Cryopreservation is the preservation of plant cells, tissues, and organs at temperatures around -196°C to maintain genetic diversity and biodiversity. The document discusses the limitations of conventional methods, techniques for cryopreservation, and the importance of using cryoprotectants. It also outlines applications and limitations associated with cryopreservation, emphasizing its role in conserving endangered species and facilitating pathogen-free plant storage.

1. Cryopreservation
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. Contents
• Introduction
• Reason for cryopreservation
• Selection of part of plant for cryopreservation
• Technique of cryopreservation
• Application
• Limitation
• Conclusion
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3. Introduction
• Preservation and storage of cells,tissues and
organs at temperature around -196oC or by
liquid nitrogen is known as cryopreservation.
• Germplasm means the total genetic variablity
of a particular species.
• Cryopreservation is used to preserve the
germplasm , and maintain genetic diversity
which was erosing due to new technique and
human activites.
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4. • Germplasm storage is necessary to maintain
biodiversity and endangered species.
• There are two method of conservation ex situ
and in situ.
• Ex situ includes seed storage , in vitro storage
,botanical garden and field gene banks.
• In situ includes forest reserve , reserve on farm
and conservation on garden.
• There are many methods for germplasm storage
but they have many drawbacks.
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5. Reason for cryopreservation
• Limitation of conventional method(seed)-
1. Some crops produce large and short lived
seeds , lack dormancy mechanism and can’t
bear subjection to dessication or exposure to
low temperature.
2. Seeds may be destroyed by pathogen and
pest.
3. Discrete clones cannot be maintained in the
form of seeds.
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6. 4.It is not applicable to vegetatively propogated
crops.
Limitation for nursery and other reserve
• Maintaining such large genotype in field need
extensive care from pathogens , needs man
power and loss due to environment hazards.
Limitation of in vitro culture-
• Due to serial subculture microbial contamination
come due to human error and uneconomical.
• Long term culture(callus and suspension) affect
regeneration capacity , biosynthetic properties
and genetic makeup.
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7. Cryopreservation
• In cryopreservation, plant material is frozen
and maintained at the temperature of liquid
nitrogen(-196oC).
• In this temperature, cells are inactive state.
• The part used for cryopreservation are shoot
apices , embryos, young plantlets.
• But generally from culture cell or suspension
culture cryopreservation is not due to
following reason.
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8. 1.Genetic instability of long term callus and
suspension culture , callus arise from non
meristematic tissue show polysomaty.
2.Several crop cultured cell do not have
totipotency and some will develop organ and
whole plant but lose potentiality.
3.Haploidy which is highly unstable in callus and
suspension culture.
4.Cells of shoot tip and young embryos are small
and meristematic , they are better suited than
larger cell to survive LN, thawing and freezing.
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9. Factor that affect cryopreservation
• Factor that affect cryopreservation are:
1. Nature of plant material- In general, small, richly
cytoplasmic meristematic cell survive in -196oC
than large vacuolated cell.
2. Pre freezing treatment-there are 3 types;
• Pre culture-the shoot apices will survive
supercoiling only when they are precultured
before storage.
eg-potato
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10. • Desiccation-cryopreservation is the exclusion
of freezable water from the cells before
freezing.
• Vitrification - This is a physical process in
which concentrated aqueous solution cooled
to low or ultralow temperature directly
solidifies into an amorphous glassy state.
• Cryoprotectants - Cryoprotectants are used
because to protect cells from this toxic effect
of concentrated intracellular concentration of
cell, they also prevent formation of large
crystal.
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11. Technique of cryopreservation
• Broadly cryopreservation occur in 4 steps:
1.Freezing
2.Storage
3.Thawing
4.Reculture
1. Freezing-the material suspended in the culture
medium treated with suitable cryoprotectant is
transferred to sterile polypropylene with a screw
cap and frozen by one of the following methods:
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12. 5/6/2020 12

13. • There are different method for different
species
1.Slow cooling method: In this method 0.5-4oC
i.e. cooling rate for changing temperature of
plant material from 0 to -100oC and then
transferring into liquid nitrogen.
2.Rapid cooling method-In this method, we can
cool rapidly by direct plunging into liquid
nitrogen but some does not survive, so first
gradually decrease temperature to -15oC and
then immerse into liquid nitrogen so cooling
rate is 1000oC per min.
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14. 3. Pre freezing method:In this method material
is first cooled at the rate of 1oC gradually or
5oC stepwise to an optimum temperature(-
30oC to -50oC) for 30 mins and then rapidly
cooling by plunging into liquid nitrogen.
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15. Storage
• Storage is as important as freezing at -196oC.
• Long term storage require liquid nitrogen
refrigrator.
• Refrigrator storing 4000 ampoules of 2 ml
each is estimated to consume 20-25lt of
nitrogen per week.
• In theory long term storage is easy to maintain
little care is needed.
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16. Thawing
• Rapid thawing of material frozen at -196oC by
plunging it into water at 37-40oC whereas
thawing rate is 500-750oC per min.
• After 90 sec the material is transferred to an
ice bath and maintained there untill reculture
or its viablity is tested.
• Slow thawing at room temperature has
generally proved fatal.
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17. Reculture
• Reculture of different species need different
type of treatment.
• But generally washing of thaw material does
not give better regenration and removal of
cryoprotectant is required.
• The dilution of cryoprotectant may avoid
injury to cell.
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18. Application
1.Maintain biodiversity and endangered species- it
is easy method to maintain endangered and
genetic biodiversity of plant.
2.Maintain pathogen free plant-this is easy method
to maintain pathogen free plant.
3.Easy transport to other countries-because it is
pathogen free and need little space for storage
and easy to transport.
4.Cryopreservation serve as large storage and when
we needed grow it.
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19. Limitation
1. It is technically demanding and generally
require special instruments for cooling
storage.
2. It require preculture with cryoprotectants.
3. There is always the possiblity of severe
damage due to ice crystal formation.
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20. Conclusion
• Cryopreservation involve cost effective due to
removal of cryoprotectant,but if it is removed,so
it is easy single step process and not so costly.
• Cryopreservation cause arrest of metabolic
activity,DNA replication interrupted before
completion cause cellular or genetic damage.
• Ionizing radiation also cause damage to genetic
material and storage of damage material has no
importance.
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21. Reference
• Plant Tissue Culture- S.S. Bhojwani and M.K.
Razdan
• Plant Cell and Tissue culture-Indra K. Vasil and
Trevor A. Thorpe
• Biotechnology by V. Kumaresan
• Biotechnology by U. Satyanarayana
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