chapter ii development of germplasm conservation technique...

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45 Chapter II Development of Germplasm conservation technique for Dioscorea prazeri 2.1Introduction 2.1.1 Methods of Germplasm Conservation 2.1.2 Cryopreservation and its Significance 2.1.3 Cryoprotective agents 2.1.4 Conservation of Yams: Need for Cryopreservation protocols 2.2Materials and method 2.2.1 Plant material 2.2.2 Cryopreservation 2.2.3 Cryopreservation of Dioscorea prazeri by technique of Vitrification 2.2.4 Cryopreservation of Dioscorea prazeri by technique of encapsulation dehydration 2.2.5 Cryopreservation of D. prazeri by technique of vitrification of encapsulated beads 2.2.6 Genetic fidelity assessment of regenerated plantlets 2.2.7 Statistical analysis 2.2.8 Isolation of Genomic DNA 2.3Results 2.3.1 Comparitive study and optimization of cryopreservation techniques for germplasm conservation of D. prazeri 2.3.2 Genetic fidelity assessment 2.4Discussion 2.5References II

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Chapter II

Development of Germplasm conservation technique for

Dioscorea prazeri

2.1Introduction2.1.1 Methods of Germplasm Conservation2.1.2 Cryopreservation and its Significance2.1.3 Cryoprotective agents2.1.4 Conservation of Yams: Need for Cryopreservation protocols

2.2Materials and method2.2.1 Plant material2.2.2 Cryopreservation2.2.3 Cryopreservation of Dioscorea prazeri by technique of Vitrification2.2.4 Cryopreservation of Dioscorea prazeri by technique of encapsulation dehydration2.2.5 Cryopreservation of D. prazeri by technique of vitrification of encapsulated beads2.2.6 Genetic fidelity assessment of regenerated plantlets2.2.7 Statistical analysis2.2.8 Isolation of Genomic DNA

2.3Results2.3.1 Comparitive study and optimization of cryopreservation techniques for germplasm

conservation of D. prazeri2.3.2 Genetic fidelity assessment

2.4Discussion

2.5References II

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2.1 Introduction

Conservation of genetic diversity in the face of rapidly depleting natural resources has

considerable significance and worldwide importance. Indiscriminate clearing of forests

and agricultural land has led to the drastic loss of plant genetic resources. If the

destruction continues at the same pace, up to 60,000 plant species may become extinct or

threatened by the middle of this century. The incidence is more conspicuous in the

tropical and sub-tropical regions where the richest and most important genetic resources

on the earth exist. Therefore, immediate efforts are required to safeguard these

germplasms, to ensure their continued availability for present and future use. This is also

India’s national obligation following ratification of legal binding ‘Convention on

Biological Diversity'. Moreover, the prevalence of genetic diversity provides great

opportunity for crop improvement today and in distant future, when confronting

situations would demand reconstruction of new cultivars and hybrids for sustaining

higher production.

Medicinal plants growing at high altitudes have slow growth and poor seedling

establishment due to harsh environmental conditions. Conventional methods of

propagation are not sufficient and especially for endangered species, attempts for

conservation using both in situ and ex situ methods are immediately required

(Hemantlata, 1997). It is therefore, imperitive to recognize the problem and to develop

strategies for the conservation and rational exploitation of Himalayan herbs (Rawat,

1989).

2.1.1 Methods of Germplasm Conservation

Cryopreservation i.e. non-lethal storage of plant tissue at ultra-low temperature usually

that of liquid nitrogen (-196°C) is the only available method for long-term conservation

of germplasm of problem species. The major advantage of storage of biological material

at such a low temperature is that both metabolic processes and biological deterioration

are considerably slowed or even halted (Kartha, 1987).

Germplasm conservation include establishment of genetic reserves/ national parks

and gene sanctuaries (in situ conservation), seed gene banks, field gene banks, herbal

gardens, in vitro repositories (ex situ conservation) etc. In situ conservation has practical

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limitations associated with shrinking of natural habitats, urbanization, industrialization

and changing government policies. Ex situ conservation of crop germplasm bearing

orthodox seeds is conventionally carried out in seed gene banks by way of reducing

moisture content and storing at sub-zero temperature (usually at -20°C). However, many

economically important plant species produce recalcitrant seeds (desiccation and freezing

sensitive) or these are predominantly vegetatively propagated. Conservation of

germplasm of these groups thus posses’ serious problems. Due to high moisture content

in plant propagules, conservation of these problem species, under the conditions of seed

gene banks is not possible. However, maintenance of germplasm in field gene

banks/clonal repositories is labor intensive, space oriented and prone to loss of

germplasm due to pest/pathogen attacks and natural calamities. Biotechnological

approaches, including in vitro conservation, have been proposed recently, as an adjunct to

field gene bank for these problem species because it can help in conservation and

exchange of disease free germplasm. In vitro conservation strategies can be divided into

two categories like in vitro conservation under slow growth (IVAG- in vitro active gene

bank) and cryopreservation (IVBG- in vitro base gene bank). In vitro slow growth has

been used at various national and international research centers (CIP-International Potato

Center, IITA-International Institute of Tropical Agriculture, NBPGR- National Bureau of

Plant Genetic Resources) for conservation of vegetatively propagated germplasm. This

method can satisfy only short to medium term conservation strategy but management of

large collections through this method is problematic. Moreover, collections maintained

under in vitro slow growth are prone to losses due to contamination and genetic

instability. Since the last decade cryopreservation has been used successfully to various

crops, more recently, for conservation of plant germplasm (Sakai, 1997). Potentially

valuable techniques are now available on cryopreservation of cultured plant tissues for a

few species (Sakai, 2000). However, majority of crops that were worked out for

cryopreservation belong to the temperate region, which has inherent capacity to tolerate

low temperature. Cryopreservation methods are relatively less investigated with tropical

species (Engelmann, 2000), though rich diversity of crop germplasm is predominant in

this region. Yams (Dioscorea spp.) belong to the same group of tropical plant species and

are one of the most important tubers crops used for both food and / medicine (as they are

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commercially used for extraction of Diosgenin which is the precursor of steroids). Hence

development of protocol for cryopreservation is important (Scottez et al., 1992).

Problems with the in vitro techniques include high costs for maintaining a large

number of stocks, space requirements, and risks of contamination and somaclonal

variation over time. Cryopreservation is an alternative choice for a long-term

conservation of germplasm. Cryopreservation at -196ºC in liquid nitrogen (LN) has been

considered to be an ideal tool which offers long-term storage capability, maximal

stability of phenotypic and genotypic behaviour of stored germplasm, and minimal

storage space and maintenance requirements (Suzuki et al., 2008).

Pretreatments were crucial for the survival and regeneration of plant tissues after

cryopreservation. Since at present, cryoprotectants alone cannot provide enough

protection to untreated cells or tissues for high rates of survival, pretreatment techniques

are needed to condition the cells to withstand the stresses imposed by freezing at ultra-

low temperatures (Halmagyi1 A., 2010). Micropropagation and cryopreservation are

tools with multiple applications and benefits within an integrated plant conservation

research program. Biotechnological tools like in vitro culture, cryopreservation, and

molecular markers offer a valuable alternative to plant diversity studies, management of

genetic resources and ultimately conservation (Paunescu A., 2009)

2.1.2 Cryopreservation and its Significance

Cryopreservation is the storage of viable biological material at ultra-low temperatures,

which provides a means for the long-term stable storage of plant germplasm.

Cryopreservation is a safe and cost-effective technique for preservation of germplasm

and management of in vitro produced material for biotechnological application (Dixit et

al., 2004).

To ensure reproducible results and continuity in research and biomedical process,

scientist faced the task of genetically stabilized living cells. Serial subculturing is time

consuming and lead to contamination or genetic drift. The genetic stability of

cryopreserved plants can be assessed by analyzing them at phenotypic and molecular

level analysis with a range of techniques. DNA-based markers have been routinely used

for monitoring genetic stability of these species on cryopreservation (Dixit et al., 2003).

Safe and long term preservation of the production source is essential for any commercial

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applications as it secures the investments for the producer of raw material; it secures the

investment for product development for the dealer of a specific brand and it is an

essential suggestive requirement of regulatory aspects for approval and patent protection

(Heine-Dobbennack, 2009).

There are many difficulties encountered in maintaining field collections of

endangered and vegetatively propagated species and severe losses were observed during

national collections over time. Use of botanical seeds for conservation of true type

germplasm is limited. Cryopreservation has been identified as the best option for long-

term conservation of germplasm (Engelmann, 2000; Reed, 2001). Development of

cryopreservation technique is highly required for recalcitrant species as it offers a

possibility for long-term storage with maximal phenotypic and genetic stability (Harding,

1999) and aids in exchange of disease free germplasm.

It scores advantages over other conservation strategies as it reduces the risk of

contamination, cost of maintenance and cost of labour (Taylor, 1998). Challenges in

cryopreservation relates to the transition to and from the exposure to cryopreservation

temperature and time period thereof. There is a growing awareness of the need to

conserve plant genetic resources, not just to maintain biodiversity, but also to support

plant breeding and biotechnology programs. Approach for conservation of this precious

natural wealth is urgently required on several fronts.

2.1.3 Cryoprotective agents

A complex phenomenon unfolds during Cryopreservation. Freezing occurs external to the

cells before the intracellular ice form during slow cooling. So water is removed from the

extracellular environment and an osmotic imbalance occurs across the cell membrane

leading to the water migration out of the cells. The increase in solute concentration

outside the cells as well as intracellular level can be detrimental to cell survival. Damage

due to ice crystal formation and recrystallisation during warming can occur in case of

excess water remain inside the cell. The rate of cooling has a dramatic effect on these

phenomena. Cooling rate should be standardized according to the nature of cells.

Cryoprotective additives or chemicals that protect the cells during freezing can minimize

the detrimental effects of increased solute concentration and ice crystal formation

(Simone, 1998). The growth cycle of the plant is an important factor for cell recovery,

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explants and the size of explants, preculturing conditions, period of each treatment and

the hormonal treatment are highly significant in the recovery growth and the regeneration

of the species being preserved. Combinations of cryoprotective agents are more effective

than the agents used singly. The cooling rate is important and sometimes a two-step

freezing before liquid nitrogen temperature is beneficial. Rapid thawing is preferred, but

there is evidence that slow warming is just as effective in some cases.

Many cryoprotective agents have been tested either alone or in combination,

including sugars, serum and solvents. Glycerol and DMSO have been widely used and

observed to be the most effective cryopreservative, on optimization with species

(Simone, 1998). The optimum concentration of these cryoprotective agents depend upon

the species and cell type and the highest concentration the plant can tolerate. It is highly

advantageous to determine the sensitivity of the plants to increasing or decreasing

concentrations of cryoprotective agent to obtain the optimum.

2.1.4 Conservation of Yams: Need for Cryopreservation protocols

There is an urgent need to conserve the native species of D. prazeri an important

medicinal yam, threatened and endemic to India. No reports are currently available on

cryopreservation and germplasm conservation in these species. Preservation only in field

is risky, as valuable germplasm can be lost (genetic erosion) because of pests, diseases

and adverse weather conditions. There are many difficulties in maintaining field

collections of Dioscorea species (Degras, 1993). Losses from various national collections

over time have been severe (Ng and Ng, 1997). Botanical seeds are of limited use for

conservation of true type germplasm, owing to limitations in flowering and problems in

maintaining viability in case of yams. Conventionally, for conservation in field gene

banks the accessions are vegetatively propagated through planting sets (including

minisets) of underground tubers or aerial tubers. Plants are grown in fields for the entire

growing season, which lasts from six to nine months depending upon the species and

genotype. However, maintenance in the field requires vine staking and takes up a great

deal of space but the gene bank is cost and labour intensive. Furthermore, the collection

is exposed to a lot of hazards, both in the field and during tuber storage, which may lead

to genetic erosion. Diseases such as anthracnose, nematodes, and yam beetles infestation

are major field disease and pest problems of concern to field maintenance of yam

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germplasm. If an anthracnose epidemic occurs at an early stage, it could cause the

complete loss of susceptible yam germplasm. Also, severe losses (up to 100%) have been

reported, caused by various pathogens in field in different regions (Ikotun, 1989; Kahl et

al., 1991). During tuber storage, bacterial and fungal infections on tubers are serious

threats to germplasm.

Slow growth regimes are used as a medium-term storage option. These techniques

enable subculture intervals to be extended to, between 12 months to 4 years for many

species, thereby reducing dramatically the laboratory space and time required for

maintenance of the cultures. Recent reports on slow growth indicate that a variety of

techniques are still being utilized, with no obvious optimal techniques emerging. For

example, shoots and microtubers of Dioscorea spp. are stored at 28C on minimal

medium with no plant growth substances (Malaurie et al., 1993; Mandal and Chandel,

1996; Ng and Ng, 1997). Slow growth itself has limitations in maintaining large

collections owing to risk of loss of germplasm because of contamination. Furthermore,

the technique is relatively expensive and genetic stability of cultures requires further

analysis (Ashmore, 1997). Therefore, storage technique, which can eliminate

requirements for transfer and other disadvantages, has obvious attractions.

Diosgenin is among the ten most important sources of steroids and is also the

most often prescribed medicines of plant origin (Fowler, 1984). The cryostorage

protocols are mostly species specific or clonal specific in nature, so development of these

procedures for D. prazeri is highly imperative.

Hence, 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. Cryopreservation of Shoot tips of

Dioscorea prazeri can be helpful as an alternative approach for conservation of

germplasm and significant in certain recalcitrant seed and vegetatively propagated

species. Cryopreservation of germplasm is a promising tool to avoid loss of embryogenic

potential and maintaining genetic stability of highly significant medicinal plant, D.

prazeri. It is important to consider that before plant regeneration the explants have been

exposed to a range of experimental conditions including tissue culture, cryoprotection,

freezing, thawing, recovery growth, and all these stages have the potential influence on

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genetic stability. (Ahuja et al., 2002) Therefore before utilizing cryopreservation as

conservation strategy for any plant material it is essential to verify that the

cryopreservation protocol developed does not induce any somoclonal variation in plants

regenerated from shoot tips/axillary buds after cryopreservation. Genetic fidelity of the

regeneranted plants post-cryopresrvation would be analyzed using DNA markers to

confirm genetic stability.

Cryopreservation of yams is a new initiative and still in its infancy. Although

attempts have been made to develop protocol for cryopreservation of yam germplasm

applicable to various genotypes/species of Dioscorea, none of the reported studies could

observe uniformLy adequate survival of plant regeneration from frozen shoot apices

(Mandal et. al., 1996 a; Malaurie et. al., 1998). In the area of yam cryopreservation, one

of the first reports was on cell cultures of D. deltoidea (Butenko et al., 1984). The first

report of successful cryopreservation of shoot tips in Dioscorea species- D. alata,

D.bulbifera, D. rotundata (Mandal et. al., 1996 a; Malaurie et. al., 1998; Kyesmu and

Takagi, 2000), D. floribunda and D. wallichii (Mandal et. al., 1996 a) has paved the way

towards advancements in yam cryopreservation.

Dioscorea plants have been used as a herbal remedy for many years as an

antispasmodic, analgesic, aphrodisiac, diuretic, and a rejuvenative tonic (Tang et al.,

2006). Reports indicated that steroidal saponins have hemolytic (Santos et al., 1997;

Zhang et al., 1999), hypocholesterolemic (Malinow, 1985; Sauvaire, 1991),

hypoglycaemic (Kato et al., 1995), anti-thrombotic (Zhang, 1999; Peng, et al., 1996),

antineoplastic (Hu et al., 1996; 1997), antiviral (Aquino et al., 1991) and anti-cancer

(Sung et al., 1995; Huang et al., 2006) activities. Diosgenin (the aglycon part of the yam

steroidal saponin) obtained after hydrolysis of yam saponins is used as industrial starting

material for the partial synthesis of steroidal drugs, e.g. progesterone and testosterone

(Chen and Wu, 1994; Morgan, 1997; Savikin-Fodulovic et al., 1998).

Although reports of cryopreservation of shoot tips for Dioscorea species exist,

cryostorage protocols are stated as mostly species or even clonal-specific in nature

(Butenko et al., 1984; Mandal and Chandel 1996; Malaurie et al., 1998; Kyesmu and

Takagi, 2000). There is no report available on cryopreservation (shoot tips/ axillary buds

desirable for conservation purposes) of D. prazeri, which is one of the most important

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medicinal yams having high content of steroidal saponins, threatened and endemic to

India.

Hence, development of a reliable cryopreservation and regeneration protocol

specific to D. prazeri is imperative for preservation of this species for future work and

commercial applicability. Various temperature conditions, growth regulators and pre-

treatment have been examined in this study to minimize desiccation and freezing damage,

thus ensuring high propagule recovery.

2.2 Materials and method

2.2.1 Plant material

Dioscorea prazeri is a native of North-Eastern Himalayas. Healthy plants for the study

were obtained from the tubers (Mungpoo, Darjeeling, West Bengal). The plants were

established in 1:1:1 proportion of farmyard manure, soil and sand. It was maintained at a

temperature below 25±2 °C, watered to sustain moisture level. It has been deposited in

Herbarium of Avesthagen Limited, India; under the voucher specimen number 35(A).

2.2.2 Cryopreservation

The objective of the cryopreservation procedures was to reduce the freeze thaw damage

during sub-lethal levels of temperatures. The optimising factors were highly significant

for the survival and healthy growth of cryopreserved explants. Standardisation of

optimizing factors like (i) Starting material, (ii) Pre-treatment, (iii) Cryopreservation

procedures (iv) Post thaw treatment, were studied for germplasm conservation of D.

prazeri.

i. Starting material

The explant material for cryopresrvation was obtained from in vitro grown plantlets and

from the healthy elite plants of Dioscorea prazeri from the green house. The explants

excised from acclimatized plants from green house were cleansed with tap water and

surface sterilized it with tween 20 for 15 minutes, subsequently the explants were treated

with cetrimide (1000 ppm) for 10 minutes then with bavistin (1000 rpm) for 20 minutes

and treated with 0.1% Mercuric chloride for 5 minutes. These were rinsed thoroughly

with sterile water after every sterilisation treatment. The explants were excised to 1-2 cm

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in length and cultured prior to cryopreservation experiments. The sterile plant material

was obtained from exponentially growing in-vitro raised plantlets of Dioscorea prazeri

.It was cultured on sterilized Murashige and Skoog medium (Murashigue, Skoog, 1962)

(pH 8) supplemented with factorial combination of 0.5 mgl-1 N6 Benzylaminopurine

(BAP) and 0.01 mgL-1Naphthaleneacetic acid (NAA). They 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.

Subcultures of the plantlets were performed every 15-17 days. The explants used were

leaves, internodal explants, nodal explants, petiole and shoot tips from actively growing

healthy plants analysed by DNA markers for genetic fidelity. Cryopreservation allows

virtually indefinite storage of Dioscorea prazeri shoot tips without deterioration over an

extended time scale. The growth phase in which the cells are at initial stages of the

procedure is an important factor for the survival and it was experimented.

ii. Pretreatment of the explants

The capacity of the plant cells to adapt the environments stress was employed in

cryopreservation period prior to cryopreservation procedure. The most accepted

pretreatment for cryopreservation of explants was standardized by pre-culturing the

explants in low concentration of sugars from 0-0.9M for different time periods for

increasing tolerance levels of explants for cryopreservation.

iii. Procedures experimented for germplasm conservation of D. prazeri

Various cryopreservation techniques experimented for cryopreservation of D. prazeri as

(a) Encapsulation dehydration

(b) Vitrification

(c) Vitrification of encapsulated bead

iv. Post thaw treatment

Post thaw treatment was one of the significant factors for the successful cryopreservation

of plant cells. The temperatures were experimented for post thawing ranging from 4 ºC to

65 ºC. The explants were treated with multiple concentrations of growth regulators for

recovery growth and regeneration with high efficiency. It was having a great impact on

survival rate and for the healthy growth of explants

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2.2.3 Cryopreservation of Dioscorea prazeri by technique of Vitrification

The cryopreservation of the explants excised from 6-8 weeks old in vitro plantlets of

Dioscorea prazeri was carried out by vitrification. The healthy explants obtained were

cryopreserved at a temperature reduction to the level of Liquid Nitrogen (-196˚ C) in

Cryovials. The dehydrated explants in the ‘unfrozen fraction’ that remains between the

masses of ice will reach a stable glassy state, or ‘vitrify’. Various conditions were

experimented to optimize the vitrification and regeneration of D. prazeri explants to

obtain healthy explants on cryopreservation. The explants were precultured for 0 to 5

days in normal regeneration medium and pretreated on filter paper soaked with Liquid

MS medium containing 0.3M sucrose in sterile petridishes for 3 hrs to 16 hrs in dark in

BOD at a temperature of 25ºC ± 2 ºC to optimize the stress tolerant level while exposing

to lower temperatures. Subsequently the explants were treated with cryoprotective agents

(CPA) for the water content to get lowered before cooling. The explants were exposed to

loading solution (Liquid MS supplemented with 2 M glycerol and 0.4 M sucrose) for 10

to 30 minutes at 25ºC in 1.8 mL cryovial. The loading solution was removed after the

exposure and dehydrated the shoot tips with Plant Vitrification Solution 2 (PVS2) {liquid

MS supplemented with 30% (w/v) glycerol, 15 % ethylene glycol (w/v), 15% DMSO

(w/v) and 0.4 M sucrose}. The explants were treated with PVS2 for 30 - 90 minutes at

0ºC, in PVS2 followed by cryopreserving the explants, which is in cryovials, by plunging

into a temperature reduction to the level of liquid Nitrogen at -196º C for a minimum

period of 1 hour. It gives the rapid cooling rates and prevents nucleation and growth of

ice crystals and facilitates vitrification of the surrounding medium as well as the cell

contents. The thawing was performed quick enough to prevent devitrification during

thawing and tried various temperatures to optimize the exposure conditions during this

process. The CPA concentration of vitrification solutions were minimised by using very

high rates of cooling and thawing. PVS2 was replaced with unloading solution (MS

medium supplemented with1.2 M sucrose) for 10 – 30 minutes at 25ºC.Various growth

phases, incubation period and pre culturing conditions were tested to enhance survival

rates of cryopreservation than prior experiments reported. The explants were transferred

to filter paper soaked with MS medium supplemented factorial combination of hormones

in dark at 25ºC ± 2 ºC in Biological Oxygen Demand incubator (BOD incubator).

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The experiments were conducted for studying the effect of various growth

regulators and various thawing temperatures while cryopreservation experiments for

enhancing the recovery growth. The viable explants obtained were cultured on sterilized

growth recovery medium constituted of Murashige and Skoog basal medium (1962) (pH

8) supplemented with factorial combination of N6 Benzylaminopurine (BAP),

Naphthalene acetic acid (NAA) and Gibberllic acid (GA3) for optimizing the medium for

regeneration following cryopreservation. The explants were incubated in BOD for 3 to 15

days and then they were grown at 25 ± 2 ºC under a photoperiod of 16 hrs light/8 hrs dark

with a photon dose of 36 mmol m-2 s-1.

2.2.4 Cryopreservation of Dioscorea prazeri by technique of encapsulation

dehydration

The explants were transferred to sterile Sodium alginate solution (Table 2.1). The

constituents of sodium alginate solution were weighed in required concentration as

mentioned. The components except sodium alginate were weighed and dissolved in

sterile distilled water. The pH was set at 5.8 and then boiled it in a magnetic stirrer with

continuous stirring and added sodium alginate a little by little to avoid the formation of

clumps. It was boiled with continuous stirring till it dissolved completely and autoclaved

at 121 ºC and 20 psi for 1.0 hr.

Table 2.1...Media composition for the preparation of sodium alginate solution

Media composition Concentration required/100mL

MS macro (20x) 5mL

MS micro (100x) 1mL

MS iron (100x) 1mL

Thiamine (10mgmL-1) 10l

Nicotinic acid (10mgmL-1) 5l

Pyridoxine (10mgmL-1) 5 l

Myoinositol 10 mg

Sucrose (0.15 M) 5.1345 g

Sodium alginate 3 g

NB. The pH has to be 5.8. The sodium alginate should be added periodically to the boiling media

without getting clumped.

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The alginate solution with the explants were added drop wise to 100mM of sterile

Calcium chloride solution to form fine Calcium alginate beads and incubated for half an

hour. The Calcium alginate beads were pre-treated for several days in various

concentration of sucrose ranging from 0.1 to 0.9 M for period of 1 to 9 days of

incubation. Control synthetic seeds were incubated in distilled water instead of Liquid

MS media with sucrose, even the untreated actively growing explants were used as

control explants and the ‘Test’ constituted of Liquid MS medium with various

concentration of sucrose containing synthetic seeds. The pretreated encapsulated beads

were desiccated in laminar airflow for different time periods of 1 to 4 hours. Beads that

were not pre- treated with Liquid MS medium at required time period were shrunken

completely. Dehydrated beads were taken in a cryovial and cryopreserved in a

temperature reduction in level of liquid Nitrogen at -196C for a minimum period of 1

hour or more (experimented with Dioscorea prazeri up to 3.5years). These beads were

thawed at various temperatures for obtaining the optimized temperature to avoid

intercellular ice crystal formation and for enhanced regeneration frequency. These were

blot dried and inoculated to the growth recovery medium constituted of basal Murashige

and Skoog medium (MS medium) supplemented with factorial combination of N-6

Benzyl adenine (BA), Naphthalene acetic acid (NAA) and Gibberllic acid (GA3) for

standardising the growth conditions for efficient regeneration. The explants were

incubated in BOD for 3 to 15 days and then they were grown at 25 ± 2 ºC under a

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

2.2.5 Cryopreservation of D. prazeri by technique of vitrification of encapsulated

beadsA combined technique was carried out for cryopreserving the explants of D. prazeri. The

encapsulated beads were vitirified and regenerated in the recovery growth medium and

reestablished in natural habitat. The explants were encapsulated with 3.0% sodium alginate

solution in 0.1M of calcium chloride solution to form fine beads and they were pre-

cultured in MS medium with high concentration of sucrose. These were dehydrated with

loading solution and vitrified with Plant Vitrification Solution 2 and preserved in liquid

nitrogen. The beads were thawed at standardized temperatures in a water bath followed

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by replacement of PVS2 with unloading solution (MS medium supplemented with1.2 M

sucrose) at optimized temperature, 25ºC. The conditions of the treatment were

experimented to regenerate the explants efficiently. The explants were transferred to the

recovery growth medium for the re-establishment in their natural habitat. The results

obtained from these techniques of cryopreservation were compared for developing the

most appropriate technique of germplasm conservation of this species.

2.2.6 Genetic fidelity assessment of regenerated plantlets

i. Random amplified polymorphic DNA analysis

DNA was isolated using slight modification (Ref. Chapter I) of lithium chloride (LiCl)

method for aromatic and medicinal plants* (Pirtila, 2001). The DNA isolated was found

to be good on quality as well as quantity basis. The DNA was quantified by gel

electrophoresis (1% agarose gel) and using Nanodrop spectrophotometer (ND-

1000/Eppendorf) and subjected to RAPD analysis (Dixit, 2003; Narula, 2007). RAPD

analysis was carried out by amplification of 50 ng of template DNA by polymerase chain

reaction (Table 2.2) using decamer oligonucleotides (Microsynth, Singapore) (Table 2.3).

Each amplification reaction mixture contained template DNA of 50 ng, 1.0mM of dNTP,

1.0x Taq buffer, 2.0 units of Taq polymerase, 1.0 picomoles of primer, 2.5 mM of MgCl2

and sterile HPLC grade water (Table 2.4). The PCR amplified sample was loaded on

1.5% agarose gel stained with ethidium bromide and photographed in a phosphoimager

(BioRad). The gel was scored for clearly identifiable bands.

Table 2 2 Cycling conditions well worked for amplification

Conditions Temperature Time Cycles

Initial denaturation 94˚ C 4’ 1

Denaturation 94˚ C 1’

44Annealing 35˚ C 2’

Extension 72˚ C 2’

Final extension 72˚ C 10’ 1

4˚C Forever

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Table 2.3 The oligonucleotides (Decamer) (Microsynth, Singapore) exhibited prominent

banding pattern on genetic fidelity assessment

SL. No Primer Sequence SL. No Primer Sequence

1 OPP7 5’-GTCCATGCCA-3’ 11 OPF 10 5’-GGAAGCTTGG-3’

2 OPC06 5’-GAACGGACTC-3’ 12 OPK 17 5’-CCCAGCTGTG-3’

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

4 OPN18 5’-TCAGAGCGCC-3’ 14 OPL 20 5’-GGAAGCTTGG-3’

5 OPN 8 5’-ACCTCAGCTC-3’ 15 OPO 10 5'-TCAGAGCGCC-3’

6 OPC19 5’-GTTGCCAGCC-3’ 16 OPQ 14 5’-GGACGCTTCA-3’

7 OPD 09 5'-CTCTGGAGAC-3’ 17 OPN 8 5'-ACCTCAGCTC-3’

8 OPH 14 5’-ACCAGGTTGG-3’ 18 OPQ 11 5’-TCTCCGCAAC-3’

9 OPJ 5 5’-CTCCATGGGG-3’ 19 OPL 01 5’-GGCATGACCT-3’

10 OPF 2 5’-GAGGATCCCT-3’ 20 OPG 18 5’-GGCTCATGTG-3’

Table 2.4 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 (autoclaved) 39.4μL

ii. Morphological analysis

The in vitro grown rooted plantlets were 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 the plants were watered regularly. 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 green house containing farm yard manure, vermiculate to

soil in 1:1:1 ratio at regulated temperature of 25±2ºC. The morphological characters were

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analyzed from different sets of experiments, the average value was calculated and the

data obtained was statistically analysed.

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

The standard curve was plotted with Diosgenin procured from Sigma, USA (~98% pure).

The plant extract was prepared from in vitro raised plantlets and the wild plants of D.

prazeri. The plant tubers were dried at 45ºC for two days and the pulverized tubers were

hydrolysed at 95ºC for 3½ hours in 2N hydrochloric acid. The pH of the extract on

hydrolysis was neutralized (7.0) with 2N sodium hydroxide. The extracts were

centrifuged and the residue was dried at 78ºC for 2 hours in hot air oven. The soxhlet

extraction (Biosox Unit, Techno Reach) was carried out with different solvents as

methanol, petroleum ether and hexane for the comparative study on yield of steroidal

sapogenin. 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). The samples were showed prominent peak of Diosgenin with 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.

2.2.7 Statistical analysis

Values are expressed as an average of replicates ± standard deviation of independent

experiments. The results obtained from the experiments on cryopreservation and on

enhancing the efficiency of recovery growth in MS medium, with various growth

regulators, were analysed statistically. The data was analysed by one-way analysis of

variance (ANOVA) with Bonferroni’s multiple comparison tests.

2.2.8 Isolation of Genomic DNA

Modified DNA extraction protocol specific for the medicinal plants was used as

described. The extraction buffer was made with Cetyl trimethylammonium bromide

(CTAB; 2% (w/v)), Tris - HCl (pH: 8; 100 mM), EDTA (pH 8.0; 20 mM), NaCl (1.4M),

lithium chloride (8M), Polyvinyl pyrrolidone (PVP; 2% (w/v)) Mercaptoethanol (2%

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v/v, immediately before use). The fresh plant tissue was ground into a fine powder in

liquid nitrogen using a mortar and pestle. The powder was weighed (0.5 g) in a

microcentrifuge tube (eppendorf; 2.0 mL) using pre-cooled spittle and vortex with the

pre-warmed extraction buffer. Lithium chloride solution along with the extraction buffer

in 1:1 propotion was added to the tubes and the contents were placed at 650C in a water

bath for 30 minutes and vortex 3-4 times during the incubation. Chloroform–isoamyl

alcohol (24:1) (700 L) was added to the extract and mixed thoroughly. It was

centrifuged at 13000 rpm (13,793 g) for 5 minutes at room temperature. The extraction

was repeated thrice with the supernatant obtained. The upper phase was pipetted into a

new tube. Potassium acetate (3M, pH: 4.8; 0.5 volume (300l)) was added to the

supernatant by inverting the tube carefully. The contents were placed at 13000 rpm

(13793 g) for 10 minutes at 4ºC and pipette out the supernatant into a new tube. Ice-cold

isopropanol (0.6v; 600l) precipitated the genomic DNA by inverted the tube carefully

several times to mix the layers. The contents were centrifuged at 13000 rpm (13793 g) for

10 minutes at 4ºC. The supernatant was discarded and dry the pellet at 37ºC in heating

block. Dissolved the pellets in 300 L distilled water and then add 600L of cold absolute

alcohol ethanol and centrifuge for 10 minutes at 13,000 rpm (13793 g) at 4˚C. The DNA was

dried pellet and dissolved in 50.0µL of 1x Tris EDTA (pH 8.0). The DNA was quantified by

electrophoresis on agarose gel (1%) and on spectrophotometer (Nanodrop, eppendorf).

2.3 Results

2.3.1 Comparitive study and optimization of cryopreservation techniques for

germplasm conservation of D. prazeri

The data indicate that MS media supplemented with BAP of concentration 0.5mgL-1 and

NAA of concentration 0.01mgL-1 was essential to induce favourable physiological

conditions for shoot initiation and regeneration of Dioscorea prazeri. A regeneration

frequency of 98±2% and a survival rate of >96% on field establishment was achieved

through micropropagation with 0.5 mgL-1 BAP and 0.01mgL-1 NAA (Ref. Chapter I).

Plants grown in this media showed the highest rate of multiplication and survival as

compared with hormones such as Thiadizuron (TDZ), Zeatin, Kinetin and 2iP (Ref.

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Chapter I). The protocol was developed, for recovery growth subsequent to

cryopreservation, based on the results from first phase of the project. Adequate

management of a whole culture system was observed to be a high requisite for effective

pre-culturing of dissected shoot apices, vigorous recovery of cryopreserved shoot apices

and it was efficiently achieved.

The protocols generated in this study have resulted in successful cryopreservation of

germplasm of D. prazeri. The results obtained with various explants and cryopreservation

techniques are illustrated.

i. Encapsulation Dehydration

The encapsulated explants of D. prazeri pre-treated in Liquid MS medium supplemented

with sucrose of 0.5 M concentration for 5 days prior to cryopreservation, regenerated

with higher efficiency (Fig. 2.1A and B). The explants pre-treated at this concentration

were viable and recovered on cryopreservation. It regenerated at a faster rate than rest of

the explants with other experimental conditions. The cryopreserved explants were

recovered and elongated on Murashige and Skoog basal medium (pH 5.8) supplemented

with higher concentration growth regulators of 1.5 mgL-1 BAP, 0.2 mgL-1 NAA and 0.2

mgL-1 GA3 for 3 days in BOD at a temperature of 25ºC ± 2 ºC which were subsequently

transferred to the regeneration medium (MS+0.5 mgL-1 BAP and 0.01 mgL-1 NAA)

containing 0.01 mgL-1 GA3.

The encapsulated explants were propagated on MS medium constituting 0.5 mgL-

1 BAP; 0.01mgL-1 NAA (Fig. 2.1C) achieved regeneration frequency of 75 ± 2%. It was

showing multiplication of shoots with healthy culturable segments (Fig. 2.1D). The

rooted plantlets were acclimatized and established in the soil. The beads that were pre-

treated with lower concentration of sucrose (<0.5M) for required time period were

shriveled completely during the post treatment of cryopreservation where as those in

higher concentration (>0.7M) showed stunted growth in regeneration media. The

optimum concentration of sucrose was found to be 0.5M and explants responded well.

High sensitivity of explants and ice crystal formation were observed in untreated

encapsulated beads on post-cryopreservation as compared to sucrose treated ones (<0.5M

to >0.7M) (Fig. 2.1E). The concentration of sucrose during the treatment had high effect

on regeneration frequency on recovery growth and regeneration (Table 2.5) (Fig. 2.2).

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Fig. 2. 1 Cryopreservation of D. prazeri by encapsulation dehydration method.

(A) Air dried encapsulated shoot tips of D. prazeri that were viable after cryopreservation by

technique of encapsulation dehydration (microscopic picture); (B) Growth recovery and initiation

of regeneration of shoot tips pre-treated with sucrose of 0.5M for 5 days; (C) Regeneration of

cryopreserved encapsulated shoot tips; (D) Multiplication of encapsulated shoot tips after

cryopreservation experiments; (E) Ice crystal formation in untreated control bead post

cryopreservation experiment.

Fig. 2. 2 The effect of concentration of Sucrose on encapsulation dehydration method of

cryopreservation of explants.

The data indicates the regeneration frequency of D. prazeri obtained on treatment with a range of

sucrose concentration with various incubation periods.

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Table 2.5 Effect of preculturing with various concentration of sucrose on regeneration

effiecieny of D. prazeri

Concentration ofSucrose used (encapsulationdehydration experiment)

No of days Regeneration frequency (%)(Out of 30 explants eachinoculated;Replicates of 3sets)

0.3M 3 10± 25 13± 27 17± 29 22± 1

0.5M 3 45± 15 74± 27 64± 29 60± 1

0.7M 3 63± 15 59± 17 30± 19 27±2

0.9M 3 16± 15 11± 17 9± 29 7± 3

ii. Vitrification

The successful regeneration with highest survival rates ever reported of 92 ± 2% attained

from vitrified shoot tips on cryopreservation. The explants were obtained from 6-8 weeks

old in vitro raised plantlets of D. prazeri. The frequency of regeneration was highest in

comparison with the other techniques reported for cryopreservation. The healthy shoot

tips used as explants pre-cultured for a day in normal regeneration media and pre-treated

in sucrose of 0.3M to 0.5 M concentration for 14 hours prior to vitrification and

cryopreservation showed high frequency of recovery and best response on regeneration.

A few explants gradually turned pale when treated for more than 14.0 hours whereas

shorter duration of pre-treatment did not enhance the growth recovery. Frequency of

recovery was less when the concentration of sucrose was lowered during pre-treatment

(<0.3). At higher concentration the growth was stagnant and found to be slightly

deleterious. The explants treated with loading and unloading solution for 15 to 20

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minutes showed a higher survival rate. The optimum temperature for thawing was found

to be 40ºC for obtaining healthy plantlets. The thawing temperature had a greater effect

on regeneration efficiency. The thawing temperatures below 37 ºC and above 50 ºC were

drastically affected the regeneration of cryopresrved explants (Fig. 2.3).

Fig. 2. 3 The optimization of thawing temperature of cryopreserved explants.

It had a great impact on frequency of regeneration and the data indicates efficacy when the

explants were treated with 37 ºC to 50 ºC

The survival rate of cryopresrved plants was observed to be very low when the thawing

temperature was lowered than 37 ºC and when it was reached 55 ºC (Table 2.6).

Table 2.6 The eefect of post thawing temperature on regeneration efficieny of D. prazeri

Temperature (ºC) Duration RegenerationFrequency (AVG) (%)

25 2-3’ 10±137 2-3’ 83±240 2-3’ 93±150 2-3’ 33±155 2-3’ 17±260 2-3’ 7±165 2-3’ 2±1

The recovery and regeneration, after the cryopreservation for a period of 1.0 hour to 16

months, was highest when the explants were incubated for 3 days in 1.5mgL-1 BAP,

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0.2mgL-1 NAA and 0.2mgL-1 GA3 in dark in BOD at a temperature of 25ºC ± 2 ºC (Fig.

2.4A) and transferred to the basal medium containing 0.5 mgL-1 BAP, 0.01 mgL-1 NAA

and 0.01 mgL-1 GA3 for regeneration and elongation (Fig. 2.4B-D). The elongated

explants cultured on MS medium constituted with 0.5mgL-1 BAP and 0.01mgL-1 NAA

(Table 2.7) resulted in multiple shooting. Efficiently regenerated explants after

cryopreservation with healthy culturable nodal segments were propagated (Fig. 2.4E-G).

The various hormonal treatments, the concentration of the growth regulators and the

duration of exposure of explants to these conditions had a greater effect on regeneration.

The rooted plantlets (Fig. 2.4H) were hardened (Fig. 2.4I-J), successfully acclimatized

(Fig. 2.4K) and established in the soil. The potential technique has been successfully

developed for conserving the germplasm of D.prazeri through cryopreservation of

vitrified shoot tips (Table 2.8).

Fig. 2. 4 Cryopreservation of vitrified shoot tips of D. prazeri.

(A) Viable shoot of tip of D. prazeri in growth recovery medium of 1.5 mgL-1 N6

Benzylaminopurine (BAP), 0.02 mgL-1 Naphthalene acetic acid (NAA) and 0.2 mgL-1 Gibberllic

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acid (GA3) in dark in BOD at a temperature of 25ºC ± 2 ºC for 3 days on post vitrification and

cryopreservation; (B) Growth recovery of vitrified shoot tip of D. prazeri and initiation of growth

of shoot tip in MS medium with 0.5 mgL-1 BAP, 0.01 mgL- NAA and 0.01mgL-1 GA3; (C)

Elongation and regeneration of cryopreserved shoot tips that were vitrified; (D) A closer view of

initiation of regeneration; (E) Multiplication and propagation of vitrified shoot tips in MS

medium with 0.5 mgL-1 BAP, 0.01 mgL-1 NAA (one week after recovery);(F) Proliferation of

shoot tips; (G) Elongation of cryopreserved shoot tips on culturing; (H)In vitro rooting; Hardened

plantlets (I) acclimatized plantlets (J) and plantlets established in the soil(K) of cryopreserved

regenerants of D. prazeri

Table 2.7 Regeneration efficiency with MS media with various concentrations of hormones

after cryopreservation

Explants(Nodal explants and shoots)

Number of cryopreserved explants regenerated onvarious hormonal treat. And frequency of regeneration

CryopreservedExplants

ControlExplants (A)

Freq.Regn(%)

(B)Freq.Regn(%)

(C)Freq.Regn(%)

Set I 50 15

18 36 46 92 37 74

15 30 44 88 34 68

18 36 46 92 35 70

19 38 43 86 37 74

17 34 45 90 34 68

Set II 50 15

18 36 43 86 30 60

16 32 45 90 35 70

17 34 46 92 37 74

16 32 47 94 32 64

17 34 44 88 32 64

Set III 50 15

17 34 44 88 34 68

19 38 48 96 35 70

20 40 46 92 34 68

18 36 46 92 37 74

17 34 47 94 35 70

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Number of explants regenerated in MS media with various hormonal concentrations as mentioned (A)(MS

+ 1.5 mgL-1 BAP, 0.2mgL-1 NAA and 0.2 mgL-1 GA3); (B) (MS+ 1.5 mgL-1 BAP, 0.2mgL-1 NAA and 0.2

mgL-1 GA3 (3d) then 0.5 mgL-1 BAP and 0.01mgL-1 NAA; then 0.5 mgL-1 BAP and 0.01mgL-1 NAA; (C)

(MS + 0.5 mgL-1 BAP and 0.01mgL-1 NAA).

Table 2.8 Regeneration efficiency of D. prazeri explants with various methods of

cryopreservation and regeneration

SI no. Explants

(Nodal explants and

shoot explants)

Number of cryopreserved explants regenerated and

Frequency of regeneration (%)

Cryo

preserved

Contro

l

Vitrficat

-ion.

Freq.

Regn.

Encapsula

-tion

Freq.

Regn.

Vitfn. +

Encap.

Freq.

Regn

1 80 20 72 90 57 71.25 29 36.25

2 80 20 74 92.5 63 78.75 28 35

3 80 20 75 94 62 77.5 26 32.5

4 80 20 74 92.5 60 75 32 40

5 80 20 73 91 61 76.25 30 37.5

The statistical data for Vitrification, Encapsulation dehydration and for the Vitrification of encapsulated

beads were analysed with ANOVA showed P value: <0.0001(***: Indicates high significance); R2 value:

0.99 and the Mean Significant Difference MSD: P<0.05 and Bonferroni multiple comparison tests for

Vitrification vs Encapsulation dehydration vs Vitrification of encapsulated beads and with treatment

between columns showed High significance (***) and MSD as P<0.05

The shoot tips responded faster and produced healthier plantlets on cryopreservation

experiments in comparison with other explants used. The response of the leaf explants

was very slow and regeneration frequency was also low. Even though the petiole explants

have not given direct regeneration, slow response in callusing was observed. In most of

the experiments during standardisation, frequency of regeneration of petiole explants was

inadequate. The shoot tip explants showed stability in D. prazeri.

An efficient and reliable protocol for cryopreservation of germplasm was carried

out and shoottips /nodal explants were observed to be the most pertinent explants with

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highest regeneration efficiency and vitrification was found to be a highly reliable method

for germplasm conservation of D. prazeri.

iii. Vitrification of encapsulated beads

Regeneration of cryopreserved plants was achieved through this combined technique but

the frequency of survival obtained with this method was 38±2%, in contrast to the

efficiency obtained when treatment carried out independently.

2.3.2 Genetic fidelity assessment

The growth and morphology of the re-established plantlets of cryopreservation were

evaluated and assessed for genetic integrity using molecular markers. RAPD profile of

donor plant and the cryopreserved regenerants of D. prazeri were analyzed for genetic

integrity. The genomic DNA was isolated from the cryopreserved plants and in vitro

regenerated donor plants (Fig. 2.5). The DNA obtained was of high quality and quantified

(900ng µL-1) by gel electrophoresis and on Nanodrop spectrophotometer (ND-1000; Eppendorf).

1 2 3 4 5 6 7 8 9 10 11 12 13 M

Fig. 2. 5 The genomic DNA isolated from In vitro regenerated plants.

Lane 1-4: Mother plantlets, Lane 5-11: Regenerated plants of cryopreservation, Lane 12: λ DNA

uncut 50ng, Lane 13: λ DNA uncut 100ng, Lane M: Molecular marker

i. Genetic stability assessment: RAPD analysis

A comparison of the RAPD amplified banding pattern between the in vitro maintained

control plantlets and the cryopreserved regenerants showed identical profiles thereby

indicating genomic stability (Fig. 2.6). The cryopreservation technique developed for

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conserving the germplasm of D. prazeri was applied to other species like D. alata and D.

composite.

Fig. 2. 6A The RAPD gel profile of Donor Plant and the regenerated plants after

cryopreservation.

Lane 1-2: D. prazeri donor plant using primer primer OPK 17; Lane 3-7: D. prazeri regenerated

plants of cryoptreservation using primer OPK 17; Lane 9-10: D. prazeri donor plant using primer

OPG 18; Lane 11-15: D. prazeri regenerants using primer OPG 18; Lanes 8, 16: Negative

controls; Lane M: molecular marker

Fig. 2.6B The RAPD gel profile of Donor Plant and the in vitro regenerated after

cryopreservation (set II).

Lane 1-2: D. alata using primer OPK-17, Lane 3-4: D. prazeri donor plant using primer OPK-17,

Lane 5-7: D. prazeri regenerants using primer OPK-17, Lane 9-10: D. alata using primer OPI-08,

Lane 11-12: D. prazeri donor plant using primer OPI-08, Lane 13-15: D. prazeri regenerants

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using primer OPI-08, Lane 17-18: D. alata using primer OPN-08, Lane 19-20: D. prazeri donor

plant using primer OPN-08, Lane 21-23: D. prazeri in vitro regenerated plants using primer OPN-

08, Lane 25-26:D. alata using primer OPO-10, Lane 27-28: D. prazeri donor plant using primer

OPO-10, Lane 29-31: D. prazeri in vitro regenerated using primer OPO-10, Lane 33-34: D.

alata using primer OPQ 11, Lane 35-36: D. prazeri donor plant using primer OPQ 11, Lane 76-

39: D. prazeri in vitro regenerated plants using primer OPQ 11, Lanes 8, 16, 24, 32, 40: Negative

controls and Lane M : molecular marker

ii. Diosgenin estimation: HPLC analysis

A HPLC analysis carried out for determination and comparison of the diosgenyl saponin

content in the in vitro regenerated plants and the donor plant showed no significant

difference between these plants in terms of secondary metabolite product pattern (Table

2.9). The percentage of Diosgenin obtained from tubers was found to be as high as 2.4%.

The negative control plantlet used for the experiment, D. alata, did not reveal the

presence of steroidal sapogenin. Thus the biochemical analysis for the content of

Diosgenin levels with pulverised tubers of cryopreserved and control donor plantlets

showed that the Diosgenin content was identical (Fig. 2.7A-C).

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Fig. 2. 7 The chromatogram obtained on HPLC analysis with methanol as mobile phase at 35ºC

for steroidal sapogenin, Diogenin of donor plant and in vitro regenerated plants of D. prazeri.

(A) The chromatogram had shown the concentration of 5.0 μg of Diosgenin (Standard); (B) The

chromatogram of extract from in vitro regenerated cryopreserved plants of D. prazeri; (C) The

extraction for steroidal sapogenin, Diogenin of donor plants of D. prazeri. The retention time of

the required peak was at 7.0 minute with methanol as an isocratic solvent system.

Table 2.9 Diosgenin assay of in vitro raised plantlets of D. prazeri

Sample (μg) Type of sample Area of the peakConcentration

obtained (μg)

1.0 Standard 235935 1.0

2.0 Standard 497542 2.0

3.0 Standard 785119 3.0

4.0 Standard 1019160 4.0

5.0 Standard 1359612 5.0

Donor Plant Extract 6148784 22.6

In-vitro regenerated cryopreserved Extract 6257692 23.0

Control plant of Dioscorea alata Extract Not detected Not detected

The sample used for HPLC analysis of tuber extract of D. prazeri with absolute methanol and the

concentration of steroidal sapogenin, Diosgenin obtained based on the peak from chromatogram. There was

no significant difference observed in the secondary metabolite product pattern of donor plant and

cryopreserved regenerants. The negative control plantlet used for the experiment, D. alata did not reveal

the presence of steroidal sapogenin.

iii. Morphological analysis

The regenerated plantlets on cryopreservation were proliferated as vigorously as control

plantlets and no morphological abnormality were detected. The plantlets regenerated

directly from shoot tips after cryopreservation and the ability to produce microtubers

remained unaltered. Both in vitro regenerated plantlets and control plantlets after

cryopreservation produced healthy leaves, shoots and roots. The morphological

characters of cryopreserved, in vitro grown and donor plant were compared. The growth

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pattern and comparative study did not show any significant difference with donor plants

and were found to be stable (Table 2.10).

Table 2.10 Comparative studies of morphological characters of D. prazeri

Morphological charactersCryopreserved

plants

In-vitro grown

plantsWild plants

Length/breath ratio of mature

leaf

10.4±3.2:7.85±2.3 10.7±3.6:8.2±2.1 10.3±3.2:7.8±2.25

Number of primary stems 7.0±2.00 8.0±2.0 7.0±2.0

Petiole length 4.65±1.55 4.69±1.8 4.28±2.01

Internodal length 9.0±1.6 9.4±2.0 8.3±0.7

Lamina/petiole length ratio 10.4±3.2:

4.65±1.55

10.7±3.6: 4.69±1.8 10.3±3.2:

4.28±2.01

Comparative studies of morphological characters of cryo-derived, in vitro grown plants in green house and

the wild type did not show any significant difference in growth pattern and found to be morphologically

stable.

iv. Statistical data analysis

The data on treatment with various concentrations of BAP, NAA and GA3 with MS

media for cryopreservation experiments (Table 2.11) and various method of

cryopreservation (Table 2.12) were analyzed statistically with one-way analysis of

variance (ANOVA) and with Bonferroni’s multiple comparison test showed high

significance. A graphical representation on comparison of the different methods of

cryopreservation (80 explants each) indicated highest regeneration frequency obtained

after vitrification was 92±2%, whereas encapsulation dehydration gave 75±2% whereas

vitrification of encapsulation dehydration was 38±4%(Fig. 2.8A). The data indicated

regeneration efficiency was remarkably effective when the cryopreserved explants were

first treated with MS+ 1.5 mgL-1 BAP, 0.2 mgL-1 NAA and 0.2 mgL-1 GA3 for 3 days

and then transferred to 0.5 mgL-1 BAP, 0.01mgL-1 NAA and 0.01mgL-1 GA3 and

propagated on 0.5 mgL-1 BAP, 0.01mgL-1 NAA (Fig. 2.8B).

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Fig. 2. 8 Graphical representation of regeneration efficiency of the explants.

(A) Methods of conservation using (V) Vitrification of explants, (E) Encapsulation of explants

and (V+E) Vitrification of Encapsulated explants; (B) Effect of various growth regulators on

growth recovery and regeneration of explants. Number of explants regenerated in MS media with

various hormonal concentrations as mentioned (Conc A) (MS + 1.5 mgL-1 BAP, 0.2mgL-1 NAA

and 0.2 mgL-1 GA3); (Conc B) (MS+ 1.5 mgL-1 BAP, 0.2mgL-1 NAA and 0.2 mgL-1 GA3 (3d)

then 0.5 mgL-1 BAP and 0.01 mgL-1 NAA; then 0.5 mgL-1 BAP and 0.01 mgL-1 NAA; (Conc C)

(MS + 0.5 mgL-1 BAP and 0.01 mgL-1 NAA).

Table 2.11 Statistical analysis (ANOVA) for plant growth recovery and regeneration in MS

media with various concentrations of growth regulators

*MSD Mean Significant Difference; The statistical analysis for regeneration efficiency with various

concentrations of growth regulators with MS media were calculated using ANOVA and Bonferroni

multiple comparison tests. P value: <0.0001; ***: Indicates high significance; R2 value: 0.98

One-way analysis of variance DataBonferroni MultipleComparison Test

Data

P value < 0.0001P value summary *** A vs B; P < 0.05 Yes*MSD (P < 0.05) A vs C; P < 0.05 YesNumber of groups 3 Treatment bet. Columns 5900F 1100 P value summary ***R squared 0.98 P value summary ***

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Table 2.12 Biostatistical analysis using ANOVA on various methods of Cryopreservation

One-way analysis of variance Data Bonferroni's MultipleComparison Test Data

P value < 0.0001 Vitrification vs Encapsulationdehydration; Significant P <0.05?

YesP value summary ***

*MSD(P <0.05) Treatment between Columns 5900

Number of groups 3 P Value Summary ***F 680R squared 0.99*MSD Mean Significant Difference; The statistical analysis for Vitrification, Encapsulation dehydration

and for the Vitrification of encapsulated beads was calculated using ANOVA and Bonferroni multiple

comparison tests. P value: <0.0001; ***: Indicates high significance; R2 value: 0.99

The values obtained from statistical analysis and the graphs indicated that the above

experiments are highly significant with P value of <0.0001 and R2 value of 0.99 for the

treatment conditions and the recovery growth in MS media with 0.5 mgL-1 BAP and 0.01

mgL-1 NAA.

The shoot tips of Dioscorea prazeri were successfully cryopreserved with

subsequent regeneration using Vitrification and Encapsulation dehydration technique.

Genetic stability of plants regenerated from cryopreserved meristematic tissue was

assessed using molecular markers .The random amplified polymorphic DNA analysis of

cryopreserved-derived plants and in vitro grown control plantlets showed genetically

similar fragmentation profiles. The amplification products were monomorphic for all the

plantlets. The DNA fragments obtained from this study showed no variation in RAPD

profiles. The morphology and ability to form microtuber were also found to be unaltered

in cryopreserved plantlets. The biochemical analysis showed the metabolite content was

not showed significant alteration. Thus the Dioscorea prazeri plants were stable at

molecular, biochemical and morphological level.

Present study indicates that the cryopreservation of shoot tips of the endangered

plant; D. prazeri by vitrification has been successfully achieved for medium, short or

long-term conservation of germplasm. The regeneration frequency achieved by

vitrification technique of cryopreservation was found to be the highest reported, which

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was 92±2% and encapsulation dehydration technique showed frequency of 75±2 % with

Dioscorea prazeri. In cryopreservation technique, the explants were regenerated with

higher frequency in comparison with the past studies. The above two techniques were

combined, in which vitrifying the encapsulated beads were showed lesser regeneration

rate than the individual method of cryopreservation. The cryopreservation of D. alata and

D. composita were successfully carried out using these standardized conditions for D.

prazeri. The same procedure was tried out for other family of medicinal plant like

Jatropha curcas and given healthy plants with 83.5±3.0% of regeneration efficiency in

MS medium with same combination of hormones used for D. prazeri on cryopreservation

experiments.

The growth, morphology and the genetic integrity of the re-established plantlets

of cryopreservation were evaluated with molecular markers, which did not show any

variation among the regenerants in comparison with the donor plant. For germplasm

conservation experiments of D. prazeri, the vitrification technique of cryopreservation

was used because of healthier and highest frequency of regeneration. So the prime

interest was to conserve the germplasm of medicinally significant, indigenous

endangered D. prazeri without compromising on the various potentialities on the

phenotypic and genotypic characteristics that hold great importance. The results reported

here in D. prazeri supported the observation that cryopreservation techniques are

generally not a basis of variation process in plants, but the various concentrations of

hormonal treatments and the pre-treatments can impose a greater effect on growth

recovery and regeneration of the explants. In D. prazeri the cryopreserved shoot tips were

regenerated directly without any callus formation.

The regeneration frequency of the plantlets was highest in vitrification method

compared to encapsulation dehydration and of vitrified encapsulated beads technique of

cryopreservation. It was found to be 92±2% and successfully recovered, regenerated and

micropropagated post cryopreservation of explants on MS medium with 1.5 mgL-1 BAP,

0.2mgL-1 NAA and 0.2 mgL-1 GA3 for 3days, further on 0.5 mgL-1 BAP and 0.01mgL-1

NAA and 0.01mgL-1 of GA3 (4 days) and on 0.5 mgL-1 BAP and 0.01mgL-1 NAA. The

values obtained from statistical analysis and the graphs indicated that the above

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experiments are highly significant and MS with BAP and NAA was most approving for

obtaining the healthy plants.

Young shoot tips obtained from in vitro raised plantlets, pre-cultured in BOD

incubator in dark for a day showed the best survival rate. The treatment of explants for a

very short interval in media with higher concentration of growth regulators and recover

and regenerate the explants further in lower concentration of hormones enhanced the

frequency of regeneration to a maximum level. The plants showed high efficiency on

field establishment with healthy, stable regeneration. The micropropagated plants of D.

prazeri, which has immense pharmaceutical benefits, will be reinstated to natural habitat.

Thus the study overcomes the problems of conservation and stability of the germplasm

and availability of this endangered medicinal plant and it is applicable to other plant

species.

Fig. 2. 9 Germplasm conservation experiment conducted for D. prazeri: a complete view of the

process.

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2.4 Discussion

D. prazeri has been regenerated on cryopreservation with higher frequency for the first

time. The regeneration frequency achieved was found to be the highest reported with

92±2%, under optimized conditions while the encapsulation dehydration technique

showed regeneration frequency of 75±2 %. Vitrification has emerged as a promising

method of cryopreservation (Halmagyi, et al, 2010; Mukherjee, et al., 2009), and was

found to be the most potential technique in D. prazeri providing maximum recovery than

reported. The pre-culturing conditions had a great impact on survival rate of

cryopreserved shoot tips as stated (Yin and Hong, 2009b; Halmagyi, et al., 2010) and the

optimized conditions for D. prazeri produced healthy plantlets. The optimisation of

sucrose concentration was critical for regeneration (Leunufna, et al., 2003) and with D.

prazeri, 0.3 M to 0.5 molar for 14 hours for vitrification procedure to 5 days for

encapsulation made improved the tolerance level on cryopreservation. The effect of

temperature during PVS2 was stated to be imperative (Fowler, 2004; Yin and Hong,

2009b). The efficacy of regeneration of explants was increased on treatment with PVS2

at 0°C for D. prazeri in comparison with room temperature. It was found with this study

that the thawing temperatures of 33°C to 55°C on post cryopreservation had an impact on

establishment of high survival rate of explants. Significant improvement in survival of D.

prazeri was observed while culturing the explants in dark, on post thawing for short term.

The hormonal effect of cryopreservation on survival of species was highly significant

(Mukherjee et al., 2009; Turner et al., 2001). It was observed with this study that the

recovery of cryopreserved explants improved when explants were treated with higher

concentration for a very short interval than the usual intensities of hormonal requirement

for micropropagation. Subsequently with low concentration of GA3 (0.01 mgL-1) along

with regeneration media just for 3 days in dark helped the healthy elongation of explants,

afterwards transferring to regeneration media facilitate regeneration to a maximum level

with multiple shoots within 40 days of growth.

The regeneration of explants on cryopreservation was reported earlier with other

Dioscorea species and with other plants stated the high requisite on optimization of

treatment period and on clonal specificity (Yin and Hong, 2010; Mandal, et al, 2007;

Tanaka et al., 2004). Hence, germplasm conservation protocol was well studied here and

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established with D. prazeri. Conditions standardized for D. prazeri were successfully

used for the cryopreservation of D. alata and D. composite and achieved high efficiency

of regeneration. Similar conditions were experimented for other medicinal plants like

Jatropha curcas and acheived 83.5±3.0% regeneration efficiency. The regeneration

media used and period of incubation was found to have a significant effect on survival of

these plants as well. In addition to that this protocol can be used to conserve the

germplasm of the plants grown in remote places and provide ease of access on

requirement; can be employed to save the precious germplasms and to cross breed for

crop improvement today and in distant future for new cultivars, and hybrids for

sustaining higher production as per agricultural needs. Cryopresrvation makes the mode

of transport of germplasm easier and protects the valuable species from being extinct.

The combined technique of vitrification of encapsulated beads was a potential technique

(Yin and Hong, 2010) but in Dioscorea prazeri the regeneration rate observed to be

38±2%, in contrast with other techniques. The current study indicates that

cryopreservation by vitrification method using shoot tips is the most successful technique

for medium, short or long-term conservation of germplasm of the endangered plant

species D. prazeri.

The genetic integrity of the re-established plantlets of cryopreservation was

evaluated using RAPD analysis (Martin et al., 1998; Englemann, 2004; Dixit, et al.,

2003), showed integrity in D. prazeri. The growth, morphology and biochemical stability

was assessed and it confirmed constancy for the regenerated plants. The prime concern

was to conserve the germplasm of the endangered species of D. prazeri without

compromising the various potentialities on the phenotypic and genotypic characteristics

and was successfully achieved. The results reported here in D. prazeri confirmed that

optimized cryopreservation techniques are generally not a source of somaclonal variation.

This cryopreservation techniques studied were observed to produce a favorable effect on

growth, recovery and regeneration of the explants using experimented concentrations of

hormonal and pre-treatments. In D. prazeri the cryopreserved shoot tips were regenerated

directly without any callus formation. The nodal explants were stored upto 16 months in

liquid nitrogen and showed high efficiency on field establishment with healthy, stable

regeneration. It was reported that buds stored over 10 years in liquid nitrogen still

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maintained high viability (Volk, et al., 2008). The micropropagated plants of D. prazeri,

which have immense pharmaceutical benefits, can be reinstated to a natural habitat. Thus

the study helps overcome issues concerned with safety and stability of the germplasm and

provides easy accessibility and availability of this endangered medicinal plant. In vitro

culture, cryopreservation, and molecular markers present important techniques for

management of genetic resources and ultimately, conservation of species.

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