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 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 clonally maintained germplasm Primary site Field, greenhouse, laboratory/growth chamber Back-up Field in another location, growth chamber in another location, cryopreservation

Cryopreservation Requires species/genotype specific protocols Time and labor consuming Costly in initial stages HOWEVER, once in LN 2 - cost of long-term storage is low Relatively safe back-up

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

Collaboration with Curators is CRITICAL for 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

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

Candidate collections for DB cryopreservation 1.Large number of accessions (>40) 2.At risk 3.Temperate or cold zone origin

TC protocols under lab adjustment/learning Pycnanthemum (Virginia mountain-mint) Vaccinium (highbush blueberry) TC High cost to establish and place in LN 2 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 germplasm maintained clonally (Dec. 2009) Species Common name Expl. No. of accession s A. sativum garlicam89 Fragariastrawberryam111 Humulushopam55 Ipomoea sweet potato am59 Malusappledb2,545 Menthamintam43* Musabananaam4 Prunus sour cherry db74 Pyruspearam170* Ribesgooseberryam75* Rubusraspberryam122* Salixwillowdb24 Solanum PVP potatoam100 Vacciniumblueberryam3 am-apical meristem; db-dormant bud; (+number-viability test not completed);* cryopreservation done wholly or in part by CorvallisSpecies Common name Expl. No. of accession s Corylus sp. hazelnutam4* Cydoniaquinceam1* Cynodongrassam24* Loliumryegrassam15* Zoysia zoysia (grass) am5* Total accessions 3,523

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

NCGRP, Fort Collins, CO Thank you We are looking forward to collaborating with YOU