Intracellular and Extracellular Cryoprotectants in Plant Cryopreservation-Juniper Publishers
Journal of Agriculture Research- Juniper Publishers
Due to the constant extractivism and changes in the
environment, destruction of the habitats of orchids, many species have
been extinct, and some others are on a near extinction list. Some
species have economic importance, such as vanilla, and others with
medicinal properties and some with ornamental value, due to the beauty
of its flowers and variety of species, being used in the landscaping. An
alternative for the preservation of these species would be through the
germplasm bank. Cryopreservation in liquid nitrogen is a widely studied
and diffused method in several areas. Several protocols are studied with
the purpose of reducing the rate of deterioration after these storage
procedures and thus increasing the storage time of the seeds with
quality. Therefore, this work aims to present intra and extracellular
cryoprotectants found in the literature for cryopreservation of orchids.
However, in order to obtain quality materials, the type and type of
cryopreserved material and storage time must be considered.
Keywords:Germplasm; Cryopreservation; Agriculture; Dehydration techniques
Introduction
Cryopreservation is a very important method and has
been increasingly used in order to preserve plants that are in danger of
extinction due to the destruction of their natural habitats with the
advancement of agriculture and extractivism. It is a conservation
process in which the biological material subjected to ultra-low
temperatures using liquid nitrogen (-196 ºC).
Germplasm banks are important tools, the simplest
form of conservation and maintenance of genetic material. However, for
the structures to be recovered after cryopreservation, the addition of
cryoprotective substances is necessary. cryoprotectants are classified
according to [1] as penetrating (intracellular) and non-penetrating
(extracellular).
The protocols should cover seed collection and
disinfection procedures, dehydration techniques and ideal water contents
for each species, ways to protect the cells against injury, freezing
and thawing techniques, seed germination until acclimatization and
seedling production.
In view of this, the use of cryoprotectants is
necessary for the protection of cellular structures, especially of the
membranes, in order to be successful at the end of the cryogenic
process. The objective of this work was to present intra and
extracellular cryoprotectants found in the literature for
cryopreservation of orchids.
Development
Cryopreservation in liquid nitrogen is a potentially
studied method to reduce the rate of deterioration, thereby increasing
seed storage time, ensuring the preservation of plant genetic sources,
and reducing costs and loss of viability. Damage to cell membranes due
to dehydration and increased solute concentration is responsible in
addition to the formation of ice crystals, disruption of membranes and,
until death of the cell [2]. Emphasizes that the thawing should also be
conducted in a judicious way avoiding to the maximum the conditions for
the formation of the crystals in the cellular interior. The survival
rates of explants after freezing are mainly related to physiology, water
content in cells and the use of cryoprotectants [3].
In view of this, the use of cryoprotectants is
necessary for the protection of cellular structures, especially the
membranes, in order to be successful at the end of the cryogenic process
[4]. In the literature there are several types of cryoprotectants used
and are divided into intracellular and extracellular
Intracellular cryoprotectants are organic solutes
responsible for protecting the organelles from cells during cooling.
They are molecules with low molecular weight and in this way can cross
the cell membranes with relative ease.
The mechanism of action according to [5] are based
on structures that promote hydrogen bonds with water molecules These
bonds change the orientation of the water molecule in the
ice crystals, creating a less harmful environment for the cells.
The most commonly used are ethylene glycol, propylene glycol,
dimethyl sulfoxide (DMSO), glycerol, methanol and ethanol [6].
The mechanism of action of non-penetrating cryoprotectants
is based on the protection of the cells against the osmotic effects
during the freezing process, promoting a hypertonic medium that
induces water to escape from the cells leading to dehydration,
that is, they act in the extracellular environment, thus reducing
the possibility of intracellular ice crystal formation. Extracellular
cryoprotectants are macromolecules and sugars, proteins, lipids
and some amino acids, with functions to reduce the formation
of ice, allow dehydration of cells and protect the cell membrane,
have high molecular weight cannot penetrate the cells. The most
used are sucrose, glucose, lactose, trehalose, polyvinylpyrrolidone
(PVP) and mannitol [7,8]. Cryoprotectants in high concentrations
can cause toxicity to the cells causing irreversible damage. The
effects of toxicity can be minimized through brief exposure to
cryoprotectants or through rapid cooling [9].
Conclusion
Cryoprotectants are used to prevent the formation of ice
crystals during freezing, and the concentration, exposure time
and type of material to be cryopreserved must be checked before
choosing the most suitable methodology to be used.
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