By –Pranita SharmaM.Phil Bioscience

SOS in Life SciencePt. Ravishankar Shukla University

• Introduction

• Cryopreservation– Preparing germplasm for cryopreservation– Pretreatments– Vitrification

• Summary

• Conclusion

• References

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 technologyB. Molecular diagnosticsC. Tissue culture (in vitro technology)D. Cryopreservation

Integrating biotechnology in conservation projects

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.

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.

PREPARING GERMPLASM FOR CRYOPRESERVATION

• Applied to germplasm before cryoprotection : enhance survival when used in combination with other cryoprotectivestratergies.

• 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).

PRE-TREATMENTS

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.

• 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.

Vegetative Tissues

GermplasmCollection

Seeds

Surface SterilizationEmbryo Rescue

Culture Initiation

Pre established culturesDessication

Pre growthTreatments

CryoprotectionTraditionalVitrification Controlled- Rate

“Programmable Freezing”

Direct (rapid) immersion inliquid nitrogen

Long term Storage Simplified Freezing

Cold Hardening

• 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.

Vitrification

• 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.

- 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.

DesicationAir Drying

Encapsulation/ Dehydration

Silica gel

Osmotic reagent

Desicationsensitivity

PVS2

Toxicity

De-vitrificationCryoprotective

additivesEncapsulated vitrification

Pathways to Vitrification

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 1hour 0.5 DMSO + 1M

Sucrose + 0.5 M glycerol (applied on

ice

From 0˚C at a rate of -1˚C/min controlled

rate freezing

to -35˚C hold 30-40 min transfer to -196˚C

Rapid re-warming at 45˚C waterbath

Transfer to fresh medium

B. A PVS2 vitrification method for shoot tips (adapted from Sakai et.al. in 1990)

Pre growth 1.2M sorbitol

medium

Sterpwiseaddition

PVS2 solution on ice over

20-30 minute

Direct plunge -

196˚C

Rapid rewarm

Un – loading in 1.2 M sucrose transfer to fresh

medium

30% glycerol + 15% ethylene glycol + 15% DMSO in medium with 0.4 M sucrose

SUMMARY

CONCLUSION1. 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.

REFERENCES

1. Plant Conservation Technology – Erica E. Benson.2. Cryopreservation of Phytodiversity: A Critical Appraisal

of Theory & Practice , Erica E. Benson3. 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/productsdetail_11152___11954381_-1

5. http://en.wikipedia.org/wiki/Cryopreservation

THANK YOU