Cryopreservation of Anti-Diabetic Plants
15
M. R. Rohini, Marcos Edel Martinez Montero, and P. E. Rajasekharan
Abstract
Medicinal plant use and trade has seen a dramatic increase over the years owing to
the increased realization about its health benefitting effects. Limited commercial
cultivation has forced the industries and traders to rely on the wild collections to
meet the growing demand. The situation has threatened the survival of many
species including those having anti-diabetic potential. These difficult to conserve
species need biotechnological techniques for their long-term conservation and
sustainable utilization. For non-orthodox and vegetatively propagated species,
cryopreservation in liquid nitrogen (LN) at 196 C offers the most successful
and economical technique for long-term conservation. Traditional techniques
based on freeze-induced dehydration and recent techniques based on vitrification
have been efficiently used for cryopreservation of all types of the explants.
Cryopreservation offers multiple advantages over other conservation strategies
as it minimizes the risk of contamination, cost of maintenance and cost of labour.
The process of cryopreservation exposes the cell or tissue to various physical,
chemical and physiological stresses which may result in cryoinjury or genetic
level changes sometimes. The analysis of such morphological, structural, genetic
or functional changes is important to assess the genetic integrity of cryopreserved
germplasm to see if they are ‘true to type’ after cryopreservation. This can be
studied at the phenotypic, histological, cytological, biochemical and molecular
levels. The chapter will throw light upon the importance and status of cryopreservation, different methods of cryopreservation adopted in major anti-diabetic
plants along with the pre and post cryotreatments and regeneration protocols,
M. R. Rohini · P. E. Rajasekharan (*)
Indian Institute of Horticultural Research, Hessaraghatta Lake PO, Bengaluru, Karnataka, India
M. E. M. Montero
Centro de Bioplantas Universidad de Ciego de Ávila Máximo Gómez Báez, Ciego de Ávila, Cuba
# The Author(s), under exclusive license to Springer Nature Singapore Pte
Ltd. 2021
S. Gantait et al. (eds.), Biotechnology of Anti-diabetic Medicinal Plants,
https://doi.org/10.1007/978-981-16-3529-8_15
437
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M. R. Rohini et al.
infrastructure requirements for cryobank. The concept of root cryobanking and
cryobionomics dealing with genetic stability and the reintroduction of
cryopreserved plants into the environment is also covered.
Keywords
Cryopreservation · Cryobank · Cryobionomics · Liquid nitrogen · Vitrification
15.1
Introduction
Efficient conservation of genetic resources is fundamental for achieving food and
nutritional security globally. Plant genetic resources are the reservoir of genes
essential for crop improvement programmes so as to develop varieties adapted to
socio-economic requirements and changed environments. Safeguarding these
genetic resources is an enormous task and will require prudent conservation strategy.
The traditional approaches of ex situ conservation through seed and field gene banks
have made remarkable contribution in the conservation of plant germplasm. But in
the case of vegetatively propagated plants and recalcitrant seed species, conservation
efforts has been hampered due to problems faced during the application of their
conventional method of ex situ conservation. Thus, efforts have been made to
develop biotechnological approaches for conservation. Biotechnological methods
of conservation include medium term conservation using in vitro slow growth
cultures and long-term conservation using cryopreservation. The complementary
application of the new and conventional approaches has increased the efficiency of
conservation system. The appropriate conservation method to be adopted depends
on the breeding nature of the species and their seed storage behaviour. For annuals,
which are propagated by seeds and have orthodox storage behaviour, seed gene bank
conservation is the most convenient and reliable method. For recalcitrant seeded
species, especially perennial tree species and vegetatively propagated species which
are considered as ‘difficult to conserve species’ cryopreservation is a promising
technique for long-term conservation.
15.2
Cryopreservation
Cryopreservation involves the conservation of biological tissue at ultra-low temperature by immersing in liquid nitrogen (LN) at 196 C. Due to storage at such low
temperatures, cell divisions and metabolic activities are arrested and thus, plant
material can be stored for unlimited periods of time. Cryopreservation employs the
use of tissue culture techniques in conservation of germplasm. Different types of
explants can be used for cryopreservation like seeds, zygotic embryos, embryonic
axes, pollen, dormant buds, shoot apices etc. Cryopreservation is the most reliable
method of choice for ensuring the long-term storage of non-orthodox seeds, clonally
propagated species and biotechnologically important plant cell lines. Many studies
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439
have confirmed that it is economically more viable than other conservation methods
as the cost of maintaining an accession in LN for the long term (over 20 years) is
substantially lower than that of in the field or in vitro, particularly when dealing with
a large number of accessions. Cryopreservation is advantageous as it requires only
minimum space and also eliminates the need for frequent subculturing because of
which chance of somaclonal variation is reduced. In terms of simplicity and the
applicability to a wide range of genotypes, cryopreservation is always reported to be
a reliable, safe and cost-effective method over most other conservation methods used
for long-term conservation of germplasm.
15.2.1 Principles of Plant Tissue Cryopreservation
The most critical factor in cryopreservation is the removal of intracellular water thus
preventing the formation of ice crystals during freezing. According to Crowe et al.
(1988), 95% of the water present in biological tissues is free and will convert to ice
during freezing causing irreparable damage. There are chances that the cell may get
injured during both the processes, i.e., dehydration (removal of water) and intracellular freezing. Too much removal of water results in cell injury, possibly as a result
of membrane stress and too little dehydration results in intracellular freezing. Thus,
the different techniques of cryopreservation are aimed at removing the intra cellular
water in such a way so as to prevent the dehydration injury and freezing injury. The
fundamental process occurring during cryopreservation, keeping in view of which
cryopreservation protocols should be optimized for better viability and survival of
tissues has been schematically presented in Fig. 15.1.
Tolerance to
temperature
Removal of water to
avoid crystallization
Excess
dehydration
Less
dehydration
Physical damage
to cell contents
Intracellular
freezing
Cell injury
Slow freezing
Vapour pressure
equilibrium
Cell survival
Fast freezing
Excessive
dehydration
Cell injury
Fig. 15.1 Schematic diagram showing principle of cryopreservation
No
crystallization
Cell survival
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M. R. Rohini et al.
Before implementation of any conservation method, the preliminary step is to
determine the seed storage behaviour of the species. Seed storage behaviour is
analysed by studying seed morphological and seed physiological features especially
desiccation and freezing sensitivity along with seed longevity. Roberts (1973)
classified seeds into two broad physiological categories; (1) Orthodox and
(2) Non-orthodox (recalcitrant and intermediate) based on storage characteristics.
Seed gene banks are generally used for the conservation of orthodox seeds and field/
in vitro or cryogene banks are used for the conservation of non-orthodox seed
species.
Orthodox Seeds Seeds that can tolerate low moisture and low temperature storage
without losing viability. Such seeds can be desiccated to low moisture contents, i.e.,
below 5%, and can be stored at sub-zero temperatures. Viability is prolonged in a
predictable manner by such reduction in moisture and storage temperature.
Recalcitrant Seeds Seeds that are desiccation sensitive, short lived and are generally intolerant to low temperatures. Seeds of recalcitrant species maintain high
moisture content at maturity (often more than 30–60% and are sensitive to desiccation below 12–30%, depending on species. They rapidly lose viability under any
kind of storage conditions.
Intermediate Seed Seeds that can tolerate desiccation to some extent, i.e., up to
12–15% moisture content but are sensitive to low temperature.
Cryopreservation is achieved either through classical technique which is based on
freeze-induced cell dehydration and vitrification-based method. The techniques
employed and the physical mechanisms upon which they are based are different in
classical and new cryopreservation techniques (Withers and Engelmann 1998).
Classical cryopreservation-based techniques involve slow cooling down to a defined
pre-freezing temperature followed by rapid immersion in LN. They are generally
operationally complex since they require the use of sophisticated and expensive
programmable freezers. In some cases, their use can be avoided by performing the
freezing step with a domestic or laboratory freezer (Kartha and Engelmann 1994). In
the new vitrification-based procedures, cell dehydration is performed prior to freezing by exposure of samples to concentrated cryoprotective media and/or air desiccation. Vitrification can be defined as the transition of water directly from the liquid
phase into an amorphous phase or glass, while avoiding the formation of ice crystal
(Fahy et al. 1984). This is followed by rapid freezing. As a result, all factors which
affect intracellular ice formation are avoided. Vitrification-based procedures are
more appropriate for complex organs like shoot tips and embryos which contain a
variety of cell types.
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15.2.2 Classical Techniques
In this method, the tissues are treated with cryoprotectants followed by gradual
cooling at a controlled rate (0.5–2 C/min), usually with a programmable freezing
apparatus, down to about 40 C. After holding at this temperature for a short time
(30 min), tissues are immersed in LN. The most commonly used cryoprotectants
such as Dimethyl Sulphoxide (DMSO), glycerol, Polyethylene glycol (PEG), sorbitol etc. are added singly or in combination. To prevent excessive super cooling, the
ice inoculation step (seeding) is performed when temperature as low as 7 to 10 C
is reached. Seeding is performed either automatically if the programmable freezer
possesses such an automatic seeding facility or by touching the outside wall of the
cryovials with the forceps pre-cooled in LN. This method is applicable to a range of
in vitro plant culture system but is most successful with culture systems that consist
of small units of uniform morphology such as protoplast cultures, exponentially
growing cell suspension cultures or fragmented callus cultures.
15.2.3 Vitrification-Based Techniques
In this method, cell desiccation is performed either by exposure of samples to
concentrated cryoprotective solutions or by air desiccation. Cryoprotectants include
dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, polyethylene glycol, sugars
and sugar alcohols used either alone or in combination to protect living cells against
damage during freezing and thawing. They act by lowering the freezing point and
also alter the crystalline structure of ice. They also prevent solute accumulation at a
given level of dehydration so that cell injury is prevented. Careful selection of
cryoprotectants is essential since most of the cryoprotectants exhibit varying degree
of cytotoxicity especially at higher concentration and temperature. This is followed
by rapid freezing through direct immersion of the samples in LN. Vitrification-based
procedures are simpler than classical technique. These methods are suitable to for
cryopreservation of organized structures such as shoot tips, axillary buds etc.
A range of protocols has been widely applied to cryopreserve plant species using
diverse cell and tissue types: cell cultures and suspensions, calluses, apices, somatic
and zygotic embryos, pollen and dormant buds. Some approaches are based on a
singular technique, whereas others use a combination of them. The majority of the
cryopreservation methods involves multi-stage steps. Their common essential steps
include preculture, cryoprotective treatment (under air or in a cryoprotectant solution), freezing, rewarming, unloading (removing of cryoprotectant solutions) and for
one to several days.
15.2.4 Preculture
Preculture of explants on medium with low concentration of cryoprotectants is
recommended before the freezing process. Process of preculture allows explants to
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recover effectively from the dehydration stress. Preculture on medium with
cryoprotectants (often sucrose or sorbitol) also induces freezing tolerance through
sugar uptake and dehydration of explants (Sakai 2000).
15.2.5 Cryoprotective Treatment
In this process, explants are subjected to dehydration either by air desiccation or by
exposing to cryoprotectants. The nature as well as concentration of cryoprotectants,
time of treatment and temperature conditions needs to be optimized for specific
species. Numerous cryoprotective chemicals are used with plant tissues, commonly
used ones are sucrose, DMSO, ethylene glycol, PEG and glycerol. The concentration
of cryoprotectant solution depends on the cryopreservation technique used. It ranges
from 0.5 to 1 M in classical freezing protocols to up to 7 M with vitrification
protocols. The treatment duration also varies with the technique used, from
10–40 min in the case of vitrification (Sakai 2000) to around 1 h in classical freezing
protocols (Kartha and Engelmann 1994). In any case, the process of dehydration is
essential for successful cryopreservation to avoid intracellular freezing and irreversible injury of cells caused by the formation of ice crystals.
15.2.6 Freezing
Freezing rate and pre-freezing temperature are the two parameters which need to be
optimized in the freezing step. In vitrification-based methods, freezing is rapid by
direct immersion of explant in LN. For zygotic embryos or embryonic axes of
recalcitrant species, increasing the freezing rate will help to successfully cryopreserve samples with high water content. After freezing, explants should be stored at
sufficiently low temperature (vapours of LN at 150 C or immersed in LN at
196 C).
15.2.7 Rewarming
Rewarming of cryopreserved samples should be done as fast as possible to avoid the
possibility of damaging ice recrystallization which can cause irreversible damage to
the tissues. It is usually done by rapid immersion of cryotubes in water bath at
37–40 C. In air desiccation technique, rewarming of the samples may be done at
room temperature also.
15.2.8 Recovery
After rewarming, samples should be placed under optimal conditions for rapid and
direct growth. Samples are usually placed in the dark or under reduced light for
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several days to reduce detrimental photo-oxidation phenomenon. The culture
conditions also need to be optimized for each species. Explants are transferred
back to standard culture conditions after a few days or weeks.
15.2.9 Air Desiccation
Cryopreservation using desiccation involves dehydrating the plant material down to
a suitable water content (critical moisture content) followed by rapid freezing
through direct immersion in LN. In desiccation, the physiological maturity of
seeds is an important factor for the successful cryopreservation of embryo/embryonic axes extracted from seeds. Dehydration of the plant material is done either by
exposing them to sterile air flow in a laminar flow chamber or over activated silica
gel for different duration of time until the critical moisture content is obtained. Air
desiccation or dehydration is considered as the simplest technique because it does
not require any cryoprotectant chemicals or expensive equipment’s like programmable freezer. Plant material is usually recovered by the rewarming of samples under
ambient conditions. In samples with very high water content, ultra-rapid drying is
adopted which involves flash drying by exposing cells to a stream of compressed dry
air (Berjak et al. 1989; Wesley-Smith et al. 1992). As of now, this is the most
followed method of cryopreservation practiced for routine storage of germplasm in
gene banks handling a greater number of germplasms per day.
15.2.10 Encapsulation/Dehydration
In this method, alginate beads are used to encapsulate the plant material and then
partially desiccated in laminar flow or over silica gel followed by direct plunging in
LN. The process involves preculture of the explant material in sucrose overnight.
This is followed by encapsulation of the explant in calcium alginate beads, then
partially desiccated by pre-culturing in liquid MS medium with different sucrose
concentrations (0.3, 0.5 and 0.75 M) at 100 rpm for 20 or 40 h followed by
transferring the beads to cryovials and plunging the cryovials in LN. Rewarming
is done by exposing the cryopreserved samples at room temperature for 15 min to
half an hour and then cultured on the standard media. The technique has been
successfully applied to conserve the embryonic axes of Poncirus trifoliata, neem,
Citrus sinensis etc.
15.2.11 Vitrification
This process involves the treatment of tissues with cryoprotectants in vitrification
solution followed by fast freezing. In vitrification, initially the explant material is
precultured on basal media supplemented with 0.3 M sucrose and 2 M glycerol
overnight. Next day, the material is transferred to cryovials containing loading
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M. R. Rohini et al.
Fig. 15.2 Schematic diagram showing the process of desiccation, encapsulation and vitrification
for embryonic axes
solution which is a combination of 0.4 M sucrose and 2 M glycerol. After 25 min, the
loading solution is replaced by plant vitrification solution (PVS2: 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% (w/v) dimethyl sulphoxide) with 0.3 M
sucrose for different duration of time followed by rapid immersion in LN. Rapid
thawing is done by immersing frozen cryovials in water bath (38 1 C) for 5 min.
After thawing, PVS2 solution is drained off and unloading solution is added (1.2 M
sucrose) for 20 min to remove the effect of PVS2. This is followed by culturing of
the explant material.
The above three process, i.e., desiccation, encapsulation-dehydration and
vitrification in the case of cryopreservation of embryonic axes of Citrus species
have been illustrated in Fig. 15.2.
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Cryopreservation of Anti-Diabetic Plants
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15.2.12 Droplet Freezing
The technique combines the droplet-freezing method with the vitrification procedure
(Sakai et al. 1990). This method consists of pretreatment of the tissue in liquid
medium supplemented with cryoprotectants followed by transferring them into
drops of cryoprotective medium placed on aluminium foil followed by rapid freezing
in LN. It is of two types:
15.2.12.1 DMSO Droplet Freezing
This method was initially developed for the cryopreservation of shoot tips of cassava
by Kartha et al. (1982) and was later applied to conserve potato shoot tips (SchäferMenuhr et al. 1996). The method involves the treatment of the explant material with
the cryoprotectant DMSO for 1–3 h and then transferring the explants to aluminium
foils containing a drop of DMSO solution (2.5 μL). The foil is then placed in the
cryovial and rapidly immersed in LN. This method also needs rapid thawing of
samples by exposing the cryovials in water bath at 38 1 C for 5 min followed by
culturing of the sample.
15.2.12.2 PVS2 Droplet Freezing
This is similar to the above method except that in place of DMSO, PVS2 solution is
used for cryoprotection. Rapid LN plunging is followed by rapid thawing and
culturing.
Droplet methods have the disadvantage of ultra-rapid freezing of the tissues
because aluminium foil permits heat transmission faster than normally used
cryovials and also because of the minute volume of cryoprotectant (Benson et al.
2013).
15.2.13 Encapsulation Vitrification
As the name suggests, this method combines two techniques of cryopreservation,
i.e., encapsulation/dehydration and vitrification. In this, first the explant material is
encapsulated in alginate beads and then exposed to cryoprotectant treatment by
PVS2 solution followed by immersion in LN. Rapid or slow thawing is done
followed by removing of vitrification solution and culturing of the beads
(Martinez-Montero et al. 2012).
15.2.14 Cryoplate Methods
This is the recently developed method of cryopreservation which makes use of the
cryoplates. Cryoplates are small aluminium plates with embedded wells for holding
the explant material. Cryoplates explant inoculation in the plates and its immersion
in LN is illustrated in the Fig. 15.3.
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M. R. Rohini et al.
Fig. 15.3 Diagram showing
the cryoplate method
In V-cryoplate method, explant material is precultured in 0.3 M sucrose and
transferred to cryoplates after encapsulation in alginate beads and then treated with
loading solution (2 M glycerol +0.6–1 M sucrose) for 30 min. Dehydration is
performed by treating with PVS2 solution followed by rapid freezing. Frozen
cryoplates are rapidly thawed by transferring to 1 M sucrose (unloading solution)
for 15 min followed by culturing of the same. In D-cryoplate method, the steps
followed are similar to the ones in V-cryoplate method except that after treating with
loading solution, instead of PVS 2 solution explants are dehydrated in laminar flow
cabinet for suitable period. Cryoplate methods are advantageous because of their
user friendliness and ease of handling samples on the aluminium plates. Successful
cryopreservation using cryoplates methods has been reported for many plant species
including strawberry, sugarcane, date palm, mat rush and potato.
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15.3
447
Root Cryobanking
Root cryobanking is emerging as a potential method of cryopreservation especially
for medicinal plants. Roots are uniquely suited for long-term conservation of plant
genetic resources due to their ease of excision and in vitro maintenance, and the
ability to produce new lateral roots and/or shoots. Cryopreservation and regeneration
of root tips in potato cultivars was first attempted by Bajaj (1978), but thereafter only
few studies are available on root cryopreservation when compared with cryopreservation of other plant organs and tissues (Normah et al. 2019; Agrawal et al. 2019).
But, with the boom in the in vitro micropropagation of medicinal plants for secondary metabolite production, successful cryopreservation of root explants which are
isolated from in vitro cultures as well as adventitious and hairy roots has been
successfully attempted. Cryopreservation of adventitious and hairy roots offers
scientific as well as commercial advantage because they are considered as the
alternative sources for many important secondary metabolites thus having potential
application in pharmaceutical, cosmetic and natural health product industries
(Popova et al. 2020). The cryopreservation process does not affect the biosynthetic
or genetic stability of the roots keeping their potential same as that of field grown
roots. Root cryopreservation was successfully achieved using encapsulation as well
as vitrification methods. Encapsulation of root tips protects them from mechanical
damage and vitrification method provides fast cooling and warming.
15.4
Pollen Cryopreservation
A pollen cryobank with diverse pollen collections provide long-term security for
wild flora, ornamentals, fruit and vegetable crops, medicinal herbs and endangered
species. Recently for so many anti-diabetic species pollen has been successfully
cryopreserved, for example, Momordica doica Momordica sahyadrica. The technique of pollen cryopreservation has the potential to overcome different challenges
facing in crop breeding programs, such as flowering asynchrony between different
parent genotypes, and the production of insufficient pollen in the case of many wild
species. The technique of pollen cryopreservation is simple enough to be used
routinely in research, plant breeding and in complementary m conservation
strategies of plant genetic resources (Dinato et al. 2020). A pollen cryobank become
an important component of national gene banks can provide a constant supply of
viable and fertile pollen to allow supplementary pollinations for breeding
programmes. Pollen banks, where large quantities of pollen are cryopreserved in a
relatively small area, are available to breeders for use in crop improvement programs
and to supplement plant genetic resources (Rajasekharana and Ganeshan 2019). The
technology for the establishment of pollen cryobank has been optimized at the
in vitro Conservation and Cryopreservation Laboratory of Division of Plant Genetic
Resources, ICAR-IIHR, Bangalore. Cryopreservation is now an accessible
conservation option for a wide range of users and it has the potential to support
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M. R. Rohini et al.
both small- and large-scale laboratories involved various research programmes and
centres involved in conservation of plant genetic resources.
15.5
Current Status of Cryopreservation
Cryopreservation is now considered as the safest, low-cost and efficient long-term
conservation strategy applied to seeds of many orthodox and recalcitrant species,
dormant buds and embryos of many tree species and pollen of several difficult to
conserve species. Cryopreservation protocols are now available for cell and tissue
types of around 200 species including both differentiated as well as undifferentiated
explants (Dulloo et al. 2010). Many economically important crops including root
and tuber crops, fruit crops, ornamentals and plantation crops of temperate and
tropical origin are successfully cryopreserved using various techniques (Engelmann
2000; Kaczmarczyk et al. 2012). Cryogene banks are now an integral part of many of
the institutes Consultative Group on International Agricultural Research (CGIAR)
like International Potato Centre (CIP) at Peru, International Transit Centre for Musa,
International Institute for Tropical Agriculture, International Centre for Tropical
Agriculture. Table 15.1 shows the germplasm holdings maintained at National
Plant Germplasm System of USDA-ARS. In India, major thrust on crop
Table 15.1 Status of germplasm in USDA-ARS, National Plant Germplasm System (NPGS), as
of 2017
Primary NPGS repositories
National Arid Land Plant Genetic Resources, Parlier,
CA
National Clonal Germplasm Repository, Corvallis, OR
National Clonal Repository for Citrus and Date,
Riverside, CA
National Clonal Germplasm Repository for Tree Fruit/
Nut Crops and Grapes, Davis, CA
National Germplasm Repository, Brownwood, TX
North Central Regional Plant Introduction Station,
Ames, IA
Ornamental Plant Germplasm Centre, Columbus, OH
Plant Genetic Resources Conservation, Griffin, GA
Plant Genetic Resources, Geneva, NY
Subtropical Horticulture Research Station, Miami, FL
Tropical Agriculture Research Station, Mayaguez, PR
Tropical Plant Genetic Resources Management, Hilo,
HI
United States Potato Gene Bank, Sturgeon, Bay, WI
Western Regional Plant Introduction, Pullman, WA
Woody Landscape Plant Germplasm, Washington, DC
Number
of genera
13
Number of
species
70
Total
accessions
603
64
9
664
10
8754
1789
21
248
7257
1
152
12
622
2328
2456
54
45
7
327
285
19
300
196
103
802
483
58
1013
1823
4410
2790
1152
709
1
24
172
92
64
701
836
542
1493
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Cryopreservation of Anti-Diabetic Plants
449
Table 15.2 Status of germplasm in cryogene bank of NBPGR (as on March 31, 2019)
Categories
Recalcitrant and intermediate
Fruits and nuts
Spices and condiments
Plantation crops
Agroforestry, industrial crops, medicinal and aromatic plants
Orthodox
Dormant buds
Pollen grains
Genomic resources
Total
Total number of accessions
6782
3520
152
88
3022
3902
387
572
1934
13,577
cryopreservation activities is carried out at NBPGR, New Delhi maintaining approximately 14,000 crop accessions in different forms (Table 15.2). Cryopreservation
protocols have been in anti-diabetic plant species like Allium, Bacopa, Dioscorea,
Morus, Musa, Picrorhiza etc. Major success has been achieved in two genera,
namely, Allium and Musa. The NBPGR cryogene bank (in vapour phase of LN)
has 13,363 accessions of diverse crops conserved in the form of seeds, embryos,
embryonic axes, pollen, budwood and DNA. Apart from NBPGR, Central Potato
Research Institute at Shimla holds pollen collection of potato germplasm (6 nos.) and
Indian Institute of Horticultural Research, Bangalore holds pollen collection of many
horticulture crops including medicinal plants (675 nos.).
15.6
Relevance of Cryopreservation in Medicinal Plants
with Special Emphasis on Anti-Diabetic Plants
Plants have been the only source of life saving drugs available to mankind since
ancient time until the modern system of medicine emerged. In developing countries
like India, plant-based medicines still hold a very significant place in the system of
medicine and now with the growing realization of the health benefitting properties of
medicinal plants there has been an increased demand for plant-based products over
the synthetic drugs. Plant-based drugs being safe with less side effects have exponentially increased the global market for medicinal plants. Diabetes mellitus (DM) is
one of the most rapidly increasing lifestyle disorder affecting approximately 2.8% of
the global population, is expected to increase to 5.4% by 2025, and this number will
be ca. 440 million people worldwide in 2040. Large number of plants are reported to
have anti-diabetic or hypoglycaemic property and are used in ayurvedic and other
plant-based systems for the treatment of diabetes. The plants with anti-diabetic
property can be broadly classified as one which are purely medicinal possessing
anti-diabetic property like Gymnema sylvestre, Aloe vera, stevia, Dioscorea
bulbifera, neem etc. and the other category plants belong to fruit, vegetable or spices
group possessing anti-diabetic property like bitter gourd, jamun, fenugreek, garlic,
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Citrus sinensis etc. In this chapter, we will be covering the cryopreservation status of
both these groups. More than 35% of compounds for diabetic treatments are
extracted from leaves of these anti-diabetic species. However, the fruits of many
plants with anti-diabetic potential can be consumed orally in the form of juices.
These plants contain various phytoconstituents such as flavonoids, terpenoids,
saponins, carotenoids, alkaloids and glycosides, which may possess anti-diabetic
activities. The most serious concern with majority of medicinal plants is that they are
accessed from their natural habitats such as forest or natural ecosystems. Unlike very
few commercially cultivated anti-diabetic medicinal species like Aloe vera, all others
are sourced from wild thereby threatening their survival in the natural habitats.
Unscrupulous collection from wild, destruction of natural habitats, climate change
scenarios has transformed the status of many native plants into rare, endangered and
threatened category. About 15,000 plant species are threatened with extinction by
the overharvesting of plant resources and their natural habitat destruction. In view of
this rapidly eroding native flora, conservation efforts have gained momentum
nationally and internationally to conserve them. For the conservation of wild genetic
resources like medicinal plants, in situ conservation through biosphere reserves are
advocated and adapted depending upon the need and situation. In situ conservation
efforts aims at the conservation of genetic resources in its natural habitats by
preventing encroachment activities, but of loss of plant populations due to factors
like biotic or abiotic stresses, climate change is inevitable in the in situ areas. Thus,
the in situ conservation strategies should be complemented with ex situ conservation
efforts for long-term conservation of the targeted species. Majority of the medicinal
plants are categorized under wild, rare, endangered and threatened group for which
cryopreservation is considered as the most viable long-term conservation strategy.
Moreover, seed banking cannot be applied to species that produce few or no seeds,
or for which the seeds are inaccessible for collecting which is the case with most of
the medicinal species.
Recently, Walters and Pence (2020) reviewed that lifespan of many samples is
reduced after storage. Triacylglycerols present in seed changes owning to different
storage conditions and thereby reducing their shelf life. This is the case with many of
the anti-diabetic plants (e.g. Juglans regia, Carya, Castaena and Corylus) and
tropical species (e.g. Cuphea, Elais and Hevea), as well as seeds from numerous
Hawaiian species. These seeds with high triacylglycerols become viscous and
crystallizes at low temperatures and therefore they require alternative conservation
approaches.
15.7
Infrastructure Requirements for Plant Cryopreservation
Cryopreservation techniques are now being used increasingly for the long-term
conservation of germplasm worldwide. Before adopting cryopreservation strategy
for any system, it is essential to examine the effects of different parameters on the
germplasm conserved. Cryopreservation protocols are already available for more
than 500 species of plant tissues, at the same time it is essential to develop and
15
Cryopreservation of Anti-Diabetic Plants
451
optimize standard protocols for specific plant species and or tissue types for effective
conservation. Cryopreservation is labour and resource intensive procedure; therefore, prioritization of plant species for conservation is important. Secure storage
location and complete inventory of the germplasm stored is vital for the successful
recovery of conserved germplasm whenever needed.
Basic facilities required for initializing the experimental procedures in small scale
laboratories are:
• Tissue culture facility.
– Washing room: For washing and storage of glassware, plasticware and other
labware.
– Media preparation room: For preparation and storage of nutrient media.
– Autoclave room: For sterilization of media, water and instruments.
– Inoculation room: For aseptic manipulation of plant material.
– Culture room: For maintenance of cultures under controlled conditions of
temperature, light and humidity.
• Storage facility
LN tanks (small capacity 10–30 L). If the number of accessions is more, wide
mouth LN tanks of 1000 L capacity can also be used.
• Green house facility
Green house facility is essential for the initial establishment of dormant plant
propagules. It is also essential for acclimatization of in vitro grown plantlets as a
part of viability testing process.
15.7.1 Laboratory Requirements
Setting up of a long-term cryopreservation facility is a one-time investment and
involves the following laboratory equipment.
• Large capacity cryotanks ranging from 60–1000 L capacity with electronically
controlled filling system along with alarm.
• Transportation containers without canisters used for transporting LN.
• Diverse inventory system specific to the cryotanks.
• Pressurized LN vessel.
• Programmable freezer.
• Small biological containers with canisters.
• High Precision electronic balance.
• Controlled temperature water bath.
• Fan assisted hot air oven.
• Cryovials and cryomarkers.
• Safety gadgets—gloves, apron, face shield or goggles.
• Heavy duty trolleys for transportation of tanks.
452
M. R. Rohini et al.
15.7.2 Storage Containers
• All types of explants are to be finally packed in airtight containers before
cryostorage.
• Glass containers can shatter when warmed from sub-zero temperatures.
• Polypropylene screw cap cryovials of different sizes (1 mL, 2 mL, 5 mL, 50 mL)
depending upon the size as well as the quantity of the seeds/embryonic axes/
dormant buds/pollen grains are used.
• Pollen desiccated to suitable moisture content can be stored in aluminium packets
or gelatin capsules.
• Large seeds and twigs are to be stored in heat-sealable polyolefin tubing with cork
stoppers or in goblets in sleeves.
15.8
Maintenance of Cryotanks
Cryotanks are self-contained, vacuum-insulated double walled stainless steel
vessels. They essentially have a LN reservoir at the base and samples are suspended
on a stationary or rotating tray which partitions the tank. It is important to choose a
suitable storage system, i.e., material containers, holding supports etc. Careful
planning needs to be done taking into account:
(a)
(b)
(c)
(d)
Explant size.
Number of explants to be stored.
Accessibility.
Vapour or liquid phase storage.
If explants are of different sizes, then cryovials of different sizes (1–10 mL)
should be used. Small seeds, embryos, embryonic axes, shoot apices and meristems
can be easily stored in large numbers in cryovials of 5 mL and 10 mL capacity. Each
tank has a static holding time which is the maximum time for which a tank can hold a
particular quantity of LN, after which more LN have to be replenished. This is
dependent on the rate of evaporation and volume of the cryotank. Evaporation rate of
LN for most tanks is 0.5–1.5% of its capacity per day and accordingly, replenishment of LN would be requires about 2 times per week. Equipment which are used
routinely for cryopreservation of plant material is depicted in the Fig. 15.4.
15.9
Management of Cryopreserved Collections: Practical
Issues
Adoption of cryopreservation techniques for conservation depends upon technical
feasibility, conservation procedures and relation between cost and benefits (Kartha
and Engelmann 1994). These all are in turn influenced by the objectives of conservation, needs and types of materials to be conserved and available resources.
15
Cryopreservation of Anti-Diabetic Plants
453
Fig. 15.4 Equipment used for cryopreservation
15.10 Prioritizing the Materials for Cryopreservation
Cryopreservation is often considered as a secondary storage strategy which is
complementary to the gene bank, field gene bank and in situ conservation. In such
a case, it is important to prioritize the materials, i.e., to be conserved, as it will
depend on the available infrastructures and national needs of the country and
institutions. The categories of anti-diabetic medicinal plant species or any plant
species for that matter which needs cryopreservation includes:
(a) Species producing intermediate and recalcitrant seeds: For example, citrus
species, neem and Jamun.
454
M. R. Rohini et al.
(b) Threatened and endangered plant species with critically small population size:
For example, Rauwolfia serpentina, Costus speciosus, Gymnema sylvestre etc.
(c) Wild species and wild relatives of crop plants: Almost all the anti-diabetic
medicinal plants will come in this category.
(d) Registered germplasm: Germplasm holding any unique value in terms of morphological or economic traits.
(e) Released varieties and genetic stock.
For embryogenic cultures that may lose their capacity for embryo formation with
time, cryopreservation provides primary storage, with cultures revived and used to
produce more embryos at a later date.
15.11 Selection of Explants for Cryopreservation
Explants for cryopreservation may be either seeds, pollen, shoot tips, dormant buds,
embryos, embryonic axes, callus or cell cultures depending on the species. Seeds are
mostly used for seed propagated orthodox species, pollen is used when the plant
breeder wants to conserve and use the part of genetic diversity in easily accessible
form and also for recalcitrant seed species. Dormant buds are used mostly in the case
of temperate budded or grafted trees. Shoot tips can be used for any type of
temperate or tropical plants.
Whatever be the explant material to be used, it has to be freshly obtained and
processed fast to retain the viability. Seeds collected should be used within few hours
to few days, similarly shoot tips, embryos or embryonic axes should be freshly
extracted, in the case of pollen, it should be collected from flowers with freshly
dehisced anthers. For collection of buds 60 cm long twigs of last 1 year’s growth
having dormant buds are to be harvested from plants growing at the field gene bank
in winter seasons. Table 15.3 shows different types of explants which have been
used for the cryopreservation in some of the anti-diabetic species.
15.12 Size of Germplasm to be Cryopreserved
The optimum sample size for cryopreservation depends on many factors like the
reproductive biology and pollination behaviour, seed storage behaviour, availability
of the plant species, its recovery potential, size of the explant used etc. For selfpollinating species, minimum of 2000 seeds may be stored and for cross-pollinating
species, a minimum of 4000 seeds needs to be stored. For embryos, embryonic axes,
shoot tips, meristems and pollen, there is no standard recommendation for minimum
number of explants to be stored. It depends on the availability of material and
percentage of survival. Storage should ensure the availability of enough propagules
to regenerate sufficient plants whenever required and also additional vials can be
used for periodic viability testing. The type of explant used also determines the size
because seeds and dormant buds need more space as compared to embryos and cell
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Cryopreservation of Anti-Diabetic Plants
455
Table 15.3 Different types of explants used for cryopreservation of anti-diabetic plants
Sl
no
1
2
3
4
5
6
Species
Citrus
sinensis
Citrus
sinensis
Dioscorea
bulbifera
Dioscorea sp.
Allium
sativum
Morus alba
Explant
Nuclear cells
Method
Classical slow freezing
Embryonic axes
Air desiccation, vitrification,
encapsulation-dehydration
Encapsulation-dehydration
Somatic
embryos
Shoot tips
Vitrification
Shoot tips
Vitrification
Winter hardy
buds
In vitro grown
nodal segments
Desiccation
7
Rauwolfia
serpentina
Vitrification
8
Azadirachta
indica (neem)
Embryonic axes
Vitrification
9
Musa
acuminata
Zygotic embryo
Desiccation freezing
Reference
Kobayashi et al.
(1990)
Malik et al.
(2012)
Mandal et al.
(1999)
Mandal and Dixit
(2000)
Makowska et al.
(1999)
Nino et al. (1992)
Ray and
Bhattacharya
(2008)
Malik and
Chaudhury
(2019)
AbdelnourEsquivel et al.
(1992)
cultures. Recovery potential of a stored accession will also determine its storage size,
as species with high recovery can be stored less in number in fewer vials and difficult
species has to be conserved in more numbers in more vials. A formula proposed by
Dussert et al. (2003) can be used for determining the number of propagules needed
for long-term recovery of plant materials in culture based on binomial distribution
using the number of controls tested, the recovery of those control plants, the number
stored and the probability of recovering one growing sample. The number of
propagules stored may vary from fewer than 100 for dormant buds to many
thousands for pollen grains.
15.13 Optimization of Protocol
While developing cryopreservation protocols for species with orthodox storage
behaviour, the strategy would be to examine the response of seeds of different
genera, species and accessions to initial LN exposure. Once it is established that
survival values tested after 18–24 h of LN storage are fairly uniform, routine
application can be taken up. For orthodox seeds, simple desiccation of seeds to
5–7% moisture content followed by placing them in cryotanks in vapour phase of
LN will ensure their indefinite storage. For recalcitrant, intermediate and vegetatively propagated species, either new cryopreservation protocols should be
456
M. R. Rohini et al.
developed or already existing protocols should be optimized for higher viability and
survival. It is possible to do preliminary tests with 3–5 genotypes and, if successful,
apply the technique to the remaining collection. Accessions with low recovery will
require improvement in the plant materials or modifications of the technique to
improve recovery following LN exposure (Reed 2008).
15.14 Germination/Viability Standards
In a cryobank, the accessions are checked for ensuring high germination and
viability. At least 80% germination should be ensured for the stored samples. This
is particularly important for genotypes where there is a chance to obtain rare genes or
alleles. The germination or viability standards can be set still high if the genes are
reported to be extremely rare in a population and even a slight deterioration of
viability can lead to loss of such potentially valuable genes. Lower germination rates
are acceptable for released varieties because they are genetically more homogenous
than landraces or wild species.
15.15 Recovery Growth of Cryopreserved Germplasm
The success of any cryopreservation process depends on the recovery of viable
propagules (growing shoots, germinating pollen, multiplying cells) after
cryostorage. Development of optimum rewarming protocol and selection of the
appropriate regrowth medium is very crucial in determining the recovery of the
propagules. Recovery of true-to-type plants and actively growing cultures requires
the best regrowth conditions. Regrowth of cryopreserved tissues in in vitro should be
without a callus phase to retain the genetic stability.
15.16 Sample Labelling and Data Storage
Proper and accurate labelling of the cryopreserved accessions is very important and
critical especially when the gene bank holds large collections. Appropriate labelling
should include accession number, name, date of storage and initial viability %.
Bar-coded labels are also now available for cryovials. Clear labelling and numbering
of canes or boxes is important for easy retrieval of specimens. Linking of
cryopreserved samples to its passport information is crucial for generating any
information pertaining to the sample. Complete protocol information for each of
the sample should be stored like the information on pre-growth of the plant material,
pre-treatments, cryopreservation protocol, thawing method and the recovery
medium because these are vital to recovering living plants. A computerized database
with the passport data and storage location is to be compulsorily maintained in the
cryogene bank for easy access and utilization of the cryopreserved germplasm.
Some items needed for the database:
15
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Cryopreservation of Anti-Diabetic Plants
457
Accession identifying number or code.
Accession name.
Cane or rack location.
Number of vials stored.
Storage date.
Standard culture medium (reference).
Preconditioning treatments.
Preculture conditions.
Osmoprotection.
Cryopreservation technique (reference to the technique).
Cryoprotectant used.
Rewarming protocol.
Regrowth medium.
Data from control regrowth.
Care should be taken to store the active collections or test samples which have to
be taken out at regular intervals separately from the base collections.
15.17 Monitoring Genetic Stability of Cryopreserved Germplasm
Plant cryopreservation is a complex, multistage process which requires optimization
of various steps involved right from the selection of quality donor material,
preculture process, cryoprotection technique to be used, thawing, recovery medium,
regrowth etc. Successful cryopreservation is the overall result of all these processes
to achieve a higher recovery along with genetic stability. Even though cryopreservation has proved to be the most amenable way for the long-term conservation of
recalcitrant species, there are an increasing number of difficulties encountered in the
process to achieve better regrowth rate (Normah et al. 2012). The field of
cryobionomics deals with cryoinjury, genetic stability and regrowth studies of
cryopreserved tissues. Cryobionomics is an interdisciplinary subject covering the
morphological, biochemical, histological, cytological as well as molecular study of
an organism to identify any type of cryoinjury or loss of metabolic or reproductive
functions in them because of cryopreservation. It also studies the change in gene
expressions causing disruption of normal regulatory mechanisms, growth and developmental sequences (Harding 2004). Cryobionomics uses high-throughput omics
technologies such as genomics, transcriptomics, proteomics, metabolomics and
other omics platforms to study the potential impacts of cryoinjury on the genome,
transcriptome, proteome and metabolome of a species.
The development of advanced biomolecular or ‘omics’ technologies has enabled
a better understanding of the fundamental biological process underlying cryopreservation system (Morrison et al. 2006). It can readily be applied to the same sample,
enabling a detailed investigation into biomolecular changes that accompany the
cryopreservation of plant materials (Martinez-Montero and Harding 2015). Thus,
cryobionomics provides a conceptual framework to examine the significance of cell
458
M. R. Rohini et al.
signalling mechanisms on cellular functions, the impacts of cryoinjury and
cryogenic/non-cryogenic stress factors on germplasm viability and the implications
for genetic stability following cryostorage.
Cryopreservation exposes the stored material to a variety of stresses because of
which the genetic stability of the conserved material will be hampered. Such stresses
may be due to the exposure of tissues to different chemicals and storage conditions.
It has been studied that in most of the cryopreserved tissues, very little or no change
is seen after short-term cryostorage, i.e., within 1-month duration (Wang et al. 2017).
But many workers like Castillo et al. (2010), Maki et al. (2015) and Pence et al.
(2017) showed that some genetic changes occur after long duration of storage. These
types of changes are found to be epigenetic. Genetic changes occur more frequently
due to DNA methylation rather than change in DNA sequences. DNA methylationinduced changes are reversible also (Johnston et al. 2009; Peredo et al. 2008; Kaity
et al. 2013).
Most common type of variation, i.e., observed during the regrowth in in vitro
condition is somaclonal variation. These kinds of variations lead to the loss of true to
type character of stored germplasm and fail to implement the whole purpose of
cryopreservation. Different types of injuries or changes that an explant material
undergoes during different steps of cryopreservation have been depicted in Fig. 15.5.
Thus, it is important to ensure the genetic stability of the cryopreserved material.
Assessment of genetic integrity of conserved material is done by comparing it to the
same material before conservation (Engelmann 2011). Numerous studies on genetic
stability analysis have been already done in many crops and revealed that no
cytological or genetic alterations occur due to cryopreservation. Different techniques
including biochemical, molecular, cytological and morphological methods can be
used to assess the genetic stability of cryopreserved germplasm. Morphological or
phenotypic method is the traditional method of comparing the morphological traits
of conserved and the original plant to see if there is any variation. Any kind of
chromosomal changes during cryopreservation can be detected by cytological
methods and biochemical methods can be used to detect any changes in metabolite
or enzyme profiles. In medicinal plants, it is important to retain the quantity of
bioactive compounds in the cryopreserved samples as it is present in the original
sample. Biochemistry techniques involving HPLC, GC, LC, MS etc. can be used for
analysing the biochemical profile of medicinal plants. Molecular techniques employ
a range of molecular markers such as RAPDs, AFLPs, RFLPs, SSRs, SNPs etc. to
study the variations occurring at the DNA level. These conventional markers will
analyse only a small part of the genome, but with the advancement of plant genomics
and rapid progress in next generation sequencing (NGS) technology, more and more
plant genomes are getting sequenced. The availability of NGS has speeded up the
analysis of plant genomes and can be used to test the genetic stability of
cryopreserved germplasm effectively. Morphological and biochemical variations
should be supplemented with the latest molecular techniques for confirmatory
results. Overall, genetic changes from cryopreservation appear small and relatively
rare, and the value of holding a sample in a cryostorage facility to guard against
15
Cryopreservation of Anti-Diabetic Plants
459
Fig. 15.5 The multistage process of cryopreservation signifying the possible events that leads to
cellular damage and genetic instability
extinction likely outweighs any risk of small genetic changes. Few of the genetic
stability studies carried out in some anti-diabetic plants are mentioned below:
Hasan et al. (2015) used AFLP technique to study the genetic stability in shoot
tips of Ziziphora tenuior L., before and after cryopreservation and found that there
were no genetic variations between the two. Agrawal et al. (2014) studied the genetic
stability of Musa plants recovered from cryopreserved and regenerated meristems
using 11 phenotypic (biometric) characters and 21 simple sequence repeat (SSR)
markers. Data on phenotypic traits revealed that cryopreserved plants were statistically comparable to the mother plants raised from suckers for all important growth
and yield parameters. SSR profiles of plants recovered from cryopreserved material
and control plants had a higher similarity coefficient concluding that Musa
meristems can be effectively cryopreserved for storage and regeneration of true-totype plants. Dixit et al. (2003) assessed genetic stability of plants regenerated from
cryopreserved embryogenic tissues of Dioscorea bulbifera using molecular, biochemical and morphological analysis. RAPD markers used for molecular analysis
shows similar profile for both cryopreserved and control plants. Diosgenin content
analysed using HPLC also showed same content in cryopreserved and control plants.
460
M. R. Rohini et al.
Similarly, morphology and the ability to form microtuber were also found unaltered
in cryopreserved embryo-derived plantlets. Thus, it was concluded that the
D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels. Ravindran et al.
(2005) and Yamuna et al. (2007) reported genetic uniformity was observed in
cryopreserved and recovered plants of cardamom, ginger and black pepper based
on RAPD and ISSR profiling. Choudhary et al. (2013) studied genetic stability of
Morus alba in in vitro regenerated plants of mulberry (fresh, before and after
cryopreservation) using RAPD and ISSR markers. Dormant buds of Morus alba
were used for cryopreservation using two-step freezing. In this study, the plants
regenerated directly from dormant buds (before and after cryopreservation) without
intermediary callus phase were analysed. Both markers showed monomorphic
banding patterns and did not reveal any polymorphism among the mother plant
and in vitro regenerants before and after cryopreservation, suggesting that cryopreservation, using two-step freezing, does not affect genetic stability of mulberry
germplasm.
15.18 Case Studies in Anti-Diabetic Plants
15.18.1 Syzigium cumini, Garcinia indica, Emblica officinalis
and Aegle marmelos
Malik et al. (2010) conducted seed storage and cryopreservation studies in
underutilized fruit crops having anti-diabetic property like Syzigium cumini,
Garcinia indica, Emblica officinalis and Aegle marmelos. Seed storage behaviour
studies showed that Garcinia indica and Syzigium cumini seeds were of highly
recalcitrant nature, whereas Aegle marmelos showed intermediate and Emblica
officinalis showed orthodox seed storage behaviour. Seeds, embryos as well as
embryonic axes were tested for viability as well as cryopreservation. In vitro culture
of embryos showed better germination than the seeds. This study showed that
A. marmelos and E. officinalis have orthodox seed storage behaviour as they were
viable even after desiccating to 5–7% moisture content whereas Garcinia indica and
Syzygium cumini lost the viability by 10–12% on desiccation showing intermediate
seed storage behaviour. Seeds of A. marmelos suffered from freezing sensitivity
after exposure to LN. Cryopreservation was attempted successfully in all these
species.
15.18.2 Aloe vera
Anti-diabetic activity of Aloe vera has been mentioned by Sharma et al. (2014).
Droplet-vitrification method has been developed by Josette et al. (2019) for
cryopreserving the clonal accession of Aloe vera. Apical shoot tips obtained from
in vitro grown plants was used as the explant. They were treated with loading
15
Cryopreservation of Anti-Diabetic Plants
461
solution for 20 min followed by PVS2 treatment for different duration of time and
then transferred to aluminium foil strips for immersing in LN. Thawing was done
and PVS2 solution was replaced by unloading solution for 20 min followed by
culturing in regeneration media. Preculture in sucrose media prior to freezing also
improved the recovery growth.
15.18.3 Allium sativum
Anti-diabetic activity of Allium sativum has been mentioned by Grover et al. (2002).
Cryopreservation of Allium sativum (garlic) has been standardized using vitrification
and droplet-vitrification method (Singh et al. 2018). Droplet vitrification proved to
be more successful in terms of regeneration. In vitrification method, garlic cloves
were isolated from dormant bulbs and meristem tip was extracted. Extracted meristem tips were surface sterilized and implanted in preculture medium (containing
0.3 M sucrose in MS basal medium) followed by treatment with PVS2 solution for
40 min. Meristems with PVS2 solution was plunged into LN for 1 h. Later, the
cryovials containing PVS2 solution is removed from LN and treated with unloading
solution for 20 min. For regrowth, the explant was removed from the unloading
solution and transferred to regrowth medium.
In droplet-vitrification method, after treatment with preculture media, explants are
transferred to aluminium foil strips containing PVS2 solution, waited for 40 min and
then plunged into the LN for rapid freezing. Remaining procedure was same as that
of vitrification.
15.18.4 Neem
Chaudhury and Chandel (1991) showed that seeds of neem show typical characters
of recalcitrant seeds like short life and high moisture content (about 46%) at the time
of shedding. But desiccating seeds to low moisture content of 4% and storing in LN
showed that the seeds are tolerant to desiccation and freezing showing their orthodox
storage behaviour. And possessing endocarp which played a significant role in
germination and desiccation responses, showed orthodox nature in terms of desiccation and freezing sensitivity. Seeds showed desiccation tolerance up to 4% and
freezing tolerance at 196 C for seeds desiccated between 18.5% and 4% moisture
level. Thus, it was found that cryopreservation at the ultra-low temperatures of LN
(196 C) can be utilized for long-term conservation of neem germplasm.
15.18.5 Stevia
Shatnawi et al. (2011) used vitrification method for in vitro propagation and
cryostorage of shoot tips of Stevia rebaudiana. Shoot tips were used as the explant
for cryopreservation. Several pretreatment and preculture trials were conducted for
462
M. R. Rohini et al.
maximizing the recovery after cryostorage. Maximum recovery of 68% was
obtained by preculturing the plantlets in MS medium with 30 g/L sucrose and
preculture of shoot tips in 0.4 M sorbitol followed by 80–100% PVS2 treatment
and immersion in LN. Acclimatization and regrowth of plants showed that growth
parameters were not affected by the cryopreservation process even after 6 months.
Thus, it was concluded that the protocol is applicable for long-term storage of
S. rebaudiana germplasm in LN.
15.18.6 Musa spp.
Kaya et al. (2020) developed an efficient cryopreservation protocol for the long-term
conservation of economically important Musa species. Cryopreservation of seeds
was done using air desiccation method in which seeds were dehydrated in a sterile
laminar flow cabinet for different exposure times and then they were directly
immersed in LN. The critical point was to support the initial germination of
cryopreserved seeds and this was achieved by the excision of zygotic embryos
after LN treatment that allowed the seed germination. The best moisture content
for tolerance to cryopreservation ranged from 15.8% (M. acuminata spp. zebrina) to
17.1% (M. ornata) and the maximum post-cryopreservation germination rates varied
from 86.4% (M. velutina) to 55.0% (M. ornata).
Apart from the above well-studied species, Table 15.4 lists the cryopreservation
protocol standardized by different workers in many other anti-diabetic plants.
15.19 Conclusion
Long-term conservation of difficult to conserve species using cryopreservation has
rapidly been evolving since the past two decades. Since most of the medicinal plants
comes under the category of difficult to conserve species (clonally propagated and
recalcitrant seed species), cryopreservation offers the most reliable conservation
method for these species. Cryopreservation protocols are already in place for many
medicinal and anti-diabetic species employing different methods ranging from
simple desiccation, encapsulation and vitrification to most modern methods of
droplet freezing and cryoplate method. Development of protocols specific to species
needs to be developed in terms of selection of explant, size of germplasm to be
stored, freezing and rewarming methods, recovery media and recovery method etc.
Stability of the cryopreserved material in terms of its genetic and biochemical
content is most important in the case of medicinal or anti-diabetic plants. Morphological, biochemical, cytological and molecular methods are routinely employed for
the genetic stability studies to ensure the true-to-type nature of conserved material
and so far, no alterations have been observed after cryopreservation. Thus, cryopreservation has evolved as the most promising method for the viable long-term conservation of potential medicinal crops with intact genetic and biochemical stability.
15
Cryopreservation of Anti-Diabetic Plants
463
Table 15.4 Cryopreservation of plants with anti-diabetic property
Species
Allium sativum
Allium cepa
var.
aggregatum
Explant
Shoot tips
Shoot tips
Cryopreservation
method
Vitrification
Droplet
vitrification
Beta vulgaris
Shoot tips
Vitrification
Citrus sinensis
Embryogenic
calli
Shoot tips
Cryoplate method
Ipomea batatas
Picorhiza
kurroa
Cicer arietinum
Arachis
hypogea
Asparagus
officinalis
Morrus sp.
Poncirus
trifoliata
Dioscorea
bulbifera and
Dioscorea alata
Carica papaya
Acacia nilotica
Rauwolfia
sepentina
Vandenbussche
et al. (2000)
Souza et al.
(2017)
Hirai and Sakai
(2003)
Sharma and
Sharma (2003)
Bajaj (1995)
Bajaj (1995)
Shoot tips
Encapsulation
vitrification
Vitrification
Seeds
Seeds
Vitrification
Vitrification
Embryogenic
suspension
cells
Dormant buds
and shoot tips
Vitrification
Nishizawa et al.
(1993)
Droplet
vitrification
Seeds
PVS2 vitrification
Shoot tips
Vitrification
El-Homosany
and Farag
(2020)
Wang et al.
(2002)
Mukherjee et al.
(2009)
Shoot tips and
seeds
Seeds
PVS2 vitrification
Desiccation,
vitrification
PVS2 vitrification
Juglans regia
In vitro
grown nodal
segment
Suspension
cultured cell
lines
Embryogenic
cell culture
Pollen
Ficus carica
Shoot tips
Vitrification
Psidium
guajava
Pollen
Desiccation
Catharanthus
roseus
Reference
Niwata (1995)
Wang et al.
(2020)
DMSO + sorbitol
treatment
Desiccation
Azimi et al.
(2005)
Jebelli et al.
(2015)
Ray and
Bhattacharya
(2008)
Tony et al.
(1984) and
Fatima et al.
(2009)
Luza and Polito
(1988)
El-homosany
and Sayed
(2020)
Vishwakarma
et al. (2020)
Anti-diabetic
property
Reference
Ozougwu (2011)
Mootoosamy
and
Mahomoodally
(2014)
Murthy and
Manchali (2013)
Shakthi Deve
et al. (2014)
Akhtar et al.
(2018)
Joy and Kuttan
(1999)
Wei et al. (2017)
Akter et al.
(2014)
Hafizur et al.
(2012)
Hasani-Ranjbar
et al. (2008)
Jia et al. (2016)
Ghosh et al.
(2012) and Wang
et al. (2011)
Juárez-Rojop
et al. (2012)
Saha et al. (2018)
Azmi and
Qureshi (2016)
Muralidharan
(2014)
Hosseini et al.
(2014)
Deepa et al.
(2018)
Oh et al. (2005)
(continued)
464
M. R. Rohini et al.
Table 15.4 (continued)
Species
Momordica
doica and
M. sahyadrica
Explant
Pollen
Cryopreservation
method
Desiccation
Reference
Rajasekharan
et al. (2010)
Anti-diabetic
property
Reference
Sharma et al.
(2018) and Singh
et al. (2011)
15.20 Future Prospects
Several base collections using cryopreservation have already been established in
many national and international crop-based research institutes. Yet, their application
is still limited to few species. This is mainly because of the lack of infrastructure and
lack of expertise in developing the protocols. The success or recovery rate of
cryopreserved samples can still be improved by manipulating various parameters
such as the maturity stage of explant, duration and rate of desiccation, rate of
freezing and the recovery media. More and more protocols need to be developed
for more number of crops especially the wild medicinal species which are difficult to
conserve. Basic studies pertaining to biochemical and metabolic activities of the cell,
their response to dehydration and freezing stress should be carried out so as to
develop the cryopreservation protocols effectively. As per the previous reports,
success rate of cryopreservation is more in intermediate species as compared to
recalcitrant species. Much improvement is expected by exploring different technical
approaches to improve the efficiency and increase the applicability of cryopreservation techniques to recalcitrant species. To conclude, the development of cryopreservation protocol should come out from the framework of academic purpose and
should become a routine programme of conservation projects so that it can be
implemented for all the species.
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