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S. Abou Taleb, I. Yates, B.W. Wood, and M.M. Fouad

A study was conducted to develop protocol for the preservation of pecan genetic variability by cryogenic storage of zygotic embryos and subsequent in vitro plant regeneration. Parameters evaluated for their influence on embryo survival included the amount of intact kernel, liquid nitrogen (LN) treatment, desiccation, and genotype specificity. Optimum germination with minimum contamination occurred with 12% of the kernel intact. Treatment of explants with LN reduced the percentage of embryos developing into intact plants. `Curtis' and `Shoshoni' had a significantly higher morphogenic response in shoots only than all other cultivars. In summary, cryogenic storage of pecan zygotic embryos was determined to be a feasible means for preservation of pecan germplasm. However, the procedures used in the current study should be altered to increase the probability of embryo survival.

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Erik J. Sacks and Dina A. St. Clair

The influence of cryogenic pollen storage on fruit set and seed production in tomato (Lycopersicon esculentum Mill.) was investigated. Flowers pollinated with pollen samples stored for 5 weeks at –80C, with or without 20 h precooling at 4C, had similar fruit set and number of viable seed per fruit as those pollinated with fresh pollen. Pollen samples, which were repeatedly cooled (–80C) and warmed (to 22 to 24C) for up to six cycles, continuously maintained the same viability as the fresh pollen. When cryogenically stored pollen of L. esculentum 2-837, LA359, LA3198, and LA3199 were used to pollinate LA359, the number of viable seed formed per fruit differed significantly. Results of this study suggest that pollen cryopreservation can be used successfully for tomato breeding and germplasm storage.

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Cameron Northcutt, Daniel Davies, Ron Gagliardo, Kylie Bucalo, Ron O. Determann, Jennifer M. Cruse-Sanders, and Gerald S. Pullman

uncovered vial and then covered and reweighed to obtain dry weights for water content calculations. Seed samples were placed in Nalgene cryogenic storage vials and either rapidly immersed in liquid N 2 or cooled to –35 °C at a rate of 0.33 °C/min using a

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L.X. Zhang, W.C. Chang, Y.J. Wei, L. Liu, and Y.P. Wang

Cryopreservation of pollen from two ginseng species —Panax ginseng L. and P. quinquefolium L.—was studied. Freezing anthers that served as pollen carriers to –40C before liquid N storage affected pollen viability little after liquid N storage. Anther moisture content affected pollen viability significantly when stored in liquid N. The ideal anther moisture content to carry pollen for liquid N storage was 32% to 26% for P. ginseng and 27% to 17% for P. quinquefolium. Viability of pollen from P. quinquefolium anthers with 25.3% moisture content changed little after 11 months of liquid N storage.

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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.

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Natalia R. Dolce, Ricardo D. Medina, and María T. González-Arnao

brevicuspis plant after 12 months of pot culture in a greenhouse, bar = 10 cm. Discussion Avoidance of intracellular ice formation during exposure to cryogenic temperatures is a prerequisite for successful cryopreservation of living organs, tissues, and cells

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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.

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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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Antonieta N. Salomão and Rosângela C. Mundim

The effects on germination of two lots of Carica papaya seed of dehydration at 25 °C, followed by exposure to -20 °C or -196 °C, were evaluated with and without gibberellic acid (GA3) treatment. In the absence of GA3 treatment, dehydration increased subsequent germination only in seed lot 1 when moisture content (m.c.) was reduced from 59% to 6.0% and 5.3%. In seed lot 2, dehydration followed by exposure to -196 °C increased germination compared with dehydration alone. Treatment with GA3 enhanced germination rate in all treatments. Dehydration to 5.3% (lot 1) or 6.9% and 6.8% m.c. (lot 2), followed by exposure to subzero temperatures and treatment with GA3, were the most favorable combined treatments to enhance papaya seed germination. The results suggest that papaya seed presents an orthodox behavior, permitting germplasm conservation in conventional and cryogenic genebanks.

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Derek N. Peacock and Kim E. Hummer

We contrasted the effect of liquid nitrogen (LN2), sulfuric acid (H2SO4), and a nontreated control on the germination of six Rubus species. We also were interested in determining if LN2 could be an effective mechanical scarifying agent for these species. Seeds of each species were treated with three 3-minute dips in LN2 with alternating 10-minute thaws, with H2SO4 for 30 minutes, or left untreated. The percent germination of R. multibracteatus A. Leveille & Vaniot, R. parviflorus Nutt., R. eustephanos Focke ex Diels, R. leucodermis Douglas ex Torrey & A. Gray, R. ursinus Cham. & Schldl., and R. chamaemorus L. treated with LN2 was not significantly different than the control. Germinated seedlings from the LN2 treatment of each species showed normal development upon planting, indicating that long-term cryogenic preservation of these Rubus species seeds may be possible. The H2SO4 treatment significantly increased the rate and percentage of germination in R. parviflorus, R. eustephanos, R. leucodermis, and R. ursinus over that of the control and the LN2 treatment. The alternative LN2 application techniques that have been attempted thus far have not significantly improved Rubus seed germination compared with that of the control.