Germplasm Preservation In Vitro Approaches

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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.

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Germplasm Preservation In Vitro Approaches

• Potential advantages of in vitro methods
• little space needs
• plants are free of pests, pathogens and viruses
  (and will remain so)
• no transfer labor (under storage conditions)
• stored cultures can be used as nuclear stock for
  vegetative preservation
• international shipping restrictions are lessened
• no soil
• pest-free plants

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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)

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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

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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

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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)

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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 _at_ 20 C

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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

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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

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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

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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

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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

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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