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- Author or Editor: Philip L. Forsline x
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.
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.
Abstract
In a greenhouse experiment, Malus hupehensis (Pamp.) Rehd. seedlings were treated weekly with simulated acid rain solutions ranging from pH 2.25 to pH 7.0. Necrotic lesions developed on leaves at pH 2.25 and pH 2.50 immediately after the first application at the 8-node stage. Following the 9th weekly application on seedlings with 23 to 26 nodes, lesions developed at pH levels up to 3.25. At final destructive harvest, 20% of the leaf area at pH 2.25 and 8% of the leaf area at pH 2.50 was injured. Significant growth reduction occurred at these 2 pH levels. Regression analysis indicated extensive growth inhibition at <pH 3.0, no growth inhibition around pH 3.5, some inhibition between pH 4.5 and pH 5.6, and normal growth at pH 7.0 in comparison to the unsprayed control. Growth was negatively correlated with lesion formation at 3 destructive harvest dates. Relative growth rates were reduced only at pH 2.25 and pH 2.50 and reduction in the unit leaf rate was also observed. Lesion development continued to increase on the basal leaves through the 6th weekly application but leveled off during the final applications. Negative correlations of photosynthesis rate to lesion percentage and dry weight to lesion percentage were observed.
Reduced availability of water for agricultural use has been forecast for much of the planet as a result of global warming and greater urban demand for water in large metropolitan areas. Strategic improvement of water use efficiency (WUE) and drought tolerance in perennial crops, like fruit trees, could reduce water use without compromising yield or quality. We studied water use in apple trees using ‘Royal Gala’, a relatively water use-efficient cultivar, as a standard. To examine whether genes useful for improving WUE are represented in a wild relative genetically close to M. ×domestica, we surveyed Malus sieversii for traits associated with WUE and drought resistance using material collected from xeric sites in Kazakhstan. This collection has been maintained in Geneva, NY, and surveyed for various phenotypes and has been genetically characterized using simple sequence repeats (SSRs). These data suggest that most of the diversity in this population is contained within a subpopulation of 34 individuals. Analysis of this subpopulation for morphological traits traditionally associated with WUE or drought resistance, e.g., leaf size and stomata size and arrangement, indicated that these traits were not substantially different. These results imply that some of the genetic diversity may be associated with changes in the biochemistry, uptake, and/or transport of water, carbon, or oxygen that have allowed these trees to survive in water-limited environments. Furthermore, genes responding to drought treatment were isolated from ‘Royal Gala’ and categorized according to the biological processes with which they are associated. A large fraction of upregulated genes from roots were identified as stress-responsive, whereas genes from leaves were for the most part associated with photosynthesis. We plan to examine expression of these genes in the M. sieversii population during water deficit in future studies to compare their patterns of expression with ‘Royal Gala’.
The USDA–ARS germplasm collection of cold-hardy Vitis held at the Plant Genetic Resources Unit, Geneva, N.Y., has over 1300 clonal accessions maintained as field-grown vines. Security back-up using field-grown or potted vines at remote sites or via in vitro methods is costly. Cryopreservation offers a safe, cost-effective alternative. While we routinely employ cryogenic storage of dormant buds of Malus, dormant buds of Vitis generally do not appear to tolerate the desiccation levels required by our current cryopreservation protocol. Since tolerance to desiccation and cold appear to be correlated in Vitis, we tested desiccation tolerance of 60 germplasm accessions selected from the core subset to represent a range of cold hardiness. Budwood was collected in December 1995 in Geneva, stored at –4°C in sealed bags, and systematically desiccated to 30% and 20% moisture. In some treatments, additional desiccation was imposed by slow freezing to –25°C. Microscopic examination of rehydrated buds indicated 60% of accessions tolerated desiccation as low as 20% moisture. Freeze-desiccation at –25°C after desiccation at –4°C neither increased nor decreased viability in these accessions. Only slight modification so current protocols should be necessary for cryopreservation of this class. Of the remaining accessions, 25% tolerated desiccation to 30% moisture, but 15% were intolerant to any desiccation level tested. Techniques must be developed to successfully cryopreserve both these classes of accessions.
Blue mold of apples, incited by Penicillium expansum, causes extensive losses on stored apples worldwide. Despite the severity of this problem, apple breeders do not evaluate their crosses for resistance to this disease, because there has been little resistance to blue mold in the gene pool of the germplasm used. A new apple germplasm collection from the center of origin in Kazakhstan, maintained in Geneva, NY, and representing a much broader gene pool, was evaluated for resistance to blue mold. Apples were harvested from the Elite collection trees that were clonally propagated from budwood collected in Kazakhstan and from seedling trees originating from seeds of the same trees as the Elite budwood or from other wild seedling trees in Kazakhstan. Fruit from 83 such accessions were harvested at the preclimacteric to climacteric stage, wound-inoculated with P. expansum at 103, 104, and 105 mL−1 conidial suspension, incubated for 5 d at 24 °C, and evaluated for decay incidence and severity. Two accessions were classified as immune (no decay at 103 and 104 mL−1), four as resistant (no decay at 103 mL−1), 53 as moderately resistant (lesions less than 10 mm at 103 mL−1), and 24 as susceptible. There were positive correlations (r = 0.92, 0.86, and 0.91) between decay severity and all three inoculum levels. Our results indicate a greater genetic diversity among the Kazak apple collection than among cultivated apples as evidenced by their broad range of fruit maturity, quality, and disease resistance patterns. The immune and resistant accessions may serve as a source of resistance in breeding programs and can be useful in explaining the mechanism of resistance to blue mold in apples.
There are several Central Asian Malus species and varieties in the USDA-ARS National Plant Germplasm System (NPGS) apple collection. Malus sieversii is the most comprehensively collected species native to Central Asia. Other taxa such as M. sieversii var. kirghisorum, M. sieversii var. turkmenorum, M. pumila, and M. pumila var. niedzwetzkyana have primarily been donated to the collection by other institutions and arboreta. We sought to determine if genetic and/or phenotypic differences among the individuals that make up the gene pools of these taxa in the NPGS exhibit unique characteristics. Genetic data, based on microsatellite analyses, suggested that the diversity within each taxa is significantly greater than that among taxa. Trait data also revealed very few differences among taxa, the primary characteristic being the dark red fruit coloration and tinted flesh color of the accessions assigned to M. pumila var. niedzwetzkyana resulting from a known single-gene mutation in anthocyanin production. We found that M. sieversii is a highly diverse species with a range in genetic and phenotypic trait variation that includes the characteristics of the other Central Asian taxa of interest. We conclude that the gene pools that comprise the accessions within the NPGS Central Asian Malus collection are highly overlapping with respect to both phenotypic traits and genotypic characters.
Unlike cold-hardy apple germplasm, dormant vegetative buds from cold-tender accessions require stabilization of meristematic tissue to protect against injury during desiccation and cryopreservation. Dormant buds of six apple cultivars [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf. `Cox's Orange Pippin', `Einshemer', `Golden Delicious', `Jonagold', `K-14', and `Mutsu'] collected at specific intervals in 1993, 1994, and 1995 at Geneva, N.Y., were stabilized by encapsulation in 5% alginate, treated with step-wise imbibition of 0.5 to 1.0 m sucrose and 0.2 m raffinose solution, and desiccated with forced air at 0 °C. Sugar-alginate stabilization reduced injury during desiccation, increased cold-hardiness of the six cold-tender cultivars frozen to -30 °C, and improved recovery following cryopreservation of buds collected before optimal cold acclimation was attained. Sucrose tissue levels did not increase following stabilization treatment, but levels of glucose and fructose, and of an unknown disaccharide increased. This procedure used nontoxic cryoprotectants, and has potential to expand the scope of dormant bud cryopreservation to include cold-tender apple germplasm.
Abstract
Mature ‘McIntosh’, ‘Empire’, and ‘Golden Delicious’ apple trees (Malus domestica Borkh.) were sprayed with simulated acid rain solutions in the pH range of 2.5 to 5.5 at full bloom in 1980 and in 1981. In 1981, weekly sprays were applied at pH 2.75 and pH 3.25. Necrotic lesions developed on apple petals at pH 2.5 with slight injury appearing at pH 3.0 and pH 3.5. Apple foliage had no acid rain lesions at any of the pH levels tested. Pollen germination was reduced at pH 2.5 in ‘Empire’. Slight fruit set reduction at pH 2.5 was observed in ‘McIntosh’. The incidence of russetting on ‘Golden Delicious’ fruits was ameliorated by the presence of rain-exclusion chambers but was not affected by acid rain. With season-long sprays at pH 2.75, there was a slight delay in maturity and lower weight of ‘McIntosh’ apples. Even at the lowest pH levels no detrimental effects of simulated acid rain were found on apple tree productivity and fruit quality when measured as fruit set, seed number per fruit, and fruit size and appearance.
The U.S. Department of Agriculture, Agricultural Research Service, National Plant Germplasm System (NPGS), Plant Genetic Resources Unit apple (Malus) collection in Geneva, NY, conserves over 2500 trees as grafted clones. We have compared the genotypes of 1131 diploid Malus ×domestica cultivars with a total of 1910 wild and domesticated samples representing 41 taxonomic designations in the NPGS collection to identify those that are genetically identical based on nine simple sequence repeat (SSR) loci. We calculated the probability of identity for samples in the data set based on allelic diversity and, where possible, use fruit images to qualitatively confirm similarities. A total of 237 alleles were amplified and the nine SSRs were deemed adequate to assess duplication within the collection with the caveat that “sport families” likely would not be differentiated. A total of 238 M. ×domestica and 10 samples of other taxonomic groups shared a genotype with at least one other M. ×domestica individual. In several cases, genotypes for cultivars matched genotypes of known rootstocks and indicated that these accessions may not accurately represent the indicated named clones. Sets of individuals with identical genotypes and similar cultivar names were assigned to sport families. These 23 sport families, comprised of 104 individuals, may have mutational differences that were not identified using the nine SSR loci. Five of the selected markers (CH01h01, CH02d08, CH01f02, G12, GD147) overlap with sets of markers that have been used to fingerprint European apple collections, thus making it possible to compare and coordinate collection inventories on a worldwide scale.