INTRODUCTION The tools of modern biotechnology applied for plant diversitycharacterization and they have a major role in assisting plant conservationprograms. There are four main areas of biotechnology which can be directly assistplant conservation programs. A. Molecular markers technology B. Molecular diagnostics C. Tissue culture (in vitro technology) D. Cryopreservation
Integrating biotechnology in conservation projects
CryopreservationCryopreservation is a process where cells or whole tissues arepreserved by cooling to low sub zero temperatures, such as 77Kor -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, includingthe biochemical reactions that would lead to cell death, is in astate of suspended animation.
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.
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).
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 Surface Sterilization Embryo Rescue Tissues Culture Initiation Germplasm Collection Seeds Pre established cultures Dessication Cold Pre growth Hardening TreatmentsCryoprotection Traditional Vitrification Controlled- Rate “Programmable Freezing” Direct (rapid) immersion in liquid nitrogen Long term Storage Simplified Freezing
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.
• 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.
Desication Air Drying Desication sensitivity Silica gel Osmotic reagent PVS2 Encapsulation/ Toxicity Dehydration Cryoprotective De-vitrification additives Encapsulated vitrification Pathways to Vitrification
Summary of some frequently used cryopreservation protocolsbased on controlled rate freezing and vitrificationA. 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
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 rewarm30% glycerol + 15% ethylene glycol + 15% DMSO inmedium with 0.4 M sucrose Un – loading in 1.2 M sucrose transfer to fresh medium
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.
REFERENCES1. 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/products detail_11152___11954381_-15. http://en.wikipedia.org/wiki/Cryopreservation