Cryopreservation using the Dormant-bud technique

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 • Grape 1204 accessions • Sour Cherry93 accessions • Total 5230 accessions

3. Sour Cherry – 90 accessions Grape – 1200 accessions P. Forsline – Acting Research Leader (Curator) 1.0 FTE C. Simon – Geneticist (Molecular Characterization) 1.0 FTE A. Baldo – Computational Biologist 0.4 FTE Apple – 4000 accessions (2500 clones and 1500 seedlots from wild) 4000 wild Malus seedlings from Kazakhstan, Russia, China & Turkey

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 Control 85.3 aZ Storage one month 63.0 b Storage one year 64.2 b Storage two years 66.5 b Storage four years 68.6 b Storage after eightYyears 68.3 b ZSeparation 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 cryopreservationof Malusaccessions at NCGRP following the 4-yr pilot project 1988-1992

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

9. 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 1760 or 91% were successful 171 or 9% of total were unsuccessful – mostly those with low cold-hardiness No. of accessions in storage 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?