germplasm preservation in vitro approaches

1. Germplasm Preservation � In Vitro Approaches Suggested reading � Vasil ch. 9, Bhojwani ch 18 Importance of germplasm preservation preservation of superior genotypes preservation of "source material" Problems with conventional storage conventional seeds � viability, pathogens, pests vegetatively propagated crops � expensive in terms of labor costs and space needs, esp.

3. Germplasm Preservation � In Vitro Approaches Basic goals of an in vitro storage system to maintain genetic stability to keep in indefinite storage w/o loss of viability must be economical Two types of systems have been developed storage in liquid nitrogen (LN) (-196� C) cold storage (1-9� C)

4. Germplasm Preservation � In Vitro Approaches Methods involving LN cryopreservation (aka "prefreezing method") isolate embryos, establish callus, then suspension cultures subculture into fresh medium with a cryoprotectant (e.g., proline or DMSO) for 3-4 d, then wash culture combine chilled culture with incr. conc. of cryoprotectant

5. Germplasm Preservation � In Vitro Approaches Methods involving LN cryopreservation (aka "prefreezing method") transfer to ampoules, then to a controlled freezing apparatus and cool to -30� C at 1� C per min. hold at -30� C for 30 min., then transfer ampoules to LN (-196� C) to thaw, take out ampoules and agitate gently in water at 40� C for 1-2 min. spread cells on solid medium for regrowth

6. Germplasm Preservation � In Vitro Approaches Methods involving LN vitrification use of highly conc. solution of a cryoprotectant that supercools to v. low temps upon imposition of rapid cooling rates (and solidification w/o ice formation) caution � high conc. cryoprotectants can be toxic procedure for Brassica campestris equilibration of cells at 0� C with 1.5 M ethylene glycol (EG)

7. Germplasm Preservation � In Vitro Approaches Methods involving LN vitrification procedure for Brassica campestris equilibration of cells at 0� C with 1.5 M ethylene glycol (EG) dehydration in 7.0 M EG + 0.88 M sorbitol + 6% (w/v) bovine serum albumin (BSA) transfer of cell suspension into 0.5 ml propylene straw and quenching in LN thaw in air 10 s, then 10 s in alc. bath @ 20� C

8. Germplasm Preservation � In Vitro Approaches Methods involving LN simple freezing method eliminates programmable freezer and use of DMSO as cryoprotectant procedure for Citrus: cells placed in 2 M glycerol and 0.4 M sucrose 10 s at 25� C 0.2 ml sample is loaded into a 0.5 ml straw and placed in LN

9. Germplasm Preservation � In Vitro Approaches Methods involving LN simple freezing method procedure for Citrus: thawing is by placing straws into 40� C water bath after thawing, cells expelled into 2 ml diluent containing 1.2 M sucrose in nutrient medium at 25� C encapsulation-dehydration

10. Germplasm Preservation � In Vitro Approaches Methods involving LN encapsulation-dehydration used for storing encapsulated somatic embryos in Ca-alginate beads procedure for carrot SEs encapsulated SEs precultured in medium with 0.3 to 0.75 M sucrose dehydration in lam. flow hood 2-6 h followed by quenching in LN

11. Germplasm Preservation � In Vitro Approaches Methods involving LN cryoselection callus of non-hardy spring wheat frozen in LN w/o cryoprotectants calli that survived and grew were regenerated when rechallenged w/LN, the selected callus lines survived at much higher rates seed progeny of some lines exhibited enhanced tolerance to freezing

12. Germplasm Preservation � In Vitro Approaches Cold storage storage at non-freezing temps, from 1-9� C dep. on spp. storage of shoot cultures (stage I or II) works well for strawberries, potatoes, grapes, prob. many more spp. transferred (to fresh medium) every 6 mo. or on a yearly basis

13. Germplasm Preservation � In Vitro Approaches Cold storage advantages � simple, high rates of survival, useful for micropropagation (esp. in periods of low demand) disadvantages may not be suitable for tropical, subtropical spp because of susceptibility of these to chill injury alternative w/coffee � shoot cultures transferred to a medium w/reduced nutrients and lacking sucrose requires refrigeration, which is more expensive than storage in LN