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
46
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
47
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
48
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
49
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,
50
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
51
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
52
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
53
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
54
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
55
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).
56
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.
57
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
58
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
59
Table 2.3 The oligonucleotides (Decamer) (Microsynth, Singapore) exhibited prominent
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).
74
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 ***
75
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
76
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
77
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.
78
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
79
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
80
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.
81
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