Search Results

You are looking at 1 - 8 of 8 items for

  • Author or Editor: L.E. Towill x
Clear All Modify Search

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.

Free access
Authors: and

cryopreservation of dormant, vegetative apple buds at the National Seed Storage Laboratory is used to maintain a base collection for germplasm held in the National Clonal Germplasm Repository for apple and grape, Geneva, NY, and is performed by a method previously reported1. Growth of buds after grafting is now used to test for survival after exposure to and storage at very low temperatures (ca.-160°C). We are interested in determining if measures of respiration can be used to assess 1. the status of buds and bark used for preservation, 2. survival after different treatments related to cryopreservation, and 3. the extent of sublethal injury after treatments. A Licor 6252 CO2 analyzer was used to measure respiration. Reproducible measurements of respiration required at least 2-3 buds. Buds from winter harvested twigs (ca 45-48% moisture content) that were briefly warmed to room temperature respired at a rate of 34 umoles CO2 g-1 (dw) hr-1. Survival of buds is enhanced if twigs are dried prior to cooling. We found such treatments reduced respiration over non-dried controls. Respiration increased as the bud was rehydrated. Buds from dried twigs slowly cooled to low temperatures had levels of respiration after warming and rehydrating similar to undried, unfrozen controls. Buds from undried twigs directly placed at -196°C and warmed gave little CO2 production.

Free access

Long-term preservation of seed germplasm is a high agricultural priority. It assures that genetic diversity will be available for future generations for continued plant improvement. This experiment reports on the affect that storage temperature had on the viability of 65 selections of lettuce seed stored for 30 years. The average seed moisture content was 5.5% ± 0.5% (fresh weight basis). Fresh seed samples were placed at 5 °C storage in 1969. In 1975 they were then transferred to -18 °C storage. Viability remained at 98% ± 5% for the first 14 years of 5 /-18 °C storage, then viability declined. At 17 years storage, the average viability had dropped to 75% and continued to drop at about 4%/year. At the 17-year mark, individual samples were split, one-half remained at -18 °C the other half was placed under liquid nitrogen vapor (lnv) conditions (about -150 to -190 °C). The -18 °C stored samples continued to deteriorate to 14% viability at the 30 year test period (1999). The samples placed in lnv did not decrease further in viability and remained at 75% viability at the 30-year mark. Seed vigor was reduced in the -18 °C stored seeds that were still viable. The lnv-preserved samples were significantly more vigorous. It is clear from this experiment that lnv preservation was significantly superior to -18 °C storage and, in fact, stopped or significantly reduced the rate of viability loss in samples that are rapidly deteriorating.

Free access

Pollen from Clianthus formosus (G. Don) Ford and Vickery was tested for viability after desiccation and exposure to low temperatures. Desiccation for 3 hours before freezing at –180C was sufficient for maintaining pollen germination. Pollen dried for a longer time showed reduced germination when plated directly. However, when pollen was rehydrated by exposure to high humidity before plating, germination was not reduced.

Free access

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.

Free access

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.

Free access

Dormant buds of 64 apple accessions from the National Germplasm Repository (NGR), Geneva, NY were cryopreserved at the National Seed Storage Laboratory (NSSL), Fort Collins, Co. Initial tests after 1 mon, 1, 2, and 3 years of LN2 storage showed no decline in viability. Storage of 16 cultivars (1988/89 and 1989/90 dormant seasons) with a broad range of cold-hardiness characteristics has shown approx 45% viability by patch budding. Storage from dormant seasons of 1990/91 and 1991/92 included 48 cultivars selected for excellent cold-hardiness characteristics. With approx 85% initial viability of these cultivars, a more sensitive statistical analysis can be performed over years. Overall viability over storage duration and sampling years showed 32 had more than 80%, 55 had more than 50% and only 4 had less than 30%.

Free access

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.

Free access