1. Cryopreservation using the Dormant- bud technique Forsline, P.L., C. Stushnoff, L.E. Towill, J.W. Waddell, W.F. Lamboy and J. R. McFerson. 1998. Recovery and longevity of cryopreserved apple buds. J. Amer. Soc. Hort. Sci. 123:365-370. Towill, L.E. and P.L. Forsline. 1999. Cryopreservation of sour cherry (Prunus cerasus L.) using a dormant vegetative bud method. Cryoletters 20:215-222. Seufferheld, M.J., C. Stushnoff, P.L. Forsline, and G.H.T.Gonzalez. 1999. Cryopreservation of cold ‑ tender apple germplasm. J. Am. Soc. Hort. Sci. 124:612-618. Forsline, P.L., J. R. McFerson, W.F. Lamboy and L.E. Towill. 1999. Development of base and active collections of Malus germplasm with cryopreserved dormant buds. EUCARPIA Fruit Breeding Section Meeting, Oxford, England. Acta Horticulturae 484:75-78. Towill, L.E., P.L. Forsline, C. Walters, J. Waddell and J. Laufman. 2004. Cryopreservation of Malus germplasm using a winter vegetative bud method: Results from 1915 accessions. Cryoletters 25:323-334.

2. Vegetatively-propagated crops at USDA, ARS PGRU, Geneva, NY  Apple 3993 accessions  Grape1204 accessions  Sour Cherry 93 accessions  Total5230 accessions

3. Sour Cherry – 90 accessionsGrape – 1200 accessions Apple – 4000 accessions (2500 clones and 1500 seedlots from wild) 4000 wild Malus seedlings from Kazakhstan, Russia, China & Turkey P. Forsline – Acting Research Leader (Curator)1.0 FTE C. Simon – Geneticist (Molecular Characterization)1.0 FTE A. Baldo – Computational Biologist0.4 FTE

4. Apple cryopreservation - processing

5. Apple bud recovery and rescue Bud recovery tests in GH Rescue accessions directly in field nursery Test 2 common cultivars for normal morphology

6. Pilot project to determine protocol for cryogenic storage of Malus dormant buds: % bud recovery of 84 apple accessions (processed 1989-1992); tested after up to 8 years of storage in liquid nitrogen Treatment Recovery % Desiccated Control85.3 a Z Storage one month63.0 b Storage one year64.2 b Storage two years66.5 b Storage four years68.6 b Storage after eight Y years68.3 b Z Separation of grand means of 84 accessions at P < 0.01 by test for differences between two proportions (LSD = 7.2) Y Fifteen year testin process 2004 to 2007

7. Annual cryopreservation of Malus accessions at NCGRP following the 4-yr pilot project 1988-1992 YearM. x domesticaHybridsOther speciesTotal 1993868598 1994108726146 19951672550242 19961692449242 19972237170364 19982096664339 19991205571246 20001084684238 200352656114 200422297121 2005922637 Total12733125982187

8. Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed No. of accessions in storage 171 or 9% of total were unsuccessful – mostly those with low cold- hardiness 1760 or 91% were successful 0-10 20-30 40-50 60-70 80-90 100

9. Accession‘95’96’97’98’99‘00’03‘04 Bisbee9065 452596NA Burgundy95100 8555957170 Demir7580 40201006780 Ein Shemer8085 30551001250 Empire90100 8560958290 G. Delicious9590 050904475 M. florentina20000000NA Novosibirski Sweet95100 90501001490 Viability of Standards that are processed each year that a batch is completed

10. 11 year test of accessions processed in 1994 146 accessions processed Grand means = 70.2% in 1994 vs. 76.4 % in 2005 73 of 146 higher in 2005 than 1994 23 of 146 same in 2004 and 1994 50 of 146 higher in 1994 than 2005

11. Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed No. of accessions in storage 171 or 9% of total were unsuccessful – mostly those with low cold- hardiness 1760 or 91% were successful 0-10 20-30 40-50 60-70 80-90 100

12. Reprocessing low viability accessions Leigh, Andrea and Dave, We have the results from the 103 accessions with low viability that were reprocessed in 2004. As expected, a significant portion of these (64 accessions) performed better the second time around (now > 40%). The remainder (39 accessions) remained < 30 Explanation: You will notice that very few of the accessions in the < 30% are M. domestica. It is apparent that some of the Malus species are difficult. M. fusca - west coast NA and M. angustifolia - SE USA continue to have low viability. In fact 10 accessions had '0' viability both times processed; 1/2 of those were either M. fusca or M. angustifolia. However, 2 exceptions of those 2 species are noted in the 90% viable status (PI 590039, M. fusca and PI 613882 M. angustifolia). Maybe there is some hope with those that are still low.

13. Sour Cherry cryopreservation

14. Research on cryopreservation of dormant grape buds

15. Questions Cost savings using cryo as a back up? Other species for dormant bud technique? Particularly Prunus other than sour cherry? Grape? Corvallis methods?