Unit-IV Germplasm Storage and Cryopreservation.pptx

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.