flowers are

racemes and form axillary with 1-3 rachises from each axil. Flowers sometimes form

terminal panicle with 10-20 cm long rachis with 3mm long flowers. Female flowers are

10

bracteate, inspike, glabrous and 4 to 5 mm long. The botanical classifications are as

follows (Table 1.1).

Table 1.1 Botanical classification of Dioscorea prazeri

Classification of Dioscorea prazeri

Kingdom Plantae

Phylum Tracheophyta

Class Liliopsida

Subclass Liliidae

Order Dioscoreales

Family Dioscoreaceae

Genus Dioscorea

Specific epithet prazeri - Prain & Burkill

Botanical name Dioscorea prazeri Prain & Burkill

1.2.4 Fingerprinting analysis for Dioscorea prazeri

The parameters for fingerprinting such as Total ash test, Acid insoluble ash test, Water

soluble extractive, Alcohol soluble extractive has to be analysed as an initiation of the

whole experiment and compared with the value obtained on pharmacopeia encyclopedia.

1.2.5 Need for in vitro propagation studies

Dioscorea prazeri (Prain and Burkill) is an economically important plant and has

become very rare in natural habitat due to over exploitation inevitably resulting in virtual

extinction. The D. prazeri grown in West Bengal is a good source of Diosgenin in

comparison with the tubers grown in other regions (Asolkar and Chanda, 1979).

Declining facet of this important group of medicinal plants due to negligence and

unawareness of local communities has been a serious concern and therefore invites due

attention before the entire population becomes extinct. Great advances in research on

medicinal plant species have been witnessed in recent years but not the significant

species D. prazeri. Biotechnology has to play an important role to restore, conserve,

utilize and improve this important species in a profitable manner. So the in vitro studies

on this plant would be very useful for reestablishing this commercially and medicinally

11

important plant. Micropropagation and re-establishment of these plants are highly

needed for saving the highly valuable plant from complete extinction.

1.3 Materials and Method

1.3.1 Media Preparation

Modified MS MediumThe culture medium consisted of the mineral salts and organic nutrients of Murashige and

Skoog medium (Murashige and Skoog, 1962), 3% sucrose and 0.8% agar. The basal

medium was supplemented with different combinations of growth regulators such BAP,

NAA, GA3, Thiadizuron (TDZ), Zeatin, Kinetin and 2-isopentanyl adenine (2iP).

i. Auxins, Cytokinins and Gibberellins

The right proportion of auxins that resembles chemically to aminoacid tryptophan known

to regulate cell elongation, cell division, root initiation, delays leaf senescence inhibit or

promote leaf abscission and assimilate movement towards auxin. The biosynthesis

happens from tryptophan through transamination, decarboxylation and oxidation.

Cytokinin with structure similar to adenine that promotes cell division, stimulate

morphogenesis in tissue culture, regulate leaf expansion, inhibit root formation, activate

RNA synthesis, stimulate protein and enzyme activity. Its biosynthesis happens through

biochemical modification of adenine (Davies, 1995; Raven et.al., 1992).

Gibberellins resembles entgibberellane skeleton. The gibberellins stimulate stem

elongation through cell divison and elongation, break dormancy, induce maleness in

dioecious flowers and delay senescence in leaves and fruits. These are diterpenes

synthesised from acetyle Co-A through melvonic acid.

ii. Macro NutrientsTable 1.2 Composition of MS-Macro Nutrients for in vitro regeneration media for D. prazeri

Composition Weight in g L-1

Potassium Nitrate (KNO3) (MERCK; Cat.No. UN 1486) 38 g

Ammonium Nitrate (NH4NO3) (MERCK; Cat.No. UN 19420 33 g

Magnesium Sulphate (Mg SO4.7 H2 O) (MERCK; Cat.No. 105886) 7.4 g

Potassium Dihydrogen Phosphate (KH2PO4)(HIMEDIA; Cat.No. RM 1188) 3.4 g

Calcium Chloride (CaCl2.2 H2O) (SIGMA; Cat.No. C336) 8.8 g

12

iii. Micro NutrientsTable 1.3 Composition of MS-Micro Nutrients for the regeneration media for D. prazeri

Composition Weight in gL-1

Manganese Sulphate (MnSO4.H2O)(SIGMA; Cat.No. 7899) 1.5

Zinc Sulphate (ZnSO4.7 H2O)(SIGMA; Z-4750) 0.86

Boric acid (H3BO3)(SIGMA; B-7901) 0.62

Copper sulphate (CuSO4.5H2O) (SIGMA; C-8027) 0.0025

Coblat Chloride (CoCl2.6 H2O) (SIGMA; C-2644) 0.0025

Potassium Iodide (KI) (SIGMA; P-8256) 0.083

Sodium Molybdate (Na2MoO4.2H2O) (SIGMA; S-6646) 0.025

iv. MS-IronTable 1.4 Composition of MS-Iron for the media for D. prazeri in vitro propagation study

Composition Weight in gL-1

EDTA (C10H14N2Na208.2H2O)(SIGMA) 1.862

Ferrous Sulphate (FeSO4.7H2O) (SIGMA) 1.392

v. VitaminsTable 1.5 Composition of Vitamins used for the standardised media for D. prazeri

Component Weight L-1

Myo-inositol (C6H12O6) (HI MEDIA; RM 102-100) 100 mg

Nicotinic acid (C6H5NO2) (SIGMA; M-0761) 100 µg

Pyridoxine (C8H11NO3.HCl)(SIGMA; P-8666) 50 µg

Thiamine (C12H17CIN4OS.HCl) (SIGMA;T-3902) I. g

vi. SugarTable 1.6 The sugar used for the optimisation media for D. prazeri

Sucrose (C12H22O11) (MERCK; 116141) 30gL-1

13

vii. Solidifying agentTable 1.7 The gelling agents used for semi solid media

Components Weight in gL-1

Phytagel (SIGMA; P-8169) 2.95

Agar (C12H18O9) (SIGMA; Y-3252) 8.0

viii. Growth RegulatorsTable 1.8 The growth regulators used for standardisation for efficient regeneration of D. prazeri

Components (SIGMA)

Benzyl Amino Purine (BAP)(C12H11N5)

Naphthalene Acetic Acid (NAA) (C12H10O2)

Thiadizuron (TDZ) (C9H8N4OS)

Zeatin (C10H3NO5)

Kinetin (Kn) (C10H9N5O)

2-isopentanyl adenine (2iP) (C15H21N5O4)

Indole Butyric acid (IBA) (C12H13NO2)

Gibberellic acid (GA3) (C19H22O6)

Each constituent was added according to table1.8 and each component was dissolved using magnetic stirrer

for better dissolution.

Table 1 9 Composition of regeneration media for Dioscorea prazeri

Media composition Concentration required/100mL

MS macro (Table 1.2) 5mL

MS micro (Table 1.3) 1mL

MS iron (Table 1.4) 1mL

Thiamine (Table 1.5) 10l

Nicotinic acid 5l

Pyridoxine 5 l

Myoinositol 10 mg

Sucrose (0.15 M)(Table 1.6) 5.1345 g

Phytagel/agar (Table 1.7) 0.8 g

NB. Required concentrations of growth regulators (Table 1.8) were added to the media. pH was

maintained at 5.8.

14

The growth regulators was added in distinct and in factorial combinations according to

various set of experiments and pH was adjusted to 5.8 using Sodium hydroxide (2.0 N).

Made up the final volume using a graduated cylinder and added to appropriate conical

flask that allows the sufficient air volume to prevent the media from boiling over, with

gelling agent. The media was autoclaved at 120 º C for 15 minutes. The sterile media was

cooled and transferred to culture bottles. The thermo-labile growth regulators (filter

sterilized) were added to the media succeeding autoclaving.

1.3.2 Plant Material

i. Collection of plant material

Dioscorea prazeri is a native of North-Eastern Himalayas, collected from Research

Laboratories, Darjeeling, Mungpo, West Bengal. Those were collected during June and

were established and maintained in greenhouse under controlled conditions for healthy

growth of the plant. The former were planted in 1:1:1 proportion of farmyard manure,

soil and sand, maintained at a temperature below 25±2 °C and watered to sustain

moisture level, for the study. The tubers have been deposited in the Herbarium of

Avesthagen Limited; under the voucher specimen number 35(A).

ii. Finger printing analysis of D. prazeri

a. Total ash test: The samples from D. prazeri were dried pulverised and was

transferred to a previously ignited silica crucible.The samples were ignited

completely to carbon free ash in electric Bunsen burner. Cooled it in desiccator to

keep away the moisture and the sample weight was calculated.

b. Acid insoluble ash test: The total ash was boiled with 2N HCl in a hot plate. The

content was filtered in ash free whatman filter paper, washed till it was free from

acid. The contents were ignited completely in pre-weighed crucible for carbon

free ash for ~3hrs. The percentage of acid insoluble ash was calculated.

c. Water-soluble extractive: The extract was incubated overnight with sterile

(Milli-Q) water. Filtered the extract and made up the volume of the filtrate with

same solvent using standard flask. The extract was taken in duplicates.

15

Evaporated the solvent completely on water bath at 65ºC and cooled it in

desiccator. The contents were weighed along with previously weighed petriplate.

The percentage of water-soluble extractive was calculated.

d. Alcohol soluble extractive: The extract was incubated overnight with absolute

alcohol. The extract was filtered and made up the volume of the filtrate with

absolute alcohol using standard flask. The extract was taken in duplicates. The

solvent was evaporated completely on water bath at 65ºC and cooled it in

desiccator. The contents were weighed along with previously weighed petriplate.

The percentage of water-soluble extractive was calculated.

iii. Explant Material and Sterilisation

Healthy explants of Leaves, Internodes, Nodes, Petiole and Shoot tips of D. prazeri were

collected from actively growing D. prazeri plants in the greenhouse.

a. Preparation of Surface sterilants

Tween –20 (0.5%) was used as detergent while Bavistine (1000 ppm) was used as

Fungicide for sterilization. Cetrimide in a concentration not exceeding (1000 ppm) was

used as bactericide. Ethanol (70%) was used as a broad range sterilizing agent and

Mercuric Chloride (0.1gL-1) was used as a strong bactericide. All the surface sterilants

were weighed and dissolved in sterile water.

b. Procedure

The young actively growing Leaves, Internodes, Nodes, Petiole and Shoot tips were

cleansed with running tap water for 15 minutes as a preliminary step to avoid any surface

contaminant. The explant materials were treated with Tween-20 for 15 minutes at 100

RPM in an incubator shaker at room temperature. Explants were sterilised with Bavistin

for 40 minutes at 100 RPM. These were transferred to Cetrimide for 40 minutes at 100

RPM and disinfected with Mercuric Chloride and rinsed with Ethanol.These were blot

dried using sterile filter paper. The explant material was excised to 1.5- 2.0 cm of length

with sterile blade and inoculated. The explant materials were rinsed with sterile water on

each treatment

16

1.3.3 In vitro establishment of explants

The surface sterilised young explants were inoculated to the culture medium consisted of

the mineral salts and organic nutrients of Murashige and Skoog medium. The explants

were treated with basal medium with 3% sucrose and 2.0 % phytagel supplemented with

different combinations of growth regulators such BAP, NAA, GA3, Thiadizuron (TDZ),

Zeatin, Kinetin and 2-isopentanyl adenine (2iP) and IBA. The plants were grown at 25 ±

2 ºC under a photoperiod of 16 hrs light/8 hrs dark with a photon dose of 36-mmol m-2s-1.

The plantlets obtained were sub-cultured every 15-17 days and growth pattern was

observed and characterised on every 3rd day on the basis of treatment with various growth

regulators, period of treatment and acclimatisation conditions

1.3.4 Acclimatisation of Plantlets

The in vitro grown rooted healthy plantlets were acclimatized with combination of

acclimatization mixture containing vermiculate, coco peat mix, to red soil for 15 days.

The hardened plantlets and were established in farmyard manure, sand and soil. The

tubers were established into plantlets in in Farmyard manure, Red soil, Sand and Soil to

produce completely grown healthy plant. Acclimatised plants were transferred to the field

for further establishment. The regenerated plants were taken for morphological,

biochemical and genetic evaluation.

1.3.5 Morphological, biochemical and genetic evaluation

i. Random amplified polymorphic DNA (RAPD) analysis

The genomic DNA was isolated from in vitro grown plants of D. prazeri, donor plants of

D. prazeri. Dioscorea alata, a plant from the same family but different species, was used

as one of the control explants for RAPD analysis. The lithium chloride (LiCl) based

method for aromatic and medicinal plants were used for isolation of DNA (Pirttila et al.,

2001). DNA was quantified by gel electrophoresis (1% agarose gel) and quality of the

DNA was checked using Nanodrop spectrophotometer (ND-1000). RAPD analysis was

carried out by amplification of 50 ng template DNA using polymerase chain reaction

with 20 oligonucleotides like OPP7, OPC06, OPF 10, OPJ 13, OPK 17, OPN18, OPI 08,

OPN 8, OPL 20, OPC19, OPO 10, OPD 09, OPQ 14, OPH 14, OPB 10, OPJ 5, OPQ 11,

OPF 2, OPL 01 and OPG 18, which were decamers (Microsynth, Singapore) (Table

17

1.10). Each amplification reaction (50µL) (Table 1.11) contained template DNA (50 ng),

dNTP (1mM), Taq DNA polymerase buffer (1x), Taq polymerase enzyme (2 units),

Primer (1 picomole), MgCl2 (1 mM) and sterile HPLC grade water was subjected to PCR

using conditions shown in Table 1.12. The PCR amplified sample was electrophoresed on

a 1.5% agarose gel, stained with Ethidium Bromide, and photographed using a

phosphoimager (Bio-Rad). The gel was scored for clearly identifiable bands along with

the donor plants and with the respective control for the genetic fidelity assessment for

confirming the genetic stability. DNA fragments obtained from amplification with low

visual intensities and those that could not be readily distinguishable were not scored.

Table 1.10 The Oligonucleotides that showed clearly identifiable bands for genetic fidelity

assessment

SL.No Primer Sequence Primer Sequence

1 OPP7 5’-GTCCATGCCA-3’ OPF 10 5’-GGAAGCTTGG-3’

2 OPC06 5’-GAACGGACTC-3’ OPK 17 5’-CCCAGCTGTG-3’

3 OPJ 13 5’-CCACACTACC-3’ OPI 08 5’-TTTGCCCGGT-3’

4 OPN18 5’-TCAGAGCGCC-3’ OPL 20 5’-GGAAGCTTGG-3’

5 OPN 8 5’-ACCTCAGCTC-3’ OPO 10 5’-TCAGAGCGCC-3’

6 OPC19 5’-GTTGCCAGCC-3’ OPQ 14 5’-GGACGCTTCA-3’

7 OPD 09 5’-CTCTGGAGAC-3’ OPB 10 5’-CTGCTGGGAC-3’

8 OPH 14 5’-ACCAGGTTGG-3’ OPQ 11 5’-TCTCCGCAAC-3’

9 OPJ 5 5’-CTCCATGGGG-3’ OPL 01 5’-GGCATGACCT-3’

10 OPF 2 5’-GAGGATCCCT-3’ OPG 18 5’-GGCTCATGTG-3’

Primers used for screening micropropagated plants for genetic fidelity. The primers were chosen based on

reproducibility and number of bands indicating stability in genomic primer region. The amplified banding

patterns of the plants regenerated from nodal explants were identical to those of control plants for all the

primers used.

18

Table 1.11 Amplification profile of RAPD analysis for Dioscorea prazeri

Constituents Quantity

Template DNA 50 ng (2μL)

DNTP (10 mM) 0.8μL

10x Taq buffer (with MgCl2 of

15mM)

5.0μL

Taq polymerase 2 units (0.8μL)

Primer (10pmoles) 25ng (1.5μL)

MgCl2 (25mM) 0.5μL

HPLC grade water (sterile) 39.4μL

Total Volume 50.0 μL

Table 1.12 Cycling conditions well worked for amplification

Conditions Temperature Time Cycles

Initial denaturation 94˚ C 4 min 1

Denaturation 94˚ C 1 min

44Annealing 35˚ C 2 min

Extension 72˚ C 2 min

Final extension 72˚ C 10 min 1

4˚C Forever

ii. Morphological analysis

Combinations of the acclimatization mixtures were experimented to obtain efficient

frequency on field establishment (Table 1.13). The in vitro grown rooted plantlets were

washed and transferred to vermiculate, coco peat mix, to red soil in 3:1:1 ratio for 15 d

for hardening. The pots were covered with polythene to maintain humidity and watered

regularly. The plants were drenched once in three days with Bavistin (0.1%) to avoid

collar rot. The plantlets with well-developed root system were transferred to the

greenhouse containing farm yard manure, vermiculate to soil in 1:1:1 ratio at regulated

temperature of 25±2ºC. The morphological characters were analyzed from different set of

experiments and the average value was calculated.

19

Table 1.13 The growth regulators used for standardisation for efficient regeneration of D.

prazeri

Acclimatization mixture Ratio used

Soil to Vermiculate 3:1

Red Soil to Farmyard manure (FYM) 3:1

Vermiculate, Sand, to FYM 3:2:1

Vermiculate, Cocopeat mix to Red Soil 3:1:1

Red Soil Whole mix

Sand Whole mix

FYM, Soil to Sand 1:1:1

iii. HPLC analysis of in vitro raised plantlets of D. prazeri

The standard curve was generated with the Diosgenin procured from Sigma, USA (~98%

pure). The sample was prepared by drying the tubers of in vitro raised plantlets and wild

plants of D. prazeri at 45ºC for two days. These were hydrolysed at 95ºC for 3½ hours in

2N hydrochloric acid. The pH of the extract was neutralized with 2N sodium hydroxide

to pH 7.0, centrifuged and the residue was dried at 55 ºC for 36 hours in hot air oven. The

extraction was carried out with methanol, petroleum ether and hexane for the

comparative study on yield of steroidal sapogenin (Biosox Unit, Techno Reach). On post-

distillation the sample volume was 5mL, which was then dried using roto-evaporator at

50ºC for 20 minutes with pressure of 300psi. The completely dried plant extract was

dissolved in various solvents to obtain the better absorbance on analysis.

Chromatographic analysis was carried out on Shimadzu Series LC-20 AT liquid

chromatographic system, equipped with a diode array detector SPD-M20A. All data were

processed using LC-Solution software (Shimadzu, Japan). Prominent chromatographic

peak of Diosgenin was observed on HPLC analysis of the soxhlet extract of D. prazeri,

using methanol as mobile phase. The chromatographic peak of the samples was

compared with the standard (Diosgenin) peak. The yield of the extract was calculated.

The tuber extract of Dioscorea alata, which does not express Diosgenin, was used as a

negative control.

20

1.3.6 Statistical analysis

Values were expressed as the average of replicates ± standard deviation of independent

experiments. Statistically, the data was analyzed with one-way analysis of variance using

Bonferroni’s Multiple Comparison Test. The results were obtained from independent

experiments with replicates. The mean significant value was calculated for the frequency

of regeneration and for various hormonal treatments and the growth pattern obtained

from the experiments.

1.4 Results

1.4.1 Authentification of D. prazeri with pharmacological aspects

The pharmacological analysis of D. prazeri was studied with Total Ash Test in which the

percentage of total ash content was found as 2.75%. Comparative studies with Indian

Pharmacopoeia (Revised new edition, 2002) showed that it should be less than 6.0% for

any consumable purpose including pharmaceuticals and the results obtained were in

limits. The acid insoluble ash assessed to be 0.05%. Comparative studies with Indian

Pharmacopoeia (Revised new edition, 2002) showed that it should be less than 1.0% for

any consumable purpose including pharmaceuticals and the extract showed the confined

values. The water-soluble and alcohol soluble extractive were used for evaluating the

quality and purity of the extract containing bioactive compound. The percentage of

water-soluble extractive was characterized to be 30.4%. Comparative studies with Indian

Pharmacopoeia (Revised new edition, 2002) showed that it should not be less than 17.0%

for any consumable purpose including pharmaceuticals and the extract showed the

consumable range. Alcohol soluble extractive with the percentage of water-soluble

extractive was found to be 29.6%. Comparative studies with Indian Pharmacopoeia

(Revised new edition, 2002) showed that it should not be less than 11.0% for any

consumable purpose including pharmaceuticals and the extract showed the confined

value above 11.0%.

1.4.2 Micropropagation

High regeneration frequency of 98±2% was obtained on MS media with 0.5 mgL-1 BAP

and 0.01mgL-1 NAA, and it was found to be the most suitable combination of hormones

21

for shoot initiation and regeneration for Dioscorea prazeri. The explants grown in MS

plain media were used as one of the control in experiments. Explants grown in the media

with 0.5 mgL-1 BAP and 0.01mgL-1 NAA showed the highest rate of multiplication and

survival as compared with explants in media with growth regulators like Thiadizuron

(TDZ), Zeatin, Kinetin and 2iP (Table 1.14&1.15).

Table 1.14 Comparative study of effect on plant growth using MS media supplemented with

different growth regulators

MS+

Growth regulators

(10 to 12 weeks of growth)

Media MS+

*GR mgL-1

Frequency

of Regn.

(%)

No. of shoots/

Explants

Regenerated

Shoot length/

Culture (cm)

BAP 0.5 90 90 18.2

1.0 90 69 13.2

1.5 81 58 11.3

Kinetin 0.5 39 7 6.0

1.0 40 7 6.3

1.5 40 5 6.8

TDZ 0.5 78 40 6.4

1.0 70 30 8.1

1.5 60 24 6.9

Zeatin 0.5 60 57 5.4

1.0 35 34 6.8

1.5 25 21 7.1

2-isopentanyladenine (2iP) 0.5 60 30 7.3

1.0 40 24 8.1

1.5 30 13 7.9*GR Growth regulator

The regeneration frequency and growth pattern in MS medium supplemented with different growth

regulators were showed. Explants treated with MS+BAP of 0.5 mgL-1 resulted in higher rate of

multiplication and survival compared to explants regenerated in media (MS) supplemented with other

growth regulators like Thiadizuron (TDZ), Zeatin, Kinetin and 2iP.

22

The combination of hormones with BAP and NAA resulted in the initiation of growth < 3

days (Fig. 1.1 A). In 4 to 5 weeks of inoculation, notable growth pattern were observed

(Table. 5).

Table 1.15 Comparative study of effect on plant growth (10 to 12 Weeks) using MS media

supplemented with combination of growth regulators

Combination Media MS+*GR mgL-1

Frequency

(%)

Shoots

Regenerated/

Explant (No.)

Shoot length/

Culture1 (cm)

BAP and NAA

0.5 BAP+0.01 NAA 100 117 23.1

0.5 BAP+0.03 NAA 60 31 11.6

0.5 BAP+0.05 NAA 50 00 4.4

TDZ and NAA 0.5 TDZ+0.01 NAA 86 48 7.9

0.5 TDZ+0.03NAA 70 31 8.6

0.5 TDZ+0.05 NAA 40 04 6.9

Zeatin and NAA 0.5 Zeatin+0.01 NAA 65 64 6.2

0.5 Zeatin +0.03 NAA 35 28 7.4

0.5 Zeatin +0.05 NAA 20 12 7.8

2iPand NAA 0.5 2iP +0.01 NAA 60 47 8.2

0.5 2iP +0.03 NAA 20 26 10.4

0.5 2iP +0.05 NAA 8 15 7.8

*GR: growth regulator supplementation in mgL-1

1 Shoot length is the average length of main shoots and axillary branches. MS media supplemented with

combination of growth regulators and its effects showed that the highest regeneration frequency of 98±2%

was achieved in MS+0.5 mgL-1 BAP and 0.01mgL-1 NAA.

High multiplication rate of 21 segments that can be cultured were obtained in 10 to 12

weeks of inoculation from nodal segments. Multiple shoots were noticed (Fig. 1.1 B and

C) and up to 9 shoots were observed from single node without callus formation.

Morphogenic callus were formed while culturing for a longer period that resembled shoot

23

primordium, and adventitious buds were formed on its surface. It was removed and

cultured on MS medium with 0.5 mgL-1 BAP and 0.01mgL-1 NAA, and regenerated in a

large number of plantlets (Fig.1.1 D - F). Profuse root induction was observed with this

combination of hormones resulting in 12.5 to 14.5 cm in root length in 8 to 10 weeks

(Fig. 1.1 G) after inoculation of shoots and in vitro tubers were also obtained in 14 to 16

weeks of culturing. The sprouted healthy tubers obtained from nodal explants were used

for propagation.

Explants were regenerated in MS medium with BAP and NAA and compared to those

grown in medium with BAP, NAA and 0.01mgL-1 GA3. The results showed that a

combination of BAP, NAA and 0.01mgL-1 GA3 was useful in elongation of shoots in

initial stages of explant growth. Continued sustenance in the medium resulted in fragile

shoots due to extensive elongation attributed to the activity of GA3 (Table 1.16).

Table 1.16 Comparative study of growth pattern of D. prazeri in media containing BAP, NAA

and GA3 in 4 to 5 Weeks

Media

MS+ Growth regulators (mgL-1)

Shoot

Length1

(Cm)

No. of

Nodes2

Nodal

Length3

(Cm)

No. of

roots

Root

length

(Cm)

Basal MS 0.0 0.0 0.0 0.0 0.0

0.5 BAP+0.01 NAA 13.3 ± 1 8 ± 1 2.5 ±0.4 11 ± 2 10 ± 0.5

0.5 BAP+0.01NAA+0.01GA3 11.1±0.3 7.0±1 2.5 ± 0.4 10±2 10 ± 0.5

0.5 BAP+0.01NAA+0.02GA3 7.3±0.3 7.0±1 1.5 ± 0.5 10±2 6.8 ± 0.5

0.5 BAP+0.02 NAA+0.02GA3 *4.5±1.0 2.0±1 1.0 ± 0.2 2.0±1 0.3±0.2

1BAP+0.02NAA+0.02GA3 *---- ---- ---- ---- ----

Values are the average of 5 replicates of 30 explants each ±standard deviation.

1. Shoot length is the average of length of main shoots and axillary branches.

2. Number of nodes is the number of culturable segments on sub-culturing.

3. Length of culturable segments obtained on sub-culturing.

*----: While increasing the concentration of NAA shoots were turning brown very soon than responding

positively and resulted in stunted growth.

24

Comparative study of growth pattern on media containing BAP, NAA and GA3 resulted that explants

grown in MS+0.5 BAP+0.01NAA+0.01GA3 has given highest regeneration frequency and survival rate. It

resulted in a higher multiplication rate and regeneration of healthy explants with higher shoot, root and

nodal length, with more number of nodes and roots.

Increased concentration of BAP resulted in delay of the initiation in growth (8-12 d) and

the resultant plants were stunted. With further increase in concentration of NAA and

GA3, plantlets and shoots turned brown and dried very fast. The rooted plantlets were

hardened (Fig. 1.1 H and I) for 15 days and then acclimatized in greenhouse. The

acclimatized plantlets showed a frequency of regeneration up to 98±2% in media

supplemented with BAP and NAA as compared to the tested set of hormones. It was

observed with D. prazeri that BAP and NAA were showing highest regeneration

frequency with healthy growth in comparison with other growth regulators like Kinetin

(1.5 mgL-1 with 40% regeneration frequency; 6.8 cm of shoot length in 3 weeks), Zeatin

(0.5 mgL-1 with 60% regeneration frequency with 5.3 cm shoot length in 3 weeks) and

2iP (60% with 7.3 cm of shoot length). The explants were regenerated at frequency of

78% with 6.4 cm shoot length in MS+ TDZ medium in case of D. prazeri. The

combination of hormones enhanced the regeneration frequency as well as healthy

elongation of shoots. Combined hormonal treatment of BAP along with NAA, showed

an enhancement of 10% in frequency of regeneration than usage of BAP individually and

5cm more growth in shoot at same time period of observation. TDZ with NAA showed

8% enhancement on frequency of regeneration with an improvement of shoot length

(1.5cm more) in comparison with the results obtained only with TDZ; Zeatin with NAA

showed 5% improvement in frequency of regeneration and improvement on shoot length

(0.8 cm more) but the results obtained with 2iP did not show any variation in frequency

of regeneration from 2iP and NAA but an enhancement of 0.9 cm shoot elongation on

observation and characterisation.

The plantlets produced could be successfully transplanted to the field with high

establishment and with 96% survivability (Fig. 1.1 J). D. prazeri was propagated by

multiple shoots and healthy tubers obtained from nodal explants. The growth and

morphology of the re-established plantlets were evaluated and assessed with donor plants

for genetic integrity using molecular markers.

25

Fig. 1. 1 Regeneration and micropropagation of D. prazeri.

(A) Initiation of shoot regeneration in MS medium with 0.5 mgL-1 BAP and 0.01mgL-1 NAA

+30gL-1 sucrose; (B) Shoot regeneration and multiple shooting of D. prazeri from the nodal

explants on two weeks of culturing on MS medium supplemented with 0.5 mgL-1 BAP and

0.01mgL-1 NAA with 30gL-1 sucrose; (C) Multiple shooting of D. prazeri from the shoot buds;

(D) Growth initiation of adventitious shoots from in vitro regenerated callus of D. prazeri from

nodal segments; (E) Culturable segments formed out of adventitious shoots;(F) In vitro

regenerated tubers from the nodal explants of D. prazeri; (G) Rooting of in vitro regenerated

plantlets of D. prazeri; (H) Hardening of rooted plantlets of D. prazeri (15 days) in Coco peat

mix to red soil in 3:1 ratio and the plantlets were covered with polythene cover to maintain

humidity; (I) Hardened plantlet transferred to the pots contained 1:1:1 ratio of Farm yard manure,

sand to soil for further establishment; (J) D. prazeri- acclimatized in vitro raised plants in

Greenhouse.

1.4.3 Genetic stability assessment

i. Genetic stability analysis with molecular markers (Random polymorphic DNA

analysis)

The genomic DNA of D. prazeri from in vitro grown plants, donor plants (wild), and

control plant D. alata (obtained from same family but different species) was amplified to

assess the genetic stability of regenerated plantlets from donor germplasm .The RAPD

fragment patterns of in vitro regenerated plantlets was compared with that of donor plants

(wild).

26

The assessment of genetic stability of in vitro regenerated plantlets is highly significant

for further studies. The amplified banding pattern was analysed for genetic integrity

based on number of fragments and reproducibility. RAPD profile revealed identity

between donor and in vitro regenerated plants indicated stability in genome (Fig. 1.2).

*M Molecular markerFig. 1. 2 The RAPD gel profile of donor wild plants and in vitro regenerated plantlets.

Lane 1-2: Amplification profile of D. prazeri donor plant using primer OPJ 05; Lane 3-7: D.

prazeri in vitro regenerated plants using primer OPJ 05; Lane 9-10: Amplification profile of D.

prazeri donor plant using primer OPN 08; Lane 11-15: Amplification profile of D. prazeri in-

vitro regenerated plants using primer OPN 08; Lane 8 and 16: Negative control Lane M:

Molecular marker

The gels were scored for bands obtained from amplification of genomic DNA with 20

oligonucleotides and the RAPD gel profile was analysed for genetic stability because it is

significant to use it as a tool for biotechnological application for any plant material.

RAPD profile showed identical genomic DNA fragmentation pattern indicated stability in

genome. Out of 20 primers screened, 11 primers amplified genomic DNA with major

bands with stable staining intensity. The oligonucleotides sequenced like OPK 17: 5’-

CCCAGCTGTG-3’, OPJ 13: 5’-CCACACTACC-3’, OPI 08: 5’-TTTGCCCGGT-3’,

OPN18: 5’-TCAGAGCGCC-3’, OPO 10: 5’-TCAGAGCGCC-3’, OPD 09: 5’-

27

CTCTGGAGAC-3’, OPN 8: 5’-ACCTCAGCTC-3’, OPQ 11: 5’-TCTCCGCAAC-3’,

OPJ 5: 5’-CTCCATGGGG-3’, OPL 01: 5’-GGCATGACCT-3’, OPG 18: 5’-

GGCTCATGTG were scored for 16 to 24 amplified DNA fragments. Rest of the primers

studied was given amplification with 9 to 11 bands. RAPD analysis was carried out in

triplicates for fingerprinting analysis and these bands showed high reproducibility. The

size of the fragments obtained ranged from 250 base pair to 2250 base pair. The primer

generated monomorphic profile for the donor plants and the in vitro generated plantlets.

The amplified genomic DNA patterns derived from in vitro regenerated plants showed

absolute similarity to donor plants. D. alata, which was used as one of the control for the

study on polymorphism, from same family but different species (D. alata) showed

polymorphism on RAPD analysis (Fig. 1.3) with respect to D. prazeri fragmentation.

RAPD analysis was observed to be a significant tool for the genetic fidelity assessment.

Fig. 1. 3 The RAPD gel profile of donor plant and the in vitro regenerated plants.

Lane 2-3: D. alata using primer OPJ-13, Lane 4-5: D. prazeri donor plant using primer OPJ-13,

Lane 6-8: D. prazeri in vitro regenerated plants using primer OPJ-13, Lane 10-11: D. alata using

primer OPJ-18, Lane 12-13: D. prazeri donor plant using primer OPJ-18, Lane 14-16: D. prazeri

in vitro regenerated plants using primer OPJ-18, Lane 18-19: D. alata using primer OPD-09,

Lane 20-21: D. prazeri donor plant using primer OPD-09, Lane 22-24: D. prazeri in vitro

1 3 5 7 9 11 13 15 17 19 21 23 25

28

regenerated plants using primer OPD-09, Lane 9, 17,25: Negative control for primers OPJ-

13,OPJ-18 and OPD-09 respectively; Lane 1 and 26: Molecular marker (1 kb)

The amplified banding pattern of the plants micropropagated from nodal explants/shoot

tips were identical to those of control plants for all the primers used and this in vitro

propagation technique can be followed for conserving the germplasm of endangered

Dioscorea prazeri and even for the related species like D. alata. The minor fragments

that were unstable in staining intensity and therefore not reliable were not considered.

ii. HPLC analysis: Diosgenin assay

The high performance liquid chromatographic analysis was carried out successfully and

standardized the HPLC conditions to determine the secondary metabolite content from D.

prazeri. Various absorption spectra of steroidal sapogenin, Diosgenin ranging from 195

to 235 nm to obtain the absorption maximum and it was found to be 205 nm using D.

prazeri plant extract. The biochemical analysis showed the Diosgenin content was the

same in regenerated plantlets as that observed in donor (wild) plants. Diosgenin content

of the regenerated explants the source plants were analysed with HPLC. No significant

difference was observed between these plants in terms of steroidal sapogenin secondary

metabolite product pattern (Fig. 1.4 A-C) (Table 1.17(A, B, C).

Table 1.17(A) Diosgenin estmation data obtained on HPLC analysis with in vitro grown plant

extract

Name Concentration Rt Channel Area Height

In vitro grown 1 28.861 7.0 Ch2 205 nm 8770718 665978

In vitro grown 2 28.861 7.0 Ch2 205 nm 8770717 665976

In vitro grown 3 28.861 7.0 Ch2 205 nm 8770716 665975

29

Fig. 1. 4 Chromatogram obtained from HPLC analysis of the petroleum ether extract with Soxhlet

for contents of steroidal sapogenin, Diosgenin of donor (wild) plant and in vitro regenerated

plants of D. prazeri.

(A) The chromatogram illustrates the concentration of 1μg of Diosgenin; (B) The Soxhlet

extraction for steroidal sapogenin, Diogenin of donor plants of D. prazeri; (C) The chromatogram

of extract from in vitro regenerated plants of D. prazeri. The retention time of the required peak

was at 7.0 minute with methanol as mobile phase

Table 1.17(B) Diosgenin estmation data obtained on HPLC analysis with Donor plant extractName Concentration Rt Channel Area Height

Donor plant 1 28.065 7.0 Ch2 205 nm 8528347 664038

Donor plant 2 28.065 7.0 Ch2 205 nm 8528349 664037

Donor plant 3 28.065 7.0 Ch2 205 nm 8528346 664038

30

Table 1.17(C) Diosgenin estmation data obtained on HPLC analysis with Standard (Diosgenin)Name Concentration Rt Channel Area

Std Dg 1 1.000 7.0 Ch2 205 nm 235935

Std Dg 2 2.000 7.0 Ch2 205 nm 497542

Std Dg 3 3.000 7.0 Ch2 205 nm 785119

Std Dg 4 4.000 7.0 Ch2 205 nm 1019160

Std Dg 5 5.000 7.0 Ch2 205 nm 1359612* R2: 0.9970856; R: 0.9985418; Mean RF: 3.936323e-006; Dg: Diosgenin

The concentration of steroidal sapogenin, Diosgenin obtained from donor plant as well as in vitro

regenerated plants on HPLC analysis exhibited genetic integrity on analysis.

The retention time was analysed from the calibration curve obtained with standard

(Sigma). The tubers obtained were analysed for the secondary metabolite Diosgenin

pattern according to the age of the plants. It varied from 0.7±0.2% to 2.6±0.2% (w/v)

according to the growth phases of the plant from 12 weeks old plant to 3 years old plant.

The maximum level of Diosgenin obtained from the plants at 2.5 years of growth to 3.0

years of growth. Even the plants of 5 weeks old were indicated the presence of Diosgenin

on analysis. When compared to the biochemical analysis data obtained from the donor

plant, the level of Diosgenin content of in vitro regenerated plants did not show

significant variation at various growth phases. D. alata was used as a negative control for

the chromatographic assay, showed the absence of secondary metabolite Diosgenin.

The percentage of Diosgenin obtained from 3.0 years old plant tubers of donor

plants were found to be 2.2±0.2%(w/v) on an average. Even though D. prazeri produced

Diosgenin at younger stages, the yield of steroidal sapogenin was very low in comparison

with the older plants. The negative control plantlet used for the experiment, D. alata did

not reveal the presence of Diosgenin.

iii. Morphological analysis

The regenerated plantlets proliferated as vigorously as control plantlets and

morphological abnormality was not detected. The ability to produce microtubers

remained unaltered as both regenerated plantlets and control plantlets produced healthy

leaves, shoots and roots on propagation. The analysis on morphological characters

31

between donor and in vitro propagated plants did not show any significant difference and

the morphology was found to be stable (Table 1.18).

Table 1.18 Comparative studies of morphological characters of D.prazeri

Morphological characters In-vitro grown plants Wild plants

Length/breath ratio of mature leaf 10.7±3.6:8.2±2.1 10.3±3.2:7.8±2.25

Number of primary stems 8.0±2.0 7.0±2.0

Petiole length 4.69±1.8 4.28±2.01

Internodal length 9.4±2.0 8.3±0.7

Lamina/petiole length ratio 10.7±3.6: 4.69±1.8 10.3±3.2: 4.28±2.01

Comparative studies of morphological characters of in vitro grown and the wild type plants grown in

greenhouse did not show any significant difference in growth pattern and found to be morphologically

stable.

1.4.4 Statistical data analysis

The data obtained by treating D. prazeri explants using multiple growth regulators were

analysed statistically. For every treatment level with 5 replicates having 30 explants

each showed a mean significant difference of P<0.05 and the coefficient of variation (R2)

of 0.99. The P value summary of data obtained with hormonal treatments and the

regeneration of explants showed high significance. The treatment conditions were

analysed with one-way analysis of variance and Bonferroni’s multiple comparison test.

Graphical representations based on statistical analysis illustrated that MS with 0.5 mgL -1

BAP and 0.01mgL-1 NAA was the most suitable media for the micropropagation

experiments to obtain healthy plants (Table 1.19, 1.20 & 1.21). The analysis on

regeneration efficiency of treatment with individual hormones (Fig. 1.5), various

combinations of hormones (Fig. 1.6) and factorial combination of BAP, NAA and GA3

(Fig. 1.7) are reported. This is with respect to various parameters such as number of

shoots, frequency of shoots, frequency of regeneration, length of shoots, number of nodes

and length of nodes.

32

Fig. 1. 5 The graphical representation of statistical analysis for individual hormonal treatment.

(A) Number of Shoots formed on micropropagated D.prazeri with MS medium + Growth

regulators; (B) Frequency of in vitro regeneration of D. prazeri with MS medium + Growth

33

regulators; (C) Length of Shoots of in vitro regenerated D. prazeri with MS medium+ Growth

regulators

Fig. 1. 5 The graphical representation of statistical analysis for different combination of hormone

treatment.

34

(A) Number of shoots formed on micropropagated D. prazeri; (B) Frequency of in vitro

regeneration of D. prazeri; (C) Length of Shoots formed on in vitro regenerated D.prazeri

Fig. 1. 6 The graphical representation of statistical analysis for multiple combinations of BAP,

NAA and GA3 with MS media.

35

Table 1.19 One-way analysis of variance for individual hormonal treatment*1

One-way Analysis of Variance No. of shoots Frequency Length of

shoots

P-value <0.0001 <0.0001 <0.0001

P value summary *** *** ****2MSD P< 0.05 P< 0.05 P< 0.05

Number of groups 15 15 15

F 990 710 260

R2 1 0.99 0.98*1 The data was graphically represented by Fig. 4 *2 Mean significant difference

Table 1.20...One-way analysis of variance for combination of hormonal treatment*1

One-way ANOVA for combination of

hormones

Frequency No. of shoots Length of

shoots

P-value <0.0001 <0.0001 <0.0001

P value summary *** *** ****MSD P< 0.05 P< 0.05 P< 0.05

Number of groups 12 12 12

F 1200 1600 130

R2 1 1 0.97*1The data was graphically represented by Fig. 5 *2 Mean significant difference

Table 1.21 One-way analysis of variance for factorial combination of BAP, NAA and GA3*1

One-way Analysis of Variance for MS+ BAP with NAA and GA3

P-value <0.0001

P value summary ****2MSD P< 0.05

Number of groups 5

R2 0.99*1 The data was graphically represented by Fig. 6 *2 Mean significant difference. The statistical analysis on

various effects of hormonal treatment showed high significance.

Dioscorea prazeri, an endangered, indigenous medicinal plant was efficiently

regenerated through in vitro culture of nodal explants and propagated by multiple shoots

36

and healthy tubers resulting from it. In the present study, plantlets were successfully

induced that could be successfully transplanted to the field with high field establishment.

Using the optimized protocol this medicinally important threatened plant can be

multiplied in vitro on a large scale and re-introduced into the natural habitat. Young

shoot tips obtained from in vitro raised plantlets, precultured in BOD in dark for a very

short period showed the best survival rate. The growth, morphology and the genetic

integrity of the re-established plantlets were evaluated, which did not show any variation

among the micropropagated plants in comparison with the donor plant. The plants

regenerated were found to be similar in biochemical characteristics of the donor plant.

The regeneration and micropropagation of D. prazeri was achieved successfully with a

high frequency (98±2%) and a survival rate of >96% on field establishment (Fig. 1.8).

The micropropagation on D. prazeri was successfully established with genetic fidelity,

biochemical stability and morphological integrity. The conditions optimized in this study

for in vitro propagation were observed to be highly significant. The propagation

techniques and the analysis were further used for germplasm conservation and for genetic

transformation studies and found to be the excellent medium for regeneration and for re

establishing the plants to natural habitat. The propagation holds immense potential in

exploring the medicinally significant bioactive compound, Diosgenin. This study on D.

prazeri is imperative as it has many applications in areas of research. Sustainable

management and utilization of this valuable medicinal yam is feasible through reinstating

the healthy plants into their natural habitat.

37

Fig. 1. 7 The whole experiment conducted of in-vitro propagation of endangered source.

Dioscorea prazeri that demonstrate inoculation to genetic stability assessment of in vitro grown

plants in a nutshell

38

1.5 Discussion

1.5.1 Micropropagation

Previous studies with Dioscorea prazeri have shown multiple shoot formation with BAP

(Sharma et al., 2006; Alam et al., 2010b). In the present study on D. prazeri we

demonstrate that additon of BAP resulted in multiple shoots with a shoot length of

18.2±1cm and with a frequency of regeneration as high as 90 %. Addition of growth

regulator Naphthalene acetic acid (0.01mgL-1) along with BAP enhanced the multiple

shoot formation as well as frequency of regeneration of explants as high as 100% and

shoot length of 23.1±1 cm. It also facilitated root formation of in vitro regenerated plants

and tubers in the same regeneration media. A further increase in the concentration of

NAA (0.5 mgL-1) resulted in decreasing the regeneration frequency to 50% with stunted

growth. Several studies support the role of GA3 or combination of Kinetin and GA3 for

elongation of shoot prior to rooting (Baskaran and Jayabalan, 2005; Mohamed et al.,

2006). In our study, we observed that although GA3 in combination with BAP and NAA

resulted in elongation of the explants, high concentrations of GA3 (0.02 mgL-1) in

combination with growth regulators produced weak shoots when compared to other

hormonal treatments. Even though lower concentration of GA3 (0.01mgL-1) produced

healthy plantlets on micropropagation, BAP and NAA alone showed efficacy in

regeneration with high shoot length and root formation. The previous reports on other

monocots indicate that BAP (2.0 - 2.5 mgL-1) supplemented media is more effective

towards induction of multiple shoot buds. Similar results were also reported

(Sheelavantmath et al., 2000) in combination with BAP and NAA at higher

concentrations in case of monocots. Dioscorea prazeri also showed high frequency of

regeneration in combination with the hormones reported but at a lower concentration of

0.05 mgL-1 that resulted in the highest frequency of regeneration with strong shoots (23.2

cm, 12 weeks) when compared with 1.0 mg/L (90% regeneration frequency with 13.2 as

average shoot length) and 1.5 mgL-1 (81% with 11.3cm shoot length). At concentrations

exceeding 1.5 mgL-1 showed significant inhibition of growth of the explants. It has been

shown in other species of Dioscorea that BAP in combination with auxins leads to

generation of healthy tubers in vitro (Bhadra and Hussein, 2003) albeit at different

concentrations. The microtuber production by the combined effect of BAP (4-8 mgL-1)

39

and Kinetin (1-2 mgL-1) has been reported in other species like D. zingiberensis (Li et al.,

2000) and D. oppositofolia (Behera, 2009). It was also observed with D. prazeri that BAP

and NAA were showing highest regeneration frequency with healthy growth in

comparison with other growth regulators like Kinetin, Zeatin and 2iP and TDZ. In this

study it was noticed that rather than individual hormone treatment the combination of

hormones enhances the frequency of regeneration as well as elongation of shoots in a

healthy manner.

BAP was more responsive than kinetin in inducing multiple shoot formation in

case of Dioscorea prazeri, as reported earlier with D. wighitti (Poornima and Rai, 2007)

shoot formation was found be high with BAP (Asha and Nair, 2007). This study showed

that the combination of cytokinins and auxins were the significant rate limiting factor for

the organogenesis as explained by Skoog and Miller (1957) for the first time. But the

results obtain from this study confirms that considerable variability exists among genera,

species and even cultivars and optimisation of the concentration is highly essential for

induction of morphogenesis.

1.5.2 Genetic fidelity assessment

i. RAPD analysis

A molecular based approach for the genetic fidelity of the micropropagtion experiment is

highly essential for further research and for confirming stability of plants that have

therapeutic properties. In some instances micropropagtion was reported to induce

somoclonal variation mainly with leaf disc culture (Jain, 1997; Khoddamzadeh, 2010)

and hence it is highly essential to confirm that the conditions optimsed for the in vitro

propagation maintained the genetic integrity of the micropropagted plants. A marker

assisted genetic fidelity assessment tool is essential for in vitro studies to develop

optimised conditions for the plants. In this study on D. prazeri the primers used

demonstarted the genetic stability of in vitro regenerated plants in comparison with donor

plants. RAPD was reported as an important tool for detection of polymorphism with the

fragment analysis and was used as a genetic marker system for this study for the

detection of polymorphism if any (Dixit, 2003). A control plant DNA (D. allata) from

40

the same family but a different species was used for the confirmation of RAPD analysis,

clearly showed polymorphism with D. prazeri. It showed that RAPD analysis exhibits

polymorphism if there is any variation at genetic make up, hence it can be used for the

assessment of genetic integrity. It was reported as a well-defined approach for genetic

evaluation and characterisation with operon primers (Punia et al., 2009). The

monomorphic fragments of DNA obtained on amplification with 20 primers showed that

the conditions optimised to generate the in vitro propagated plants of D. prazeri are

reproducible and do not compromise the genetic integrity of the plant. RAPD analysis is

essential to verify genetic stability and RAPD patterns found stable on evaluation of D.

prazeri as previously reported in the study of Solanum tuberosum L (Hirai and Sakai,

2000). In vitro propagated D. prazeri plants with monomorphic bands with donor plant

ranging from 250 base pair to 2250 base pair used to analyse the genetic stability.

Molecular analysis confirmed the genetic stability of the in vitro regenerated plantlets, as

reported (Alizadeh and Singh, 2009). In another study, the plantlets regenerated were

assessed with molecular markers and monomorphic bands were well characterised in

determining genetic integrity as reported in dicot as well as monocot plants (Gupta,

2009). In this study it was shown that the plants propagated in highly favourable medium

optimised for growth will not result in any polymorphism on micropropagation based on

the electophoretic monomorphic pattern of DNA fragments obtained using RAPD

markers, which is well documented for determining genetic integrity in a number of plant

species (Devarumath, 2002; Rahman, 2001;Rout, 2002). Furthemore the use of a

molecular based assessment on stability of genome of clonal material and to certify

stability throughout the micropropagated plant system is sorely needed (Rout, 1998).

ii. HPLC analysis: Diosgenin assay

Saponins are reported to have a wide range of biological activities (Francis et al., 2002)

and isolation techniques enabling the characterization of saponins have been reported

(Gurfinkel and Rao, 2003). Since Dioscorea prazeri is an endangered medicinal plant

that has been subjected to microporpagation the stability of Diosgenin, an important

metabolite, was one of the main concerns in this study. The extraction conditions were

standardised for Dioscorea prazeri species using pulverized tubers for the extraction and

estimation of Diosgenin and to study the stability of steroidal sapogenin, of in vitro raised

41

plants. The pure fraction of Diosgenin obtained was compared with known concentration

of the standard. The Diosgenin content of the donor plant and the in vitro propagated

plants showed significant stability on analysis. The procedure developed in this study is

highly efficient with respect to time and yield of Diosgenin obtained, even from very

limited amounts (0.15 g) of dried material.

iii. Morphological analysis

The morphology of the plants regenerated were analysed with the donor plant with

respect to mature leaf, primary stems, petiole length and intermodal length and its ratio

and found to highly stable. The in vitro regeneration in standardized conditions was

found to be stable as mentioned with previous studies on other Dioscorea species (Dixit,

2003 and Ahuja, 2002). The stability in morphology on micropropagation and healthy

growth observed in D. prazeri in this study was similar to that seen in other plant species

(Annarita, 2009).

iv. Statistical data analysis

The data obtained by treating D. prazeri explants using multiple growth regulators were

analysed statistically for every treatment level having 5 replicates with 30 explants each.

This resulted in a mean significant difference of P<0.05 and the coefficient of variation

(R2) of 0.99. The hormonal treatments and the regeneration of explants showed high

significance in terms of regeneraton efficiency and on data analysis statistically. The

treatment conditions were analysed with one-way analysis of variance and Bonferroni’s

multiple comparison test.

42

1.6 References I1. Ahuja. S., Mandal, B.B., Dixit, S., Srivastava, S.P. (2002). Molecular, phenotypic and biosynthetic

stability in Dioscorea floribunda plants derived from cryopreserved shoot tips. Plant Sci. 163:971-977

2. Alam, I., Sharmin, S.A., Mondal, S.C., Alam, M.J.,Khalekuzzaman, M., Anisuzzaman, M., Alam,M.F.(2010b). In vitro micropropagation through cotyledonary node culture of castor bean (Ricinuscommunis L.) Aust J Crop Sci. 4: 81-84.

3. Alizadeh, M., Singh, K. S. (2009). Molecular assessment of clonal fidelity in micropropagated grape(Vitis spp.) rootstock genotypes using RAPD and ISSR markers. Iran J Biotechnol. 7:37-44

4. Alizadeh, S., Mantell, S.H., Viana, A.M. (1998). In vitro shoot culture and microtuber inductionin the steroidal yam Dioscorea composita Hemsl. Plant Cell Tiss Org. 53: 107-112

5. Annarita, L. (2009). Morphological evaluation of olive plants propagated in vitro culture throughaxillary buds and somatic embryogenesis methods. Afr J Biotechnol. 3:037-043

6. Asha, K.I., Nair, G.M. (2007). In Vitro Bulbil Induction in Dioscorea species. 33: 81-87

7. Asolkar,L.V., Chadha,Y.R. (1979). Diosgenin and other steroid drug precursors. Publication andInformation Directorate, CSIR New Delhi.pp.2-17

8. Ayensu, E.S. Coursey, D.G.(1972). Guinea yams. The botany, ethnobotany, use and possible future ofyams in West Africa. Eco Bot. 26: 301-308

9. Badoni, A., Bisht, C., Chauhan, J.S. (2010). Micropropagation of Hedychium spicatum Smith using invitro Shoot Tip. Stem Cells. 1:11-13

10. Bannerman, R.H. (1983). The Role of Traditional Medicine in Primary Health Care, TraditionalMedicine and Health Care Coverage." World Health Organization, Geneva pp. 318-327

11. Baskaran, P., Jayabalan, N. (2005.) An efficient micropropagation system for Eclipta Alba – Avaluable medicinal herb. In vitro Cell Dev. 41:532–539m

12. Batugal, P. A., Kanniah. J., Young, L. S., Oliver, J. T. (eds) (2004). Medicinal Plants Research in

Asia, Volume 1: The Framework and Project Workplans. International Plant Genetic Resources

Institute – Regional Office for Asia, the Pacific and Oceania (IPGRI-APO), Serdang, Selangor DE:

Malaysia

13. Behera, K.K., Sahoo, S., Prusti, A. (2009). Regeneration of Plantlet of Water Yam (DioscoreaoppositifoliaaL.) through In Vitro Culture from Nodal Segments. Not Bot Horti Agrobo. 37: 94-102

14. Bhadra, S.K., Hossain, M.M. (2003). In vitro Germination and Micropropagation of Geodorumdensiflorum (Lam.) Schltr., an Endangered Orchid Species. Plant Cell Tiss Org. 13: 165-171

15. Chu,I.Y.E.(1992). Perspective of micropropagtion industry. In: Kurata,K., Kozai,T. (eds), Transplantproduction systems. Kluwer academic, Amsterdam. pp. 137-150

16. Coursey, D.G. (1976). Yams, Dioscorea spp. (Dioscoreaceae). In: Simmonds, N.W., (ed), Evolution ofCrop Plants. Longman, London, pp. 70-74

17. Davies, P.J. (1995). Plant hormones: Physiology, BIochemisrty and Molecular Biology. Dordrecht:Kluwer

43

18. Devarumath, R.M., Nandy, S., Rani, V., Marimuthu, S., Muraleedharan, N., Raina, S.N. (2002). RAPD,ISSR and AFLP fingerprints are useful markers to evaluate genetic integrity of micropropagatedplants of three diploid and triploid elite Tea clones representing Camellia sinensis(China type) and C.assamica spp assamica(Assam India type). Plant Cell Rep. 21: 166

19. Dixit, S., Mandal, B.B., Ahuja, S., Srivastava, P.S. (2003). Genetic stability assessment of plantsregenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera Using RAPD,biochemical and morphological analysis. Cryo-Lett. 24: 77-84.

20. Francis, G., Kerem, Z., Makkar, H., Becker, C. (2002). The biological action of saponins in animalsystems: a review. Brit J Nutr. 88:587-605

21. Gupta, R., Modgil, M., Chakrabarty, S.K. (2009). Assessment of genetic stability of apple root stockplant EmLA 111, using RAPD markers. Indian J Exp Biol. 47: 925-928

22. Gurfinkel, D.M., Rao, A.V. (2003). Soyasaponins: The Relationship between Chemical Structure andColon Anticarcinogenic Activity. Nutr Cancer. 47:24-33

23. Hirai, D., Sakai, A. (2000). Cryopreservation of in vitro-grown meristems of potato (Solanumtuberosum L.) by encapsulation vitrification. In: Engelmann, F.,Takagi, H. (eds) Cryopreservation ofTropical Plant Germplasm. Current Research Progress and Application, JIRCAS-IPGRI, Rome, Italy.pp. 205–211

24. Jain, M.S. (1997). Micropropagation of selected somaclones of Begonia and Saintpaulia. J.Bioscience. 22: 585-592

25. Jasik, A., Mantell, S.H. (2000). Effects of jasmonic acid and its methylester on in vitromicrotuberisation of three food yam (Dioscorea) species. Plant Cell Rep. 19: 863-867

26. Joy, P.P., Thomas, J., Mathew, S., Skaria, B.P. (2001). Bose, T.K., Kabir, J., Das, P., Joy, P.P.(eds),Medicinal Plants. Tropical Horticulture -Vol. 2. Naya Prokash, Calcutta, pp. 449-632

27. Khoddamzadeh, A.A., Sinniah, U.R., Kadir, M.A., Kadzimin, S.B., Mahmood, M., Sreeramanan, S.(2010). Detection of somaclonal variation by random amplified polymorphic DNA analysis duringmicropropagation of Phalaenopsis bellina (Rchb.f.) Christenson. Afr J Biotechnol. 9: 6632-6639

28. Li, X., Ming, L., Hua, Z., Ke, Y., Qing, Z., Xu, X.L., Liu, X.M., Zhou, P.H., Hi, K., Zhu, Z.Q. (2000).In vitro tissue culture and rapid tuberization in Dioscorea zingiberensis. Wright, C.H., Hunan J AgriUniversity. 26: 282-285

29. Mikulik, J. (1999). Propagation of endangered plant species by tissue cultures. Biologica. 37: (27-33)

30. Nalawade, S.M., Sagare, A.P, Lee, C.Y., Kao, C.L., Tsay, H.S. (2003). Studies on tissue culture ofChinese medicinal plant resources in Taiwan and their sustainable utilization. Bot Bull Acad Sinica.44:79-98

31. Onwueme. (1978). The tropical tuber crops. John Wiley, New York, pp. 234

32. Poornima, G.N., Rai, V.R. (2007). In vitro propagation of wild yams, Dioscorea oppositifolia (Linn)and Dioscorea pentaphylla (Linn). Afr J Biotechnol. 6: 2348-2352

33. Prain, D., Burkill, I.H. (1936). An account of genus Dioscorea part I species which turn to left Ann. R.Bot. Cdn. Cal. pp. 141-210.

34. Punia, A., Arora, P., Yadav, R., Chaudhury, A. (2009). Optimization and Inference of PCR Conditionsfor Genetic Variability Studies of Commercially Important Cluster Bean Varieties by RAPD Analysis.AsPac J Mol Biol Biotechnol. 17:33-38

44

35. Quigley, F.R. (1978). Diosgenin in West Africa Dioscorea plants. Planta Medica. 33: 414-415

36. Rahman, M.H, Rajora, O.P. (2001). Microsatellite DNA somoclonal varaiations in micropropagatedtrembling aspen (Poplus tremuloides). Plant Cell Rep. 20: 531

37. Rahman, M.M., Amin, M.N., Ishiguri, F., Yokota, S., Sultana, R. S., Kazuya, Y.T., Yoshizawa, I.N.(2009). In vitro plantlet regeneration of ‘‘dwarf’’ Indian olive (Elaeocarpus robustus Roxb.): a fruitplant of Bangladesh. Plant Biotechnol Rep. 3:259–266

38. Raven,P.H., Evert,R.F., Eichhorn,S.E.(1992). Biology of Plants. New York, Worth.pp.545-572

39. Razdan, M.K. (2003). Introduction to Plant Tissue. 2nd Edition. Oxford & IBH Publishing Co. Pvt.Ltd., New Delhi, pp. 27

40. Rout, G.R., Das, G. (2002). Assessment of genetic integrity of micropropagated plant of Plumbagozeylanka by RAPD markers. Biol Plantarum. 45:27

41. Rout, G.R., Das. P., Goel. S., Raina, S.N. (1998). Determination of genetic stability ofmicropropagated plants of ginger using random amolified polymorphic DNA(RAPD) markers. BotBull Acad Sinica. 39: 23-27

42. Sengupta. J., Mitra, G.C., Sharma, A.K. (1984). Organogenesis and tuberization in cultures ofDioscorea floribunda. Plant Cell Tiss Org. 3:325-331

43. Sharma, S. (2006). In vitro regeneration and mass multiplication of Capsicum annuum L. J Food AgrEnviron. 4:205-208

44. Sheelavantmath, S.S., Murthy, H.N., Pyati, A.N., Kumar, H.G.A., Ravishankar, B.V. (2000). In vitropropagation of the endangered orchid, Geodorum densiflorum (Lam.). Schltr. through rhizome sectionculture. Plant Cell Tiss Org. 60:151-154

45. Skoog, F., Miller, C.O. (1957). Chemical regulation of growth and organ formations in plant tissueculture in vitro. Symp. Soc. Exp Biol., 11:118-131

46. Tempesta, M.S., King. S. (1994). Tropical plants as a source of new pharmaceuticals, in P. S. Barnacal(ed) Pharmaceutical Manufacturing International: The International Review of PharmaceuticalTechnology Research and Development. Sterling Publications Ltd., London

47. Thomas, T.D., Shankar, S. (2009). Multiple shoots induction and callus regeneration in Sarcostemmabrevistigma Wight & Arnott, a rare medicinal plant. Plant Biotechnol Rep. 3:67-74

48. WHO. (2002). WHO Traditional Medicine Strategy 2002-2005. World Health Organization, Geneva