Cryopreservation of dormant buds has potential to provide back-up conservation of vegetatively propagated genetic resources for fruit crop species. This system may be useful where clonal integrity must be maintained and where it is desirable to rapidly recover plants with flowers for crossing purposes. In 1988, a pilot project involving the National Clonal Apple Repository at Geneva, NY and the National Seed Storage Laboratory, Fort Collins, CO, was initiated to test handling protocols as a prelude to establishing a cryopreservation backup system for apple genetic resources. Sufficient buds have been cryopreserved to permit viability evaluation after 1 month, 1, 2, 3, 4, 5, 10, 15, 20, and 25 years storage in liquid nitrogen vapor phase storage (-150 C]. Recovery of dormant buds collected 12/12/88 and 02/06/89 after one month in LN2 was 36% and 35%, respectively, for eight different taxa. After one year in LN2, recovery was 50% and 48% for the same taxa. The difference was attributed to improved handling during dehydration prior to patch budding for viability estimation. In 1990, recovery after 1 month in LN2 was 38% for six different cultivars. The response to controlled acclimation and desiccation for 15 taxa will be presented.
Cecil Stushnoff, Philip L. Forsline, Leigh Towill, and John Waddell
Leigh E. Towill, John W. Waddell, and Philip L. Forsline
Three years ago we established a long-term cryogenic storage project for apple germplasm and utilized grafting of buds obtained from stored dormant shoot sections as the major viability assay. Grafting, however, is time consuming and requires considerable skill. Electrolyte leakage and oxidative browning tests were used as alternative viability assays. Using leakage from individual buds in a multiwell analyzer, we examined modifications of the electrolyte leakage test and analyzed the kinetics of leakage in an attempt to determine whether the test can predict grafting success. The results suggest that more buds were viable than were estimated by the grafting test. In vitro culture is being examined to test this and to determine if practical recovery is feasible for diversity within the germplasm collection.
Gayle M. Volk, John Waddell, Leigh Towill, and L.J. Grauke
Pecan [Carya illinoinensis (Wangenh.) K. Koch] trees native to northern regions are more cold-tolerant than those native to and grown in the southern United States. To identify a possible assay for cold hardiness, dormant winter twigs from 112 diverse pecan cultivars grown in Texas were surveyed using differential thermal analyses (DTA). The low temperature exotherm (LTE) from DTA was identifiable when twigs were stored at –3 °C for up to 120 d after harvest. Thirty-nine percent of the southern pecan cultivars lacked an obvious LTE, and the remaining southern cultivars had an average LTE of –32.9 °C. In contrast, only 11% of the northern pecan cultivars lacked the LTE and the remaining cultivars had a significantly lower LTE of –35.4 °C. Because twig samples were collected from trees grown in the same Texas orchard, it is suggested that there is a genetic component that affects the temperature of the LTE. Budbreak generally occurred earlier in southern cultivars than those that originated in the north. Both budbreak and LTE data can be correlated with regional origin; timing of budbreak may be preferred over DTA to predict relative cold hardiness in pecan.
Philip L. Forsline, Leigh E. Towill, John Waddell, and Loren Wiesner
The USDA/ARS collection of Malus is held by the Plant Genetic Resources Unit in Geneva, N.Y. The collection comprises ≈2500 accessions, most of which must be maintained as clones in the field to provide propagating material for distribution to the user community. Field maintenance of replicated accessions places the collection at risk from weather extremes, pests, diseases, etc. and is extremely costly. Cryopreservation of dormant buds in a base, or backup, collection could reduce risks and decrease maintenance costs. Since 1988, we have developed and implemented protocols to cryopreserve dormant apple buds at the National Seed Storage Laboratory, Fort Collins, Colo. More than 500 accessions have been placed in cryogenic storage. Buds have been successfully recovered by grafting from >70% of the first 250 accessions cryopreserved. These results, and those from ongoing recovery tests, indicate cryopreservation may be a safe, cost-effective approach to back up collections of tree fruit germplasm. It also may be used to enhance management of the active collections of Malus, Vitis, and Prunus at Geneva.
Philip L. Forsline, Leigh E. Towill, John W. Waddell, Cecil Stushnoff, Warren F. Lamboy, and James R. McFerson
Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.