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Cryopreservation of Clonally Maintained Germplasm Preparation and Viability testing

Cryopreservation of Clonally Maintained Germplasm Preparation and Viability testing. USDA-ARS, National Center for Genetic Resources Preservation, Fort Collins, CO. Maintenance of clonal germplasm. More vulnerable than seed-based accessions Requires more space

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Cryopreservation of Clonally Maintained Germplasm Preparation and Viability testing

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  1. Cryopreservation of Clonally Maintained GermplasmPreparation and Viability testing USDA-ARS, National Center for Genetic Resources Preservation, Fort Collins, CO

  2. Maintenance of clonal germplasm • More vulnerable than seed-based accessions • Requires more space • Requires special procedures and/or techniques • Easily compromised and challenged (biotic + abiotic factors) • Can require years to get established • Distribution issues • Yearly costs

  3. Preserving and backing-up of clonally maintained germplasm • Primary site • Field, greenhouse, laboratory/growth chamber • Back-up • Field in another location, growth chamber in another location, cryopreservation

  4. Cryopreservation • Requires species/genotype specific protocols • Time and labor consuming • Costly in initial stages • HOWEVER, once in LN2 - cost of long-term storage is low • Relatively safe back-up

  5. What is needed to place clonal germplasm in long-term LN2 storage at the NCGRP 1. Procedures tested and modified (as needed) at the Center 2. Collaboration with Curators

  6. Collaboration with Curators is CRITICALfor success • 1. Decide which taxa are priority • 2. Providing material/germplasm to be processed • Tissue culture, dormant budwood (DB), field derived material (i.e. garlic) • 3. If DB are used – viability testing (grafting or rooting) • During experimental protocol adjustments • During routine cryopreservation

  7. Dormant buds (DB) • Advantages: • Large number of accessions able to process in one season • Short (relatively) time from cryo to establishing a usable plant • Limitations: • Applicable only to woody and semi-woody accessions • Limited number of reliable cryo-procedures (apple, sour cherry) • Inconsistencies in viability testing/grafting

  8. Species under protocol development in the Preservation Program (DB) • Juglans • black and English/Carpathian walnut; butternut • collaboration with UC Davis, Burchell Nursery, Forest Service, UM/MO • Prunus • almond, apricot, peach, sweet cherry • collaboration with Davis repository, Burchell Nursery • Pyrus, Ribes, Vaccinium • pear, black and red currant, highbush, lowbush and rabbiteye blueberry • collaboration with Corvallis repository • Salix • collaboration with Ames (some willow accessions)

  9. Example of material needed - DB • -Experimental stage (3 rep.; 10 twig segments/rep.; 120+ segments/accession) • Controls • Not-processed • Desiccated • Desiccated and slow cooled • Pre-treatment conditioning – if any needed • LN2 exposed • -Routine cryostorage (140 segments/accession) • Control (2 rep.; 10 segments/rep) • LN2 (~ 100 segments) • Variability/longevity tests (standardization)

  10. Candidate collections for DB cryopreservation • Large number of accessions (>40) • At risk • Temperate or cold zone origin

  11. TC protocols under lab adjustment/learning • Pycnanthemum (Virginia mountain-mint) • Vaccinium (highbush blueberry) • TC • High cost to establish and place in LN2 • Low number of accessions processed in time (ca. 40 accessions/technician/year) • Require usually a long time from cryo to establishing a usable plant • In-house viability testing (+)

  12. Status of long-term cryopreserved germplasm maintained clonally (Dec. 2009) am-apical meristem; db-dormant bud; (+number-viability test not completed);* cryopreservation done wholly or in part by Corvallis

  13. DB vs. TC preserved accessions • NCGRP: 3,523 accessions in cryo • DB: 2,643 accessions (75%) • TC: 880 accessions (25%)

  14. NCGRP, Fort Collins, CO Thank you We are looking forward to collaborating withYOU

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