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A Review on the Effectiveness of Cryopreservation
as A Germplasm Management Option
Kugedera Andrew Tapiwa*
Great Zimbabwe University Masvingo, Zimbabwe
Submission: July 02, 2019; Published: August 26, 2019
*Corresponding author: Kugedera Andrew Tapiwa Great Zimbabwe University Masvingo, ZimbabwePerú
How to cite this article: Kugedera Andrew Tapiwa. A Review on the Effectiveness of Cryopreservation as A Germplasm Management Option. JOJ Wildl
Biodivers. 2019: 1(3): 555563.DOI: 10.19080/JOJWB.2019.01.555563
Germplasm management is one of the best methods of conserving wildlife species for future use and to protect extinction of wildlife resources such as trees, animals and plants. The use of cryopreservation has been noted as one of the best options because all resources kept under this method can be used after many years without any problem. Many countries have opted this method compared to keeping of live plant and animals. The only major challenge faced in the use cryopreservation is the effect of climatic changes and mutations which may take place and affect the resources. Some Germplasm resources such as seeds have life span and after that time they may fail to germinate or fail germinate but fail to suit to the climatic conditions due to climate change which causes mutation from live genetic resources. There is need to come up with a viable option of way of making cryopreserved resources suit the climatic conditions after climate change.
Cryopreservation is a perfect method for long-term conservation of plant, tree and animal genetic resources, using very low temperature (liquid nitrogen, -196°C). This method has been recognized as a practical and efficient tool for the long-term storage of Germplasm. Cryopreservation methods may provide the conditions for unlimited conservation of biological materials by reducing metabolic rates. During the cryopreservation all biochemical activities significantly reduced and biological deterioration are stopped. Conservation and subsequent sustainable use of genetic resources are essential to meet the demand for future food security. Several techniques have been developed yet to minimize the damaging effects of desiccation and freezing, ensuring high recovery of plant materials.
Techniques like cryopreservation collect and conserve plant genetic resources, especially plants with limited seed storage capability. In agroforestry tree species like Baikeae plirijuga need conservation so that they can be available for many years in research. There is only limited number of plants that cryopreservation techniques are used for their germplasm conservation, mainly because the techniques need to be adapted for each species. Therefore, continued efforts are needed in cryopreservation techniques to develop protocols for a wider range of plants. Formation of ice crystal during cryopreservation
is detrimental to cellular structure integrity and causes physical damage to the cells.
Cryopreservation is a part of biotechnology. Biotechnology plays an important role in international plant conservation programs and in preservation of the world’s genetic resources [1,2]. Advances in biotechnology provide new methods for plant genetic resources and evaluation (Paunesca, 2009). Cryopreservation, developed during the last 25 years, is an important and the most valuable method for long-term conservation of biological materials. The main advantages in cryopreservation are simplicity and the applicability to a wide range of genotypes . This can be achieved using different procedures, such as pre-growth, desiccation, pregrowth-desiccation, ED, vitrification, encapsulation-vitrification and droplet-freezing . Cryopreservation involves storage of plant material (such as seed, shoot tip, zygotic and somatic embryos and pollen) at ultra-low temperatures in LN (196°C) or its vapour phase (-1500C). To avoid the genetic alterations that may occur in long tissue cultures storage, cryopreservation has been developed Martin et al. (1998).
Cryopreservation has conservation methods which includes ex situ and in situ conservation. Today most tissue culture technologies offer two major options to ex situ conservation
of valuable plant germplasm that is slow growth storage and
cryopreservation. Cryopreservation is usually called freeze
preservation at ultra-low temperature of -196˚C that is the
temperature of liquid nitrogen. A temperature of -196˚C arrest
biochemical and most physical processes in plants making
cryopreservation suitable for conservation of meristematic cells,
seeds, pollen seeds, hard wood, softwoods and even embryos
. Cryopreservation is a long-term conservation method
where plant and tree materials can be stored for unlimited
periods whilst in good condition and suitable for use . Besides
conserving plant genetic resources, cryopreservation is also
useful for safe keeping of medicinal and alkaloids- producing
cell lines, hairy root cultures and genetically transformed and
transformation-component culture lines .
Cryopreservation of biological tissues can be successful
if intra-cellular ice crystal formation is avoided because lower
temperatures damages cell membrane destroying their partial
permeability . Avoidance of crystallization is not possible by
plants during cryopreservation. Vitrification is the only method
which can prevent crystal formation during cryopreservation
. Vitrification is the transformation of physical transition
process from aqueous solution to an amorphous and glassy
(non-crystalline) . Cryopreservation protocols mostly used
are air drying, slow freezing, dehydration and vitrification [8,9].
Air drying is commonly called flash or normal drying and is
applicable to orthodox seed, zygotic embryos and pollen of Zea
mays, Citrus lemon, Mangifera indica and other horticultural
species [10,11]. Some orthodox seeds can even withstand drying
below 3 percent moisture content without any damage and
reduction of viability. This cryopreservation protocol allows
seeds to be kept for long time whilst viable . The protocol
proved to be beneficial to recalcitrant zygotic embryos of some
plant species .
Encapsulation/ dehydration is used to conserve explants
such as meristems and embryos . Meristems and embryos
are encapsulated in alginate beads which contain mineral salts
and organic substances forming synthetic seeds (synseeds /
artificial seeds). The synseeds are treated with high sucrose
concentration, dried to a moisture content of 20 to 30 percent
and rapidly frozen in liquid nitrogen . Drying is done under
airflow or using silica gel. The presence of nutritive matrix
surrounding synseeds promotes regrowth after thawing since
the nutrients provides energy and food for respiration and
development of roots and shoots .
Vitrification is the most widely used cryopreservation
protocol in germplasm management [13,14]. Synseeds are
treated with concentrated vitrification solution for variable
periods of time and followed by direct plunge into liquid nitrogen.
Vitrification solution contains penetrating and non-penetrating
cryoprotective substances to preserve both inside and outside of
the synseeds so that they remain viable for long period of time
. This protocol can be applied to several tissues and plants
species . The most commonly applied solution is plant
vitification solution number 2 (PVS2) which contains glycerol,
ethylene glycol and sucrose. These nutrients are used by the
synseeds during regrowth so that they quickly grow and prevent
loss of viability of synseeds [7,13].
In Herbaceous species cryopreservation is done to conserve
specific features which can be lost during normal in vitro
maintenance. Cell suspensions and callus cultures are done,
and it was noted that morphogenetic potential of embryogenic
callus lines is not affected by their storage in liquid nitrogen .
Cryopreservation does not affect the expression of foreign sam
gene in transgenic Papaver somniferum cells and is beneficial for
pyrethrin biosynthesis by Chrysanthemum cinerariaefolium cell
cultures [17,18]. Cryopreseved Oryza sativum and Zea mays calli
proved to be source of regenerable cell cultures for transgenic
plant production . Cryopreservation of non-organised
cultures is done in coffee, oil palm and banana  to make sure
specific genes are maintained.
Cryopreservation of pollen facilitates crosses in breeding
programmed, distributing and exchanging germplasm among
locations, and preserving nuclear genes of germplasm . This
also facilitates studies in basic physiology, biochemistry, fertility
and biotechnology involving gene expression, transformation
and in vitro fertilization . Preservation pollen is very useful
for cross-pollination of cultivars differing in flowering periods
. Cryopreservation of pollen is very useful because their
genetic maintenance and prevents variation in plants caused
environment. Same plant can be reproduced for several years
without any change.
Cryopreservation is suitable in hardwoods, softwoods and
other several plant types where seeds, meristems and pollen
are stored in liquid nitrogen for use in future. In herbaceous
species, cryopreservation of meristematic tissues was the most
successful as preservation of non-organised tissues . Shoot
meristematic tissues are the most commonly used explants
for cryopreservation of vegetative propagated species such as
fruit trees, roots and tuber crops. These have lower chances for
soma clonal variation . Meristems can be preserved using
droplet freezing and vitrification cryopreservation protocol for
example cryopreservation of Solatium tuberosum uses both
protocols . Banana meristems are preserved using droplet
vitrification protocol , producing about one-quarter of the
world’s banana collection. The efforts for cryopreservation
are also done to preserve cassava meristems so that cassava
production increases in future . In Australia endangered
species of plants and trees are preserved using cryopreservation
so that their genetic material is not lost and production increases
in future . Cryopreservation is one of the best options for
germplasm management because germplasm can be managed at
any stage of plants and trees.
Cryopreservation is also applicable to woody species.
Traditionally germplasm preservation of woody species was
through seed and field collections . Numerous forest
angiosperms such Acer species, Quercus species, chestnut
and horsechestnut have non-orthodox seeds with very limited
storage period. Germplasm management of these species need
collection of vegetative material and cryopreservation is done
so that the species are obtainable in future . They require
conservation of huge numbers of accessions in clonal orchards
including old, new, local varieties and wild material to allow
choice during reproducing new plants . Cryopreservation
should be regarded as a strategically important support to
traditional in-field banks. Cryopreservation minimizes risks of
accidental loss of woody plant genetic resources .
In woody plant cryopreservation shoot tips, seeds and lines of
embryogenic callus are used to maintain genetic resources .
The shoot tips are differentiated organs used for preservation of
vegetatively propagated plants such as Citrus synesis and timber
tree cultivars for the maintenance of genetic resources .
Shoot tips are obtained from apical or axillary buds and excised
from in vitro grown shoot cultures. The bud is kept under sterile
conditions to obtain apical meristems which are also kept in
liquid nitrogen . Cryopreservation is done for those species
characterized by non-or sub-orthodox seeds, which cannot
be stored traditional seedbanks . Cryopreservation is the
best option for long term conservation of genetic resources for
these species [30-34]. Seeds or the isolated embryo axes are
preserved for species which produces seeds such as Eucalyptus
camaldulensis. Lines of embryogenic callus are used in nonorthodox
seeds which permit germplasm conservation of these
species. This organ type is used to prevent loss of embryogenic
potential due to repeated subculturing or allowing the storage of
transgenic material Gass et al. (1996).
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