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Leigh E. Towill

Papaya shoot tips, obtained either from seedlings or from in vitro plants, survived liquid nitrogen (-196°C) exposure using a vitrification procedure. Vitrification is a technically simple method but requires large concentrations of cryoprotectants. These were added in two steps, first slow addition of dimethylsulfoxide (DMSO) and PEG-8000, and subsequent fast addition of ethylene glycol (PG). The final concentration before cooling was 40% EG, 7.8% DMSO, and 10% PEG-8000. Both rapid cooling and rapid warming rates were required. Differential scanning calorimetry (DSC) was used to determine that the external solution vitrified upon cooling. It could not be demonstrated by DSC that cells within the shoot-tip vitrified, but since both DMSO and EG rapidly permeate plant cells, vitrification within the cells seems a likely explanation for retention of viability.

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Harrison Hughes and Leigh Towill

There are turfgrasses species that are clonally propagated; notably bermudagrass, buffalograss, and zoysiagrass. Some of the early cultivars of these species are no longer widely grown, and may eventually be lost if not preserved. In order to facilitate studies on the long-term cryopreservation of these species and specific lines of saltgrass, it is necessary to develop suitable micropropagation procedures. We have developed protocol for the isolation and establishment of clean cultures in vitro for all four species. A 1/2-strength MS basal medium with Nitsch & Nitsch vitamins, 5 mg/L of thiamine, 2 mg/L of glycine, 30 g of sucrose, 7 g of agar with varying growth regulators has been used. Explant materials are prewashed in the greenhouse prior to a 15- to 30-min soapy wash in the laboratory. After a 30- to 60-min rinse in running water, nodal sections are surface-disinfested in 10% bleach with Tween 20 for 15 min, followed by three sterile water rinses. This procedure, sometimes with PPM (a proprietary antimicrobial compound), results in 50% or greater clean cultures. Rapidly growing nodal sections work best and preferably those not established in soil. We have tested various growth regulator combinations and have found that 10 mg/L of BA results in proliferation of buffalograss and saltgrass. However, proliferation remains relatively slow, requiring 8 to 12 weeks to develop sufficiently for subculture. Although we have succeeded in obtaining clean cultures of bermudagrass and zoysiagrass, proliferation is minimal, Further research is ongoing to develop a proliferative system with these two species.

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Leigh E. Towill*

Apple cryopreservation at USDA-ARS NCGRP uses a winter vegetative bud method that incorporates desiccation prior to cooling. Although this method is valuable, desiccation is time consuming, requiring cutting nodal sections to exact lengths, moisture content estimates, and 1-4 weeks of desiccation. Processing sections without desiccation is being examined to improve the efficiency of handling Malus accessions. Vi-ability was estimated using an oxidative browning assay or a sprouting test. Sections from mid-winter collected scions were cooled at different rates to -30°C or -35 °C and transferred to the vapor phase over liquid nitrogen. Sections were warmed at + 4 °C and held for 24 h before testing viability. Some lines were processed after several months of storage at -3.5 °C. Although viability after cryopreservation occurred with a cooling rate of 1 °C/h, slower cooling (5 °C/day) was beneficial for many accessions. In tests with a limited number of lines, cooling rates ≥10 °C/h to -30 °C caused injury to buds and cambium. Scions stored for up to 8 months could survive cryoexposure. Scions from three lines tested survived three cycles of cooling from + 4 °C to LN. Extent of acclimation affected results. With non-desiccated sections cryogenic survival of `Golden Delicious' differed over years, but this has also occurred with the procedure that uses a desiccation step. It is not expected that this method is generally applicable to more tender species of Malus or other fruit genera, but the method has been successful with many lines of M. × domestica, a fairly cold hardy taxa, and with some other cold hardy Malus species. Grafting tests are needed to confirm the usefulness of the method

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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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Joyce C. Pennycooke and Leigh E. Towill

Cryopreservation offers the simplest and most economical way for the long-term conservation of germplasm and vitrification is the preferred method to accomplish this. Undefined endogenous compounds are produced during plant growth and shoot tip preculture conditions. These may influence “cryopreservability” and interact with cryoprotectants that are artificially added during the cryogenic protocol. We are beginning to examine these aspects to improve cryopreservation. Nodal segments of PI 296057 were propagated on a hormone-free modified Murashige and Skoog (MS) solid medium and were grown with 16 hr/8 hr photoperiod. Shoot tips were excised at 0, 3 or 10 hr in light after the dark period. Excised shoot tips were precultured in 0.06 M sucrose in MS for 24 hr and 0.3 M sucrose in MS for 24 hr and then treated with 0.4 M sucrose plus 2 M glycerol for 20 min or 1 hr before being dehydrated in PVS2 [30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v) dimethylsulfoxide in MS and 0.4 M sucrose[for 10, 16 or 26 min at 22°C. Shoot tips were placed on thin strips of aluminum foil, which were folded to enclose the shoot tips and then immersed in a liquid nitrogen (LN) slush. Rapid warming and dilution were achieved by transferring the foil strips from LN into 3 ml of 1.2 M sucrose at 22°C for 20 min. All cultures were incubated in darkness for 2 days then dim light for 3 days before transfer to the usual light intensity. Elimination of iron and nitrogen from MS medium in post thaw culture for 5 days increased the viability of LN-treated samples. Maximum survival after LN exposure was achieved with excision immediately after the dark photoperiod, cultured for 1 hr in 0.4 M sucrose plus 2 M glycerol and exposed for 16 min in 100% PVS2 at 22°C. Previously, Towill and Jarret (1992, Plant Cell Reports 11: 175–178) reported that surviving shoot tips developed callus and a variable percentage subsequently formed shoots. In this line all surviving shoot tips eventually formed shoots.

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Leigh E. Towill and Gayle M. Volk

Arabidopsisthaliana shoot tips provide a model to study processes important for cryopreservation. Cryopreservation was accomplished using both vitrification and two-step cooling methods. With vitrification methods, shoot formation after liquid nitrogen (LN) exposure was as high as 100% and 95% for shoot tips exposed to PVS2 at 0 °C and to PVS3 at 23 °C, respectively. A two-step cooling method also gave greater than 90% survival if shoot tips were cooled at 0.3 °C per minute to below –30 °C before immersing the samples into LN. The high levels of shoot formation after LN exposure in Arabidopsis thaliana shoot tips will allow the use of mutants to examine how alterations in biochemical, metabolic, and developmental processes affect survival and growth.

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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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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.

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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.