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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
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