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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.
Morphological traits were examined in an F3 generation derived from a cross between C. lanatus var. lanatus [(Thunb.) Matsum. & Nakai] and C. lanatus var. citroides. At least three genes, C (yellow) vs. c (red), i (inhibitory to C) vs. I (non-inhibitory to C), and y (yellow) vs. yw (white), with epistatic and inhibitory actions were found to govern the inheritance of fruit flesh color. The high frequency of yellow-fleshed fruit and low frequencies of white and red fruits can be explained by the presence of a new allele (yw recessive to y) in the multiple allele series at the Y locus. The low frequency of tan colored seeds in segregating populations could be explained by at least three genes governing inheritance of seed-coat color. Single factor analysis of variance was conducted for each pairwise combination of random amplified polymorphic DNA (RAPD) locus and fruit or seed characteristics. Several RAPD loci were identified to be loosely linked to morphological characteristics.
Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f.sp. niveum (FON), is a serious disease of the watermelon (Citrullus lanatus). Three races of this pathogen (races 0, 1, and 2) have been identified based on differential pathogenicity assays. Most commercially available cultivars are resistant to races 0 and 1. Inheritance for resistance to these races is thought to be controlled by a single dominant gene. No cultivars are resistant to race 2 and resistance is thought to be a quantitative trait. F2 lines derived from a cross between the Fusarium-resistant Citrullus lanatus PI296341, and the Fusarium-susceptible watermelon cultivar `New Hampshire Midget' were used to generate a RAPD-based map of the Citrullus genome. F2:3 families were assayed in the greenhouse for resistance to races 1 and 2. Those families that were either highly resistant or highly susceptible were used in identifying markers linked to Fusarium wilt resistance. A preliminary map of the Citrullus genome based on random amplified polymorphic DNA (RAPD) markers has been expanded with the inclusion of simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs), and isozymes.
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.
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.
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.
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.
Isozyme, randomly amplified polymorphic DNA (RAPD), and simple sequence repeats (SSR) markers were used to generate a linkage map in an F2 and F3 watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai] population derived from a cross between the fusarium wilt (Fusarium oxysporum f. sp. niveum) susceptible `New Hampshire Midget' and resistant PI 296341-FR. A 112.9 cM RAPD-based map consisting of 26 markers spanning two linkage groups was generated with F2 data. With F3 data, a 139 cM RAPD-based map consisting of 13 markers covering five linkage groups was constructed. Isozyme and SSR markers were unlinked. About 40% to 48% of the RAPD markers were significantly skewed from expected Mendelian segregation ratios in both generations. Bulked segregant analysis and single-factor analysis of variance were employed to identify RAPD markers linked to fusarium wilt caused by races 1 and 2 of F. oxysporum f. sp. niveum. Current linkage estimates between the resistance trait and the marker loci were too large for effective use in a marker-assisted selection program.
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%.