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.
Leigh E. Towill
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
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.
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.
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.
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.
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.
Philip L. Forsline, Leigh E. Towill, John Waddell, and Loren Wiesner
The USDA/ARS collection of Malus is held by the Plant Genetic Resources Unit in Geneva, N.Y. The collection comprises ≈2500 accessions, most of which must be maintained as clones in the field to provide propagating material for distribution to the user community. Field maintenance of replicated accessions places the collection at risk from weather extremes, pests, diseases, etc. and is extremely costly. Cryopreservation of dormant buds in a base, or backup, collection could reduce risks and decrease maintenance costs. Since 1988, we have developed and implemented protocols to cryopreserve dormant apple buds at the National Seed Storage Laboratory, Fort Collins, Colo. More than 500 accessions have been placed in cryogenic storage. Buds have been successfully recovered by grafting from >70% of the first 250 accessions cryopreserved. These results, and those from ongoing recovery tests, indicate cryopreservation may be a safe, cost-effective approach to back up collections of tree fruit germplasm. It also may be used to enhance management of the active collections of Malus, Vitis, and Prunus at Geneva.
I.E. Yates, Darrell Sparks, Kris Connor, and Leigh Towill
Pecan [Carya illinoensis (Wangenh.) C. Koch] pollen was stored under reduced moisture conditions to determine if pollen viability could be maintained for long periods at either 23, 5, or - 12C. Pecan pollen was oven-dried at 35C to a constant weight and stored in moisture-proof bags. Pollen maintained at - 12C for 2 years was as viable as freshly collected pollen. The duration of viability at SC was at least 2 months. Even at 23C, viability was detectable for 1 month, but at a greatly reduced level. Provided precautions are taken to reduce pollen moisture before and during storage, long-term storage of pecan pollen can be easily accomplished with commonly available supplies and equipment.
Philip L. Forsline, Leigh E. Towill, John W. Waddell, Cecil Stushnoff, Warren F. Lamboy, and James R. McFerson
Clonally propagated crops, unlike seed-propagated crops, require intense and costly maintenance, generally in ex situ field gene banks. Consequently, large germplasm collections of tree species especially, are difficult to conserve in a well-replicated fashion and are vulnerable to damage from environmental stresses. Accordingly, long-term storage in liquid nitrogen presents a viable conservation alternative. To assess effectiveness of one approach to cryopreservation, dormant buds from 64 apple (Malus ×domestica Borkh. and other Malus spp.) accessions were collected and preserved in liquid nitrogen using a dormant-vegetative-bud method. Buds were retrieved from liquid nitrogen storage, rehydrated, and grafted onto rootstocks to determine survival. Mean recovery was 76% for 40 cold-hardy accessions, 66% for 20 moderately cold-hardy accessions, and 24% for four cold-tender accessions (range: 16% to 100%). Only four accessions had ≤25% recovery while 54 accessions had ≤50% recovery and 35 accessions had ≤75% recovery. No significant decline in recovery of these accessions by bud grafting occurred after 4 years of liquid nitrogen storage.