The document outlines the 8 key steps to cryopreserve plant cell cultures: 1. Develop sterile tissue cultures from appropriate plant material 2. Add cryoprotectants like DMSO or glycerol to tissues before freezing 3. Freeze tissues using slow, rapid, or stepwise freezing into liquid nitrogen 4. Store frozen tissues in liquid nitrogen at -196°C for long-term preservation 5. Thaw tissues quickly to avoid ice crystal formation 6. Reculture tissues to remove cryoprotectants 7. Measure viability using staining or regrowth ability 8. Regenerate whole plants from surviving cryopreserved cultures

1. Unit-IV
Germ plasm
Storage And
Cryopreservatio
n

13. Process of Cryopreservation:
⚫The cryopreservation of plant cell culture
followed by the regeneration of plants
involves the following steps:
⚫1. Development of sterile tissue cultures
⚫2. Addition of cryoprotectants and pre-
treatment
⚫3. Freezing
⚫4. Storage
⚫5. Thawing
⚫6. Re-culture
⚫7. Measurement of viability
⚫8. Plant regeneration

14. step I: Development of sterile
tissue culture:
⚫ One of the important steps is the selection of
plant species with reference to
morphological and physiological characters .
⚫ It directly influence the ability of explant to
survive cryopreservation.
⚫ Any tissue from a plant can be employed for
cryopreservation e.g. meristems,
endosperms, embryos, ovules, seeds,
cultured plant cells, calluses, protoplasts.
⚫ Out of these, meristematic cells and
suspension cell cultures which are in the late
lag phase or log phase are most appropriate.

15. step II: Addition of cryoprotectants
and pre-treatment:
⚫ The compounds that can prevent the damage
caused to cells by freezing or thawing are called as
cryoprotectants.
⚫ Cryoprotectants reduce the freezing point and
super-cooling point of water.
⚫ As a result, the ice crystal formation is delayed
during the process of cryopreservation.
⚫ Cryoprotectants used are dimethyl sulfoxide
(DMSO), glycerol, ethylene, propylene, sucrose,
mannose, glucose, proline and acetamide.
⚫ Among them, DMSO, sucrose and glycerol are
most commonly used.
⚫ Generally, a mixture of cryoprotectants instead of
a single one is preferred for more effective
cryopreservation without damage to cells/tissues.

16. step III: Freezing
⚫ The sensitivity of the cells to low temperature is
variable and largely relies on the plant species.
⚫ The different types of freezing methods used are as
follows:
⚫ 1. Slow-freezing method:
⚫ The tissue or the essential plant material is allowed to
slowly freeze at a slow cooling rates of 0.5-5°C/min
from 0°C to -100°C.
⚫ Then it is transferred to liquid nitrogen.
⚫ Slow-freezing method facilitates the flow of water
from the cells to the outside.
⚫ This avoids intracellular freezing and promotes
extracellular ice formation.
⚫ Because of this, the plant cells are partially
dehydrated and can survive better.
⚫ The slow-freezing technique is successfully
employed for the cryopreservation of suspension
cultures.

17. 2. Rapid freezing method
⚫ This process is quite simple.
⚫ In this technique, the vial containing plant
material is plunged into liquid nitrogen.
⚫ During rapid freezing, reduction in
temperature from -300° to -1000°C/min
occurs.
⚫ The freezing process occurs so quickly that
small ice crystals are formed within the cells.
⚫ In addition to it, the growth of intracellular
ice crystals is also minimum.
⚫ Rapid freezing technique is applied for the
cryopreservation of shoot tips and somatic
embryos.

18. 3. Stepwise freezing method
⚫This technique is a combination of slow
and rapid freezing procedures having the
advantages of both, and occurs in a
stepwise manner.
⚫Firstly, the plant material is cooled to an
intermediate temperature.
⚫Then it is kept there for about 30 minutes.
⚫Finally, it is rapidly cooled by plunging it
into liquid nitrogen.
⚫Stepwise freezing method has been
successfully applied for cryopreservation
of suspension cultures, shoot apices and

19. 4. Dry freezing method
⚫It has been reported that the non-
germinated dry seeds can survive freezing
at very low temperature in comparison to
water-imbibing seeds which are sensitive
to cryogenic injuries.
⚫ In a similar way, dehydrated cells are
observed to have a better survival rate after
cryopreservation.

20. step IV: Storage
⚫ The frozen cultures should be maintained at the specific
temperature.
⚫ Generally, the frozen cells/tissues are maintained at
temperatures in the range of -70 to -196°C for storage.
⚫ Although, with temperatures above -130°C, ice crystal
growth may take place inside the cells which decreases
viability of cells.
⚫ The ideal storage is done in liquid N2 refrigerator at 150°C
in the vapour phase, or at -196°C in the liquid phase.
⚫ The final aim of storage is to halt all the cellular metabolic
activities and preserve their viability.
⚫ The temperature at -196°C in liquid nitrogen is regarded as
ideal for long term storage.
⚫ A regular and constant supply of liquid nitrogen to the
liquid nitrogen refrigerator is necessary.
⚫ It is essential to check the viability of the germplasm time
and again in some samples.
⚫ Proper documentation of the germplasm storage should be
done.

21. step V: Thawing
⚫ Thawing is usually performed by plunging the
frozen samples in ampoules into a warm water
(temperature 37-45°C) bath with robust
swirling.
⚫ By this process, rapid thawing (at the rate of
500- 750°C min-1) takes place, and this
preserves the cells from the damaging effects
from ice crystal formation.
⚫ As soon as the thawing occurs (ice completely
melts), the ampoules are transferred to a water
bath at temperature 20-25°C at the same
instant.
⚫ The cells get damaged if left in warm (37-45°C)
water bath for long time.
⚫ For the cryopreserved material (cells/tissues)
where the water content has been decreased to
an optimal level before freezing, the process

22. step VI: Re-culture
⚫To remove cryoprotectants, the thawed
germplasm is washed various times.
⚫Following standard procedures, this
material is then re-cultured in a fresh
medium.
⚫In some cases, the direct culture of the
thawed material is preferred without
washing.
⚫ It is so because certain vital substances,
released from the cells during freezing, are
assumed to enhance in vitro cultures.

23. step VII: Measurement of
viability:
⚫ The measurement of survival or viability of
the frozen materials can be performed at
any stage of cryopreservation or after
thawing or re-culture.
⚫ The techniques used to determine viability
of cryopreserved cells are the same as
applied for cell cultures.
⚫ The commonly used techniques are staining
techniques using triphenyl tetrazolium
chloride (TTC), Evan’s blue and fluorescein
diacetate (FDA).
⚫ The entry of cryopreserved cells into cell
division and regrowth in culture is the best
indicator to measure the viability of them.
⚫ This can be evaluated by the using following
expression.

24. step VIII: Plant regeneration
⚫The regeneration of the desired plant is
the ultimate purpose of cryopreservation
of germplasm.
⚫The cryopreserved cells/tissues have to be
carefully nursed, and grown for
appropriate plant growth and regeneration
.
⚫Along with maintenance of proper
environmental conditions, addition of
certain growth promoting substances is
often essential for successful plant
regeneratio

25. Limitations for
Cryopreservation:
⚫ An individual with good technical and
theoretical knowledge of living plant cells as
well as cryopreservation method is required.
⚫Precautions for cryopreservation:
⚫ The formation of ice crystals inside the cells
should be prevented as they are responsible
for causing injury to the organelles and the
cell.
⚫ Cells might be damaged if the intracellular
concentration of solutes is high.
⚫ Leakage of certain solutes from the cell
during freezing should be checked.
⚫ The physiological status of the plant
material is also essential.