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.

Vegetatively-propagated crops at USDA, ARS PGRU, Geneva, NY

Apple 3993 accessions Grape 1204 accessions Sour Cherry 93 accessions Total 5230 accessions

Sour Cherry – 90 accessions Grape – 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 FTEC. Simon – Geneticist (Molecular Characterization) 1.0 FTEA. Baldo – Computational Biologist 0.4 FTE

Apple cryopreservation - processing

Apple bud recovery and rescue

Bud recovery tests in GH

Rescue accessionsdirectly in field nursery

Test 2 common cultivars for normal morphology

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 eightY years 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 test in process 2004 to 2007

Annual cryopreservation of Malus accessions at NCGRP following the 4-yr pilot project 1988-1992

Year M. x domestica Hybrids Other species Total1993 86 8 5 981994 108 7 26 1461995 167 25 50 2421996 169 24 49 2421997 223 71 70 3641998 209 66 64 3391999 120 55 71 2462000 108 46 84 2382003 52 6 56 1142004 22 2 97 1212005 9 2 26 37Total 1273 312 598 2187

Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed

0

100

200

300

400

500

600

700

Categories of success based on bud recovery via grafting

0-10 %20-30 %40-50 %60-70 %80-90 %100%

No.

of a

cces

sion

s in

sto

rage

171 or 9% of totalwere unsuccessful – mostly those with low cold-hardiness

1760 or 91% were successful

0-10 20-30 40-50 60-70 80-90 100

Accession ‘95 ’96 ’97 ’98 ’99 ‘00 ’03 ‘04

Bisbee 90 65 65 45 25 96 NA NA

Burgundy 95 100 100 85 55 95 71 70

Demir 75 80 80 40 20 100 67 80

Ein Shemer 80 85 85 30 55 100 12 50

Empire 90 100 100 85 60 95 82 90

G. Delicious 95 90 90 0 50 90 44 75

M. florentina 20 0 0 0 0 0 0 NA

Novosibirski Sweet 95 100 100 90 50 100 14 90

Viability of Standards that are processed each year that a batch is completed

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

Successful cryopresevation for > 90% of accessions stored at NCGRP: those with < 30% viability will be reprocessed

0

100

200

300

400

500

600

700

Categories of success based on bud recovery via grafting

0-10 %20-30 %40-50 %60-70 %80-90 %100%

No.

of a

cces

sion

s in

sto

rage

171 or 9% of totalwere unsuccessful – mostly those with low cold-hardiness

1760 or 91% were successful

0-10 20-30 40-50 60-70 80-90 100

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.

Sour Cherry cryopreservation

Research on cryopreservation of dormant grape buds

Questions

• Cost savings using cryo as a back up?• Other species for dormant bud technique?

• Particularly Prunus other than sour cherry?• Grape?• Corvallis methods?