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Manfredo J. Seufferheld, Cecil Stushnoff and Philip Forsline

Cryopreservation of mature dormant vegetative buds is a useful method to preserve germplasm of a large number of cold hardy apple cultivars. However, cold tender cultivars have proven to be much more difficult to cryopreserve. Eight cultivars were harvested in September 1993 at Geneva, NY before developing cold hardiness naturally. The twigs were encapsulated with 5% alginate and treated with stepwise imbibition of 0.5 to 1.0 M sucrose. The samples were desiccated over glycerol at 0C. Half of the samples were plunged directly into liquid nitrogen (IN) and the other half were first cooled slowly to -30C. The twigs that had been exposed to prefreezing conditions showed the highest survival (20 to 100%). The samples that were plunged directly in LN survived poorly (0 to 20 %). Samples without encapsulation and no sucrose imbibition had 0% survival. We conclude that this protocol opens up the possibility to expand cryopreservation of cold tender apple cultivars, presently grown only in field gene banks, at great expense and inconvenience.

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Philip L. Forsline and Kim E. Hummer

The National Plant Germplasm System (NPGS) of the U.S. Department of Agriculture (UDSA), Agricultural Research Service (ARS), has greatly expanded since 1980. Foremost in this expansion was the addition of seven repositories for clonally propagated fruit and specialty crops. Many collections at state agricultural experiment station sites were in jeopardy as breeders retired. These collections can now be preserved by the NPGS. The NPGS has provided funding for plant exploration and exchange. From 1980 to 2004, 37 exploration/exchange proposals for fruit crops were funded, and over 3000 accessions introduced as a result. Crop Germplasm Committees (CGCs), established for each commodity have prepared genetic vulnerability statements and prioritized collection activities. The USDA ARS, National Germplasm Resources Laboratory (NGRL), facilitates international relationships, and the USDA Animal and Plant Health Inspection Service (APHIS), National Plant Germplasm Quarantine Center (NPGQC), tests and makes pathogen-tested germplasm available. As a result of the Convention on Biological Diversity (1993) and the International Treaty on Plant Genetic Resource for Food and Agriculture (2004), the USDA now pursues germplasm collection through the establishment of bilateral agreements of mutual benefit.

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Virgil Esensee, Cecil Stushnoff and Philip L. Forsline

There is need for backup storage of clonally propagated plant cultivars of numerous taxa. Initial tests, using a protocol developed for dormant apple buds that includes desiccation and slow freezing prior to immersion in liquid nitrogen (-196 C), was not effective with `Valiant' grape. Accordingly, replicates of V. vinifera `Riesling', V. riparia, `Valiant' and a V. amurensis × riparia cross were also tested for survival at –196 C, following desiccation to 25% & 18% water (fwb) and direct immersion into liquid nitrogan. Visual and electrolyte leakage ratings following nine days of dehydration in moist peat were used to assess viability. Direct immersion of desiccated samples resulted in survival for some buds of `Valiant' and a V. amurensis × riparia cross. V. riparia showed some survival when field hydrated and at 25% water, while all buds desiccated to 18% survived. `Riesling' did not survive desiccation, and was killed by all -196 C treatments. The apple protocol was partially effective, in combination with desiccation to 18% in `Valiant' and V. riparia. This is the first report of grape bud survival in liquid nitrogen and more detailed studies are planned.

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Leigh E. Towill and Philip L. Forsline

The dormant vegetative bud method for cryopreservation has been successfully applied to many lines of apple. We examined this method for five cultivars (Kentish, Montmorency, Meteor, North Star, Schatten Morelle) of sour cherry (Prunus cerasus L.) with the aim of developing long-term storage at NSSL. Singlebud nodal sections (35 cm) were desiccated to 25%, 30%, or 35% moisture before cooling at 1°C/hour to –30°C and holding for 24 hours. Sections were then directly placed in storage in the vapor phase above liquid nitrogen (about – 160°C). Warmed samples were rehydrated and patch budded at Geneva to assess viability. Sections that were either undried, dried but unfrozen, or dried and cooled to –30°C survived very well. For samples then cooled to –160°C, highest viabilities for each line occurred with the 25% moisture level, although fairly high viabilities also were observed at 30% and 35% moistures. Cryopreserved buds from four lines directly developed into a single shoot; buds from Montmorency formed a shoot from a lateral within the bud, suggesting that the terminal meristem died but that axillary meristems within the bud survived and formed a shoot or multiple shoots. Nineteen lines were harvested in January 1996 for long term storage of sour cherry germplasm under cryogenic conditions.

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Manfredo J. Seufferheld, Cecil Stushnoff, John Fitzpatrick and Philip L. Forsline

Cryopreservation of woody-plant, dormant buds may provide cost-effective, long-term, back-up conservation of germplasm for vegetatively propagated crops that are presently maintained as trees in field gene banks. Dormant buds can be recovered quickly by grafting to dwarfing rootstocks, thus producing flowers for breeding purposes, with minimum potential for inducing somaclonal variants. These attributes are essential to preserve the clonal integrity of unique gene combinations such as those found in tree fruit crops. Previous research has shown that dormant buds from cold-hardy apples can be recovered from storage in liquid nitrogen (LN) with high survival rates (80% to 100%) using controlled desiccation and slow freezing before immersing in LN. On the other hand, dormant buds from cold-tender taxa and buds collected at less than optimal stages for desiccation and freezing have much lower (0% to 50%) survival rates. We increased survival of cold-tender taxa by using a modified vitrification procedure. Dormant apple buds from tender and hardy cultivars were perfused with modified PVS [15% (w/v) ethylene glycol, 15% (w/v) propylene glycol, 7% (w/v) DMSO, and 15% (w/v) glycerol in 0.5 m sorbitol]. Toxicity from the PVS was reduced by dilution soaking in 1 m sorbitol, 0.2 m raffinose, and 15 mm CaCl2 before and after quench-freezing and slow-freezing cryopreservation. Up to 100% of some cold-tender taxa survived. In addition, xylem ray parenchyma tissues that supercool and are normally killed at about -40C with the desiccation protocol survived this vitrification procedure.

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Cecil Stushnoff, Philip L. Forsline, Leigh Towill and John Waddell

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.

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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.

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Philip L. Forsline, E.E Dickson and A.D. Djangaliev

The USDA National Plant Germplasm System (NPGS) sponsored a 1993 collection of wild Malus in Kazakhstan and Kyrgyzstan which followed a collection in 1989 from sites in Tajikistan and Uzbekistan. There is strong evidence that the domestic apple originated in the foothills of the Tian Shari mountains where Malus sieversii (Ldb.) M. Roem remains as a primary forest species. The goal of the recent expedition was to obtain additional genetic diversity of apple from some of the remote sites in that area with the assistance of the Kazakh hosts. While there, isolated pockets of other fruit in the wild (especially Vitis) were discovered and collected. Seed collections from the expedition are stored with the NPGS and seedling populations are being evaluated for valuable traits.

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Philip L. Forsline, Margie Luffman, John Warner and Leigh E. Towill

Based on protocols developed by the Plant Genetic Resources Unit (PGRU), Geneva, NY and the National Seed Storage Laboratory, Fort Collins, Colo., nearly 40% of the 2500-accession USDA–ARS Malus germplasm collection has been preserved cryogenically. Recent program changes require the entire Canadian Malus collection of 700 accessions at the Canadian Clonal Genebank, Trenton, Ont., be moved to a new location in Harrow, Ont., by the end of 1996. This provided an opportunity to utilize cryogenic storage during repropagation and reestablishment to develop a security backup for the collection. In a cooperative experiment, dormant buds of four Canadian Malus accessions were collected in Trenton and cryopreserved in Geneva in February 1995. Field-level moisture of dormant buds ranged from 45% to 50%. Three levels of bud desiccation were tested: 25%, 30% (current standard), and 35%. The desiccated buds were containerized and slowly frozen to –30°C, plunged into liquid nitrogen, and held for one month at Geneva prior to recovery testing by bud-grafting at Geneva and Trenton. Results were identical at both sites. We obtained 60% recovery at 30% and 35% moisture levels and 80% recovery at 25% moisture across all four accessions. Further studies on a broader range of germplasm will determine if desiccation to the 25% level is superior to the 30% level. Meanwhile, we have initiated a cooperative project to cryopreserve 350 accessions unique to the Canadian collection at Ft. Collins.

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Philip L. Forsline, James R. McFerson and Warren F. Lamboy

The USDA–ARS active collection of Malus includes over 2500 accessions maintained as field-grown trees at the Plant Genetic Resources Unit (PGRU), Geneva, N.Y. Nearly 30% of this collection is presently cryopreserved as dormant buds at the National Seed Storage Laboratory, Fort Collins, Colo., as a backup security collection. Successful bud-grafting recovery rates (≥40%) after one to four years of cryogenic storage have been documented for over 675 of 750 accessions tested. However, current protocols dictate budwood collection at PGRU from late December through early March, when buds are thought to be optimally acclimated for desiccation and slow freezing to –30°C, our pretreatment for cryopreservation. This causes a processing bottleneck. Our observations suggest temporary storage of budwood at –4°C after field harvest is possible, but we had not tested this directly. Therefore, we collected budwood from four accessions representing different levels of cold tolerance on six dates from January to March, 1995. Dormant buds were processed for cryopreservation monthly after storage in sealed bags at –4°C for 1 to 6 months. Recovery rates ranged from 55% to 100%. Neither collection date nor length of storage at –4°C affected rate of recovery. These results suggest we can significantly increase the throughput and efficiency of our cryopreservation efforts, thereby enhancing management and security of the Malus collection.