Germplasm of Ozark chinquapin (Castanea ozarkensis Ashe) can be cryopreserved by dormant winter buds.

Ozark chinquapin (Castanea ozarkensis Ashe) is a forest tree, endemic to the Ozark Mountain region in Eastern United States. Its nutritious nuts were consumed by Native Americans, European settlers, livestock, and wild animals and its wood was an important rot-resistant construction material. Once a significant tree in regional forest communities, the species was nearly eradicated by a chestnut blight caused by Cryphonectria parasitca (Murill) Barr fungus. Some individuals have survived as sprouts from adventitious root buds, but they rarely reach reproductive maturity. While some in situ restoration efforts are underway, the development of a viable ex situ germplasm preservation method is critical to the conservation of this important food-bearing species. Our experiment aimed to develop a cryopreservation method for C. ozarkensis dormant winter buds subjected to eight experimental treatments before desiccation, slow cooling, and storage in liquid nitrogen vapor. The highest post cryogenic viability was 91.2 % for dormant buds pretreated with 0.3 M sucrose for 16 h followed by 0.75 M sucrose for 3 h; this treatment is suggested for cryopreservation of dormant winter buds of Ozark chinquapin germplasm.

High regrowth of potato crop wild relative genotypes after cryogenic storage.

Potatoes are consumed by millions of people and are the survival food in several countries. Cultivated varieties of potato (Solanum tubersosum L.) are results of selection and crossing of many wild species. Only 8-13% of wild potato species used for food are preserved by either in situ or ex situ methods. The U.S. National Potato Germplasm Collection maintains over 5900 accessions, of which 75% are crop wild relatives (CWR). The objective of the study was to investigate regrowth of cryogenically stored clonal propagules (shoot tips) of selected CWR accessions maintained in the collection. Sixty-nine accessions from 30 Solanum species and six accessions that are not yet assigned to a species were cryopreserved by a droplet vitrification method at the NLGRP. The post cryopreservation regrowth varied from 40 to 100% (average 68%) but was not significantly different between the tested accessions. Regrowth of six accessions tested after 10 years of cryogenic storage was between 35 and 90% (average 66%) and was significantly different from their initial regrowth (average 87%); the largest viability loss was in S. condolleanum; but for the other five accessions the regrowth was between 45 and 90% (average 72%) and suggested at least 10 years of successful storage in LN was possible. Twelve potato wild species cryopreserved in this study were reported in literature as important for developing cultivated varieties for changed weather conditions.

Determining the effect of pretreatments on freeze resistance and survival of cryopreserved temperate fruit tree dormant buds.

Freeze resistance is critical to successful dormant bud (DB) cryopreservation, and is affected by genotype, environmental conditions, dormancy phase and processing techniques. Pretreatment induced freeze resistance may contribute to more successful and efficient protocols for cryopreserving DB. Differential thermal analysis (DTA) was used to quantify the effects of cryopreservation pretreatments on freeze resistance of dormant budwood. Low temperature exotherm (LTE) profiles created by DTA could rapidly identify pretreatments that are contributing to increased freeze resistance in tree fruit species. In this study, DTA was used to help elucidate the effects of varying pretreatments (sucrose, desiccation and their combination) on peach, a model crop in tree fruit physiology that has shown little cryosurvival using the DB method in the past. Post cryopreservation recovery trials using an antimicrobial forced bud development (AFBD) protocol evaluated the ability of selected pretreatments, that improved freeze resistance based on DTA, to improve recovery of dormant budwood of various deciduous tree fruit and nut species. Precryogenic exposure to sucrose solution (5.0 M, 96 h), desiccation to 30% moisture content (MC) and their combination tested for their efficacy on improving postcryogenic viability in peach, apricot, sweet cherry, little walnut, black walnut, English walnut, apple, and pear. Among the different pretreatments tested, desiccation to 30% MC had the greatest impact on increasing freeze resistance and cryosurvival across most fruit species tested and little walnut. Gradual reduction of MC (from 40 to 25%) levels increased freeze resistance in peach (R2=0.95) and increased some recovery outcomes (leaf, shoot and bud swell), however, this was not correlated with equal cryorecovery outcomes as severe bud cracking was observed. Overall, our approach linking freeze resistance and preconditioning treatments could help establish efficient species-specific cryopreservation protocols for a number of important temperate woody crops which could be recovered as complete plants by coupling AFBD and plant tissue culture.

Antimicrobial forcing solution improves recovery of cryopreserved temperate fruit tree dormant buds.

Dormant bud cryogenic preservation is a cost- and labor-efficient method of genetic resource backup compared to in vitro derived meristem shoots cryopreservation. While protocols have been developed for cryopreserving apple dormant buds, effective and reproducible protocols are yet to be developed for several temperate fruit and nut species. Dormant bud cryopreservation typically requires material to be grafted to evaluate viability and recover a plant. Forced bud development has been used on a very limited scale for cryostored dormant budwood recovery, however, it provides a labor-efficient alternative viability assessment. To increase the utility of this approach, regrowth must be optimized to allow complete plant recovery. We hypothesized that bacterial attacks are limiting regrowth, thus, an antimicrobial forcing solution can maximize regrowth potential. This study examined the effects of an antimicrobial forcing solution (8-hydroxyquinoline citrate and sucrose, 8-HQC) on the cryosurvival and recovery of dormant buds of fruit (Malus x domestica, Prunus armeniaca, Prunus avium, Prunus persica, Pyrus communis), and nut species (Juglans regia, Juglans nigra, Juglans microcarpa). Recovery and shoot development were significantly improved for all the fruit and one nut species (J. microcarpa) treated with the 8-HQC, compared to standard recovery under high humidity alone (P < 0.001). Additionally, this post cryo recovery approach led to successful in vitro shoot tip establishment across all surviving fruit species. 8-HQC embedded forced bud development method increased viability and efficiency for existing cryostored material and can be used as a benchmark to develop protocols for different crops that could potentially lead to complete plant recovery.

Twig pre-harvest temperature significantly influences effective cryopreservation of Vaccinium dormant buds.

Cryopreservation of temperate woody-plant material by dormant buds is less expensive than using shoot tips isolated from tissue cultured plants; however currently, dormant buds are used only for preservation of selected temperate tree and shrub species. Using dormant buds could be an efficient strategy for long-term preservation of blueberry (Vaccinium L.) genetic resources. In this study, viability of V. hybrid 'Northsky' (PI 554943) dormant buds was evaluated at 30 harvest dates over three consecutive fall/winter seasons to determine the optimal harvest time that promotes high post cryopreservation viability. Twigs with dormant buds were cut into 70 mm segments containing at least two nodes, desiccated, slowly cooled, stored in liquid nitrogen vapor and tested for post-cryopreservation regrowth. The highest regrowth of cryopreserved dormant buds was observed for buds harvested in mid-December and during the first half of January. Pearson's correlation coefficients were computed to evaluate the association between bud characteristics and viability at harvest date and logistic regression models were fit to test the ability of twig characteristics and temperatures to predict post cryopreservation bud viability. Post-cryopreservation viability was negatively correlated (p < 0.05) with average minimum, maximum and daily mean temperature preceding the bud harvest but was not correlated with the dormant bud initial and end moisture content, twig diameter, the number of dormant buds/cm of twig length and the number of days in desiccation. Regression tree analysis suggested post-cryopreservation viability to be between 52 and 80% for dormant buds harvested after a 10 day average maximum air temperature of <11.2 °C. Pre-harvest air temperature was a significant indicator of optimal dormant bud harvest time to produce adequate viability for long term preservation of blueberry genetic resources.

Medium- and long-term storage of the Pycnanthemum (mountain mint) germplasm collection.

The United States of America collection of mountain mint (Pycnanthemum Michx.) is held at the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon as seed, potted plants and tissue cultures and a long-term storage collection is preserved at the USDA-ARS National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado. The clonal collection is comprised of 34 accessions as potted plants that are duplicated with 31 accessions stored as in vitro cultures at 4 degrees C in tissue culture bags for medium-term storage at NCGR and as cryopreserved shoot tips in liquid nitrogen at NCGRP for long-term storage. This study reports on these two models of preservation of mountain mint at the U.S. National Plant Germplasm System. In vitro plants required 2 to 7 months for propagation on MS medium without growth regulators before storage at 4 degrees C. Plants remained in storage with good vigour in bags on 1/2x nitrogen MS medium without growth regulators for a mean of 2.08 y. An encapsulation-dehydration protocol was successful for cryopreservation of shoot tips from cold acclimated in vitro plants. Post-cryo viability, indicated by shoot tips with developed leaves and roots, ranged from 60 to 100 % for 27 accessions and 40 to 50 % for the other four. The encapsulation-dehydration cryopreservation method proved suitable for long-term preservation of the 31 Pycnanthemum accessions. These alternative storage forms allow for active use of the collection as well as base storage for clonally propagated accessions.