Variability Between Vials of Cryopreserved Shoot Tips from Tissue Cultured Plants

Authors: Ellis, David; Holman, Gregory; Staats, Elise; Ambruzs, Barbara - Bobbie Ambruzs; Jenderek, Maria

Submitted to: Society for In Vitro Biology Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 3/30/2007
Publication Date: 6/9/2007
Citation: Ellis, D.D., Holman, G.E., Staats, E.R., Ambruzs, B.D., Jenderek, M.M. 2007. Variability Between Vials of Cryopreserved Shoot Tips from Tissue Cultured Plants. Society for In Vitro Biology Proceedings. June 9-13, 2007. Indianapolis, Indiana. pp.5-6. Meeting Abstract.

Interpretive Summary: The safe long-term storage of genetic resources is critical to ensure the diversity needed for future crop improvement. The principal method for the long-term storage of plant genetic resources is by the storage of seed in a freezer. Unfortunately, many important crops are vegetatively-propagated and therefore storage of seed does necessarily maintain the genetic resource. Examples of crops which are vegetatively-propagated include apple, grapes, strawberries, currants, blackberries, garlic and potatoes. The long-term storage genetic resources of value to these crops is often done through field plantings which are vulnerable to drought, disease, insects, flooding, fires and changing environmental conditions. One method to store large collections of vegetatively-propagated crops is by freezing, at ultra-low temperatures (cryopreservation), the growing shoot tip of these crops. At the National Center for Genetic Resources Preservation cryopreservation is being used to safely store a wide variety important crop species. However, to be of use, the cryopreserved material must be able to be regenerated into a whole plant. This study looks at the variability in survival of the shoot tips placed in cryopreservation from three different crops. The information gained from this work will aid in the development of protocols for the long-term storage of vegetatively-propagated crops.

Technical Abstract: At the USDA-ARS National Center for Genetic Resources Preservation, cryopreservation is used as a method of long-term storage of valuable plant genetic resources from vegetatively-propagated crop plants. The main germplasm collections for vegetatively-propagated crops are maintained as field plantings, with some also maintained as tissue culture collections. While the field plantings serve as a great source of propagules for distribution of germplasm, they are acutely vulnerable to loss through both biotic and abiotic factors. Tissue culture collections are expensive to maintain and are vulnerable to equipment malfunctions as well as contamination. The loss of any of this germplasm can be catastrophic even if representatives still exist in the wild or other collections, since importation to the United States to replace the germplasm may not be possible. Therefore cryopreservation of these valuable genetic resources offers a secure and safe replicate of the collection. Already, cryopreserved material has been used to replenish Malus accessions lost in field plantings due to disease. To be of practical use, duplicate back-up collections need to be readily available and replacement of field materials should not deplete the duplicates stored in the cryobank. One question that arises when creating a cryopreservation bank to duplicate field material is how many shoot tips are needed for long-term back-up and how many vials should be pulled to ensure the regeneration of a healthy plant. We, therefore, have investigated vial-to-vial variability in viable Ribes, Fragaria and Rubus shoot tips stored in liquid nitrogen to provide baseline information in the design of protocols for a cryopreservation genebank. In Ribes, viability of cryopreserved shoot tips was very high, >80% and vial-to-vial variation in viability was extremely low. In contrast, vial-to-vial shoot tip viability was much more variable in both Fragaria and Rubus, although the variability was still within acceptable ranges for cryobank management. These studies clearly demonstrate that with ten shoot tips/vial and a minimum acceptable viability of 40%, single vials can safely be pulled to ensure success in producing a viable plant for replacement of field plantings, leaving remaining vials in crypreservation for future needs.