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