This document discusses cryopreservation techniques for conserving plant germplasm. It describes preparing plant materials for cryopreservation through techniques like surface sterilization and embryo rescue from seeds. Pretreatments like stabilizers, osmotic agents, and cold acclimation are used to enhance cell survival. Vitrification prevents ice crystal formation during freezing by solidifying plant materials into a glass-like state using high concentrations of cryoprotectants. Common cryopreservation protocols involve controlled slow freezing or vitrification with solutions like PVS2, followed by storage in liquid nitrogen. Biotechnology tools like cryopreservation can help conserve plant diversity.

1. By –Pranita Sharma
M.Phil Bioscience
SOS in Life Science
Pt. Ravishankar Shukla University

2. • Introduction
• Cryopreservation
– Preparing germplasm for cryopreservation
– Pretreatments
– Vitrification
• Summary
• Conclusion
• References

3. INTRODUCTION
 The tools of modern biotechnology applied for plant diversity
characterization and they have a major role in assisting plant conservation
programs.
 There are four main areas of biotechnology which can be directly assist
plant conservation programs.
A. Molecular markers technology
B. Molecular diagnostics
C. Tissue culture (in vitro technology)
D. Cryopreservation

4. Integrating biotechnology in conservation projects

5. Cryopreservation
Cryopreservation is a process where cells or whole tissues are
preserved by cooling to low sub zero temperatures, such as 77K
or -196˚C (the boiling point of liquid Nitrogen).
Stabilized cultures are preconditioned with osmotic agents,
frozen to -30˚C to -45˚C and subsequently stored in the vapour
(-140 ˚C) or liquid phase of nitrogen -196˚C.
At these low temperatures, any biological activity, including
the biochemical reactions that would lead to cell death, is in a
state of suspended animation.

6. PREPARING GERMPLASM FOR
CRYOPRESERVATION
• Cryopreservation can be applied to freshly collected
seeds or vegetative germplasm-shoot tips or buds
which have been sampled from the field.
• Surface sterilize the germplasm before it is placed in
liquid nitrogen.
• In recalcitrant seeds, embryo rescue is performed as
embryos are more suitable to cryogenic storage than
whole seeds.

7. PRE-TREATMENTS
• Applied to germplasm before cryoprotection : enhance
survival when used in combination with other cryoprotective
stratergies.
• Pre-treatment increase cellular viability by removing harmful
substances secreted by the cells during growth or cell death
from the culture medium.
• This include:
i ) Stabilizers-substances that may be naturally occurring or
artificially produced and can be introduced directly into the
culture medium.
• This include anti-oxidants or radical scavenger chemicals that
neutralize the deleterious effects of active oxygen species and
other free radicals (capable of damaging both internal and
external cell membranes).

8.  Eg: reduced glutathione, sodium thiosulfate, thiourea, ascorbic
acid.
 Another group of stabilizers include agents that hinder or
prevent ethylene biosynthesis and/or ethylene action. (plant
cells emit ethylene when stressed and ethylene damages cells
and leads to cell death).
ii) Osmotic Agents- reduce tissue water prior to freezing.
Eg. Sugars- fructose, glucose, maltose, mannitol, sorbitol,
sucrose and trehalose.
iii) Preculturing cells in media which contain “anti stress”
agents such as proline, abscisic acid or trehalose.
iv) Exposing temperate plant tissues to cold acclimation or
hardening regimes.

9. • Acclimated to a temperature which is reduced from culturing
temperatures, but above freezing.
• This prepares cells for the cryopreservation process by
significantly retarding cellular metabolism.
• It reduces the shock of rapid temperature transitions through
some of the more critical temperature changes.
v) Application of simple dehydrating pretreatments in
combination with sucrose and alginate bead encapsulation.

10. Vegetative Surface Sterilization
Embryo Rescue
Tissues Culture Initiation
Germplasm
Collection
Seeds Pre established
cultures
Dessication
Cold
Pre growth
Hardening
Treatments
Cryoprotection
Traditional
Vitrification Controlled- Rate
“Programmable Freezing”
Direct (rapid) immersion in
liquid nitrogen
Long term
Storage Simplified Freezing

11. Vitrification
• Vitrification is a process in which water undergoes a phase
transition from a liquid to amorphous ‘glassy state’.
• In this form water does not possess a crystalline structure.
• The major difficulty in cryopreservation of any cell is the
formation of intracellular ice crystals during both freezing and
thawing.
• Excessive ice crystal formation will lead to cell death due to
disruption of cellular membranes and organelles.
• One method to prevent ice crystal formation is to freeze the
cells rapidly such that the ice crystals formed are not large
enough to cause significant damage.

12. • Vitrification occurs when the solute concentration of a
biological system becomes so high that ice nucleation is
prevented, thus ice crystal formation and growth is inhibited.
METHODS
 Dessication of tissues to a point at which the critical moisture
content is so low that there is no water available for ice
formation and the viscosity of the cell membrane is so high
that a glass is formed.
 Achieved through:
- Treatment of germplasm with a sterile air flow (LAF) or by
drying over silica gel.

13. - Dehydrating the germplasm with an osmotic agent such as
sucrose before dessication ( Dumet et al,1993a,1993b).
- Encapsulation of tissues in calcium alginate matrix followed
by osmotic dehydration and air or silica gel drying (Fabre and
Derueddre 1990; Phunchindawan et al,1997).
- High concentration of cryoprotective additives, Plant
Vitrification Solution Number 2 ‘PVS2’ developed by Sakai
and Collegues ( Reinhoud et al,1995; Sakai et al, 1990)
• It comprise of ethylene glycol, DMSO and glycerol.
• Vitrified tissues may be directly plunged into liquid nitrogen,
without the need of controlled rate cooling.

14. Desication
Air Drying
Desication
sensitivity
Silica gel
Osmotic reagent
PVS2
Encapsulation/ Toxicity
Dehydration
Cryoprotective
De-vitrification additives
Encapsulated
vitrification
Pathways to Vitrification

15. Summary of some frequently used cryopreservation protocols
based on controlled rate freezing and vitrification
A. The Withers and King Controlled rate Freezing method for cell suspension cultures
Cryoprotection for From 0˚C at a rate of -
1hour 0.5 DMSO + 1M 1˚C/min controlled
Sucrose + 0.5 M rate freezing
glycerol (applied on
ice to -35˚C hold 30-40
min transfer to -196˚C
Rapid re-warming at
45˚C waterbath
Transfer to fresh
medium

16. B. A PVS2 vitrification method for shoot tips (adapted from Sakai et.al. in 1990)
Sterpwise
Pre growth addition
Direct
1.2M sorbitol PVS2 solution
plunge -
medium on ice over
196˚C
20-30 minute
Rapid
rewarm
30% glycerol + 15% ethylene glycol + 15% DMSO in
medium with 0.4 M sucrose
Un – loading in 1.2 M
sucrose transfer to fresh
medium

17. SUMMARY

19. CONCLUSION
1. Biotechnology is now integrated in all aspects of
plant germplasm characterization , acquisition,
conservation, exchange and genetic management.
2. Most significantly, vitrification – based protocols and
simplified procedures have made cryopreservation an
accessible and cost – effective storage option for
most laboratories who have a requirement for long
term ex situ conservation.

20. REFERENCES
1. Plant Conservation Technology – Erica E. Benson.
2. Cryopreservation of Phytodiversity: A Critical Appraisal
of Theory & Practice , Erica E. Benson
3. Black M. and Bewely J.D. (2000); Seed Technology and its
Biological Basis; Scheffield Academic Press Ltd., England;
1st edition (Chapter 10 seed substitutes from the laboratory
pp 327-358)
4. http://www.thermoscientific.com/ecomm/servlet/products
detail_11152___11954381_-1
5. http://en.wikipedia.org/wiki/Cryopreservation

21. THANK
YOU