Cryopreservation of plant tissues

1. Cryopreservation of PlantTissues and Organs
Plant tissues and organs can be frozen and stored in
liquid nitrogen (LN) at -196°C for long-term storage of
germplasm. This would be of great value in the
conservation of germplasm of those crops, which
normally do not produce seeds, e.g., root and tuber
crops produce recalcitrant seeds, or where it may not
be desirable to store seeds.

2. The preservation of cells, tissues and organs in liquid
nitrogen is called cryopreservation, and the science
pertaining to this activity is known as cryobiology. Many
studies have been carried out on cryopreservation of
plant cells and organs, and the approach appears to have
considerable promise in germplasm conservation.

3. At -196°C, since at this temperature, all metabolic
processes and growth are suppressed, and the occurrence
of genetic, karyotypic, morphological and biochemical
changes are also prevented. Cryopreservation has proved
to be the most reliable method for long-term
preservation of cell cultures.

4. Calli, cell suspensions, protoplasts, pollen shoot-tips and
embryos have all been successfully preserved.
Drawback:
A serious drawback of the technique is that a general
protocol applicable to all species and explants is not
available. In addition, survival tends to decline with
storage period in most of the cases, most likely due to
injuries sustained by cells during the freezing step.

5. Choice of Material:
Material chosen for cryopreservation should be, as far as possible, in
meristematic state. Cell cultures are generally preserved in lag or early
exponential phase of growth. Cells in the early lag/stationary phase
may be susceptible to cryoinjury because of their arrest in the G1
phase.
Cultures in the exponential phase contain cells in different stages of
mitosis and cytokinesis; such cells suffer from injury during freezing
and thawing. In comparison, cells in the late lag phase are
predominantly in G2 and ready to embark upon cell division. In some
species, it may be important to use highly embryogenic cell cultures
since nonembryogenic or poorly embryogenic cultures show poor or no
re-growth after thawing.

6. Preculture
Culture of cells/tissues/organs in the presence of amino acids like proline,
sugars like sucrose and mannitol or at low temperature prior to freezing
initiates in them important physiological changes, which increase their
freezing tolerance. These changes relate to membrane fluidity to facilitate
dehydration and accumulation of substances like proline, which protect
the cells from toxicity of high solute concentrations.
Plantlets may be hardened by growing them at a near freezing
temperature, e.g., for one week at 22°C for 8 hr and -1°C for 16 hr (each
day). Cold-hardened tissues show increased water efflux, which permits a
rapid dehydration of the cells; this prevents the formation of large
intracellular ice crystals during freezing.

7. Cold-hardening pretreatments have been quite useful in
cryopreservation of temperate plant species. Sucrose (30-
240 gl-1) is the most commonly used sugar for the
pretreatment of cultures. But mannitol can also be used. A
3-4 day- preculture on 2-4% proline either alone or in
combination with some other cryoprotectant like DMSO
markedly improves the survival of suspension cultures.

8. Sugars act as osmotically effective agents, although
they do not penetrate inside the cells. Dehydration of
cells/tissues occurs in the presence of sugars during
the preculture, which prevents lethal ice crystal
formation during freezing. Proline may act by
reducing the level of latent injury to the cells or it may
actively participate in recovery metabolism.

9. Cryoprotection
The two major sources of cryoinjury are mechanical
damage due to ice crystals and toxic solution effects
due to the excessive concentration of intracellular
solutes resulting from ice formation. Water loss during
freezing may reduce the cell volume below a critical
level necessary for survival. In addition, dislocation of
structural water, which protects the integrity of cellular
components like membranes may also be involved in
cryoinjury. In fact, a critical amount of liquid water is
essential for membrane integrity and for cells to survive
freezing.

10. Culturing of cells/tissues in the presence of some high
molecular weight substances like dimethyl- sulphoxide
(DMSO), sugar alcohols like glycerol, sorbitol and amino
acids like proline for a period of time, prepares them to
tolerate cryoinjury. In majority of freeze preservation
protocols a mixture of DMSO, glycerol and sucrose is
used. The general order of cryoprotection seems to be
proline > DMSO + glycerol = DMSO + proline > DMSO.

11. Cryoprotective compounds differ remarkably in their
structure, and possibly in their mode of action. Some
cryoprotectants like DMSO penetrate the plant cell
quickly; others like glycerol are rather slow to enter cells,
while still others like sorbitol may not enter the cell at all.
Theoretically, a cryoprotectant may protect living cells
against freezing injury at the following three locations:
extracellular, cell membrane and intracellular sites. It may
also help to stabilize the intra- and inter- molecular
arrangements of cellular components and prevent the
removal of water molecules associated with cell
membranes during freezing.

12. DMSO (5-10%) is widely used either alone or in combination
with 5-20% glycerol dissolved in water or sugar solution; it is
also used in combination with sucrose, sorbitol or other
sugar alcohols. A combination of DMSO with other
cryoprotectants may be beneficial because they may act in
complementary manne.
For example, compounds like sorbitol do not enter the cells;
these would reduce cellular water content and, thereby,
decrease the rate of initial ice crystal formation. DMSO
itself enters the cells, and it reduces cellular dehydration
during freezing. Thus both the initial ice crystal formation
and subsequent dehydration during freezing are reduced
and, as a result, the survival may be increased.

13. Importance of DMSO
DMSO is the most commonly used cryoprotectant; it is
added gradually over a period of 30-60 min and the
temperature of the cultures in maintained at around 0°C.
Often, cells and tissues are suitably pre-cultured/pretreated
before freezing; this makes them hardy and improves their
survival. The frozen cells and tissues are stored in a liquid
nitrogen refrigerator; the temperature must not rise above
– 130°C otherwise ice crystals may be formed.

14. Different tissues have different sensitivities for cooling rates.
Ideally, cooling should be such that damaging ice crystals are
not formed.
In general, there are three strategies for freezing:
(1) Slow cooling,
(2) Rapid cooling, and
(3) Freezing, following dehydration.
Freezing:

15. Slow Cooling:
The material is cooled at the rate of 0.5-4°C min-1 up to -
40° to-100°C, and held at this temperature for 20-45 min
before it is plunged into liquid nitrogen. Initial slow
cooling reduces the amount of intracellular water, since
ice is first formed outside the cell and unfrozen
protoplasm loses water due to vapour deficit between
super-cooled protoplasm and extracellular ice. This
approach is particularly suitable for cell suspensions.

16. Rapid Cooling:
Some tissues cannot survives intermediate or slow
cooling rates and require rapid cooling of 200-l, 000°C
min-1. For example, shoot apices of strawberry and
Solatium goniocalyx did not survive cooling rates below
1,000°C min-4. In rapid cooling, the critical temperature
zone of ice crystal formation is passed so rapidly that ice
crystals of lethal size are not formed.

17. Freezing following Dehydration:
Excised single node segments (5 mm) from 6 to 8 week-old
plantlets of Asparagus officinalis were pre-cultured for 2 days
on 0.7 M sucrose, and then dehydrated with silica gel before
immersing them in LN. Explants dehydrated to 20% moisture
showed more than 60% survival as compared to around 17%
in case of non-dehydrated explants. Similarly, dehydrated
(18-30% moisture) embryogenic cell masses of sweet potato
showed 1.5 to 2- fold increases in survival on rapid freezing by
direct immersion in LN.

18. Extensions of this approach are the protocols for
vitrification and dehydration following encapsulation,
where tissues are dehydrated to threshold water levels.
In vitrification, the material is dehydrated using highly
concentrated aqueous solutions of cryoprotective
agents, whereas encapsulated cells/tissue may be
dehydrated by preculture on high sucrose concentration
or by exposure in a laminar air low. The dehydrated
materials are directly transferred into LN.

19. Thawing is the bringing of cryopreserved
materials back to the normal state in such a way
that damaging ice crystal formation does not
take place. Generally, the frozen material is
plunged into a waterbath at 37-40°C for 1-2
minutes; this gives the initial warming rate of
1,450°C min-1, and of 120°C min-1 between -50°C
and -10°C.
Thawing:

20. Viability of cells and tissues is usually determined by re-
growth, but chemical tests like staining with fluorescein
diacetate (FDA) or 2, 3, 5-triphenyltetrazolium chloride
(TTC) may be used to assess cell viability immediately
after thawing. Phenosafranine has also been used for
checking cell viability.

21. The post-thaw treatments should be such as to provide the
best possible conditions for recovery of cells from cryoinjury
and for resuming growth. The cryoprotectants may be
removed either by washing with the culture medium or by
plating the thawed cultures on to a filter paper kept on the
medium. Washing of the cells is considered deleterious in
many cases, and should preferably be avoided.
Reculture:

22. The permeability of plasma membrane increases due to
freezing and essential cell constituents may leak out of
the cells into the cryoprotectant solution. Washing may,
as a result, reduce the viability of cultures. Washing of cell
suspensions and embryo-genic callus cultures of
Gossypium hirsutum prevented their re-growth. But
when unwashed cells were placed on a filter paper, they
exhibited re-growth within two weeks of plating.
Similarly, washing of Saccharum cells or their re-culture
directly on the culture medium without washing was
deleterious.
However, healthy tissue was formed when the cells were
first plated on a filter paper discs and after 5 hr the discs
along with the cells were transferred to a fresh medium.
Finally, after 24 hr the cells were scraped off the filter
paper and transferred on to a fresh culture medium.

23. Many other authors have reported the beneficial effects of a
gradual removal of cryoprotectants by use of the filter paper
disc culture technique, following their placement under low
light intensity or in total darkness for two to three weeks.

24. At a sufficiently low temperature, highly concentrated
aqueous solutions of cryoprotective agents become so viscous
that they solidify into a metastable glass state, without ice
crystal formation at practical cooling rates; this phenomenon
is called vitrification.
It eliminates the need for controlled slow freezing as cells and
meristems are first dehydrated by a treatment with a plant
vitrification solution (PVS) of suitable concentration and then
cryopreserved by direct transfer into LN. It has been used for
cryopreservation of cell cultures, shoot-tips and protoplasts.
Vitrification:

25. Vitrification solutions themselves may cause toxicity, which
depends mainly on their osmotic potential. Therefore,
formulation of a PVS of suitable osmotic potential, manipulation
of duration and temperature during exposure of cells to PVS,
and subsequent dilution procedures would minimize toxicity.
Freezing does not appear to cause cellular injury in addition to
that produced due to dehydration with PVS. The PVS toxicity to
Wasabia japonica apical meristems was markedly reduced by
use of cyroprotectants.

26. Shoot apices cryoprotected with 2 M glycerol + 0.4 M sucrose
prior to their dehydration with PVS2 showed shoot formation
in 80-90% of the explants after their immersion in LN. In
contrast, only 12.5% of the uncryoprotected shoot apices
dehydrated with PVS2 and frozen by immersion in LN showed
shoot formation.

27. Vitrification has been used to the greatest effect for
cryopreservation of germplasm of such plant species that
are recalcitrant to the traditional cryopreservation
methods based on controlled freezing, e.g., tropical crop
plants, recalcitrant seed producing forest trees and
tropical fruit crops, and certain clonally propagated
tropical fruit crops.

28. In contrast, successful temperate crop plant
cryopreservation methods are based on cold hardening
pretreatments. In addition, optimized embryo rescue
techniques used in combination with cryopreservation
and efficient re-culture procedures are promising in
conservation of recalcitrant germplasm.
Cryopreservation of plant tissues and cell cultures has
advanced to a level where it can be used as a routine tool
to conserve germplasm derived from wild relatives,
ancient and modern cultivars, and biotechnologically
derived genotypes of at least some plants species.

29. Encapsulation Dehydration:
In another approach, explants are first encapsulated in a
suitable matrix like alginate and then subjected to
dehydration. Generally, tolerance to dehydration is induced
by pre-culturing the encapsulated meristems in a medium
enriched with sucrose (about 0.7-1 M) or a mixture of sucrose
and glycerol.

30. The beads may be dehydrated under laminar air how to
critical water level, which varies among different species,
placed in a cryotube and plunged directly into LN. Survival
of encapsulated cells of Catharanthus roseus subjected to
rapid freezing increased many-fold when they were
dehydrated to 25% moisture prior to freezing. Apical
meristems, zygotic embryos and cell cultures of several
species have been cryopreserved by this method.

31. This procedure eliminates the need of cryoprotection that is
necessary when vitrification solutions are used and may
improve survival. Shoot primordia of horseradish (Armoracia
rusticana) were encapsulated in 2% alginate;, the beads were
pre-cultured on MS agar medium having 0.5 M sucrose or 1 M
glycerol for 1 day, and then dehydrated by silica gel/partial
vacuum to 70% of their weight after pre-culture.

32. The beads were then kept in cryotubes and plunged into LN.
More than 90% of shoot primordia formed normal shoots when
thawed and re-cultured. But vitrification of encapsulated shoot
primordia with PVS2 before plunging them into LN gave only
69% survival. However, in some species, dehydration of
encapsulated tissues itself may reduce survival.
Somatic embryos of Camellia japonica did not survive freezing
in either nonencapsulated or encapsulated states and subjected
to various cryoprotective treatments. However, a proportion of
zygotic embryos, with or without desiccation, survived rapid
freezing. Thus somatic embryos of C. japonica appear to be in a
physiological state not suitable for cryopreservation.

33. Shoot-tips isolated from preconditioned shoot cultures of
Holostemma annulare (H. ada-kodien), an endangered
medicinal plant of the Indian subcontinent, were encapsulated,
dehydrated and precultured in the dark on 0.5-0.75 M sucrose
and 3-5% DMSO before being cryopreserved.
Pre-culture in sucrose alone for 2-3 days resulted in 46-56%
survival and shoot regeneration in 42-52% of the shoot-tips that
survived cryopreservation. A 3-day pre-culture with 3 and 5%
DMSO enhanced the survival rates to 72-76% and shoot
regeneration rates to 67-68%, but the entire enhancement
came from cells producing callus.

34. Materials subjected to cryopreservation may show some special
requirements during re-culture. For example, shoot-tips from
freeze-preserved seedlings of tomato required GA3for
developing into shoots (they formed callus in the absence of
GA3), while normal shoot-tips of tomato do not require GA3.
Similarly, survival of carrot plantlets was greatly improved by
activated charcoal. It is, therefore, necessary to determine the
optimum conditions for re-culture of different plant species,
particularly when commonly used regimes fail.

35. Cell cultures, shoot-tips, somatic/zygotic embryos and even
plantlets of a number of species have been successfully frozen
and stored for variable periods (from a few minutes to several
months). In general, meristematic cells survive better during
freeze preservation than do mature differentiated cells. The
techniques for freeze preservation of shoot-tips are being refined
for germplasm storage over very long periods.

36. Application
s
Conservation of genetic material
Freeze storage of cells
Maintenance of disease free stocks
Cold acclimation and frost resistance