Cryopreservation of Clonally Cryopreservation of Clonally Maintained GermplasmMaintained Germplasm
Preparation and Viability testingPreparation and Viability testing
USDA-ARS, National Center for Genetic Resources Preservation, USDA-ARS, National Center for Genetic Resources Preservation,
Fort Collins, COFort Collins, CO
Maintenance of clonal germplasmMaintenance 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
Preserving and backing-up of Preserving and backing-up of clonally maintained germplasmclonally maintained germplasm
Primary site
•Field, greenhouse, laboratory/growth chamber
Back-up
•Field in another location, growth chamber in another location, cryopreservation
CryopreservationCryopreservation
•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
What is needed to place clonal germplasm What is needed to place clonal germplasm in long-term LNin long-term LN22 storage at the NCGRP storage at the NCGRP
1. Procedures tested and modified (as needed) at the Center
2. Collaboration with Curators
Collaboration with Curators is Collaboration with Curators is CRITICALCRITICAL for for successsuccess
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
Dormant buds (DB)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
Species under protocol development in Species under protocol development in the Preservation Program (DB)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)
Example of material needed - DBExample 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)
Candidate collections for DB Candidate collections for DB cryopreservationcryopreservation
1. Large number of accessions (>40)
2. At risk
3. Temperate or cold zone origin
TC protocols under lab adjustment/learningTC 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 (+)
Status of long-term cryopreserved Status of long-term cryopreserved germplasm maintained clonally germplasm maintained clonally (Dec. 2009)(Dec. 2009)
SpeciesSpecies Common Common namename
Expl.Expl. No. of No. of accessionaccessionss
A. sativumA. sativum garlicgarlic amam 8989
FragariaFragaria strawberrystrawberry amam 111111
HumulusHumulus hophop amam 5555
IpomoeaIpomoea sweet sweet potatopotato
amam 5959
MalusMalus appleapple dbdb 2,5452,545
MenthaMentha mintmint amam 43*43*
MusaMusa bananabanana amam 44
PrunusPrunus sour cherrysour cherry dbdb 7474
PyrusPyrus pearpear amam 170*170*
RibesRibes gooseberrygooseberry amam 75*75*
RubusRubus raspberryraspberry amam 122*122*
SalixSalix willowwillow dbdb 2424
Solanum Solanum PVPPVP
potatopotato amam 100100
VacciniumVaccinium blueberryblueberry amam 33
am-apical meristem; db-dormant bud; (+number-viability test not completed);* cryopreservation done wholly or in part by Corvallis
SpeciesSpecies Common Common namename
Expl.Expl. No. of No. of accessionaccessionss
Corylus sp.Corylus sp. hazelnuthazelnut amam 4*4*
CydoniaCydonia quincequince amam 1*1*
CynodonCynodon grassgrass amam 24*24*
LoliumLolium ryegrassryegrass amam 15*15*
ZoysiaZoysia zoysia zoysia (grass)(grass)
amam 5*5*
Total Total accessionsaccessions
3,5233,523
DB vs. TC preserved accessionsDB vs. TC preserved accessions
NCGRP: 3,523 accessions in cryo
DB: 2,643 accessions (75%)
TC: 880 accessions (25%)
NCGRP, Fort Collins, CONCGRP, Fort Collins, CO
Thank you
Thank you
We are looking forward to
We are looking forward to
collaborating with
collaborating with YOUYOU