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Title: Assessing Variability Between Accessions, Cryovials and Over Time of Cryopreserved Shoot Tips

Author
item Ellis, David
item Ambruzs, Barbara - Bobbie Ambruzs
item Holman, Gregory
item Staats, Elise
item Jenderek, Maria

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/1/2009
Publication Date: 4/20/2009
Citation: Ellis, D.D., Ambruzs, B.D., Holman, G.E., Staats, E.R., Jenderek, M.M. 2009. Assessing Variability Between Accessions, Cryovials and Over Time of Cryopreserved Shoot Tips. I International Symposium on Cryopreservation of Horticultural Species, April 5-8, 2009, Leuven, Belgium. pp. 40. Meeting abstract.

Interpretive Summary: Cryopreservation in liquid nitrogen (-320oF) is used to store plant tissue long-term. A common method is to cryopreserve a tiny section of the plant containing a living shoot tip. Due in part to size, these shoot tips are not stored as single entities, but rather as multiple shoot tips in a single container or vial. As is can take months to regenerate a plant from cryopreserved material, one needs to know the survival rate of the shoot tips and if removing a single vial is enough to obtain a living plant. This study looked at the variability in the survival of shoot tips in each cryopreserved vial of strawberry, current, blackberry and pear cryopreserved shoot tips. Overall variability was low supporting the notion that a single vial can be pulled with confidence to obtain at least one plant. The next question involves the stability of the shoot tips over time in cryopreservation. To access this, an experiment was set up with multiple vials which will be harvested over the next 60-110 years. Settings up experiments such as these are critical so that future scientists have material to work with to access stability of plant collections in such long-term storage conditions.

Technical Abstract: The development of a long-term cryopreservation program for plant genetic resources relies heavily on the ability to predictably reproduce plants from cryopreserved explants. Since plant explants are not generally cryostored as single entities, but rather as multiple explants in vials or tubes, this predictability is dependent on reliably being able to have at least one live explant derived from each unit (vial or tube) stored or at minimum to know how many units are needed to obtain at least one live explant after thawing and regeneration. Further, the program needs to ensure that this predicted number of vials or tubes does not change over time, or if it does change, that the program can adjust where needed the number of cryopreserved units pulled to ensure a live explant. To address the issue of how many cryopreserved units are needed to obtain a single living explant, variability between individual cryopreserved vials was accessed in cryopreserved shoot tips of Fragaria, Ribes, Rubus and Pyrus. For each species three accessions were used with 100 shoot tips distributed into 10 cryovials (10 shoot tips per cryovial) and replicated three times (300 shoot tips per accession). Overall, all vials yielded viable shoot tips, with Pyrus and Rubus showing the greatest variability in viable shoots per vial (100-50% and 100-20% respectively). These results suggest that for these species one vial can be safely pulled when needed to yield at least one viable shoot tip. To access changes in the viability of cryopreserved shoot tips over time, a set of 40 vials (10 shoot tips per vial) were set up for each of the three accessions of the species used in the cryovial variability study above. Based on the relatively low variability between cryovials, it was determined that accessing viability in 5 cryovials at each time point would suffice to determine if viability losses were occurring over time. As expected, initial shoot tip viability from the cryovials set up for this long-term study were similar to those in the cryovial variability study above. We expect to pull five vials/accession from cryopreservation after 1 year and then at each 10-20 year interval. If pulled every 10 years, the final cryovial will be accessed in 2068 (60 years from the start of the experiment) and if pulled every 20 years, the final cryovial will be accessed in 2118 (110 years after the start or the experiment).