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Using dormant buds (DB) for germplasm cryopreservation was published few decades ago and since then, dormant buds have been used to preserve genetic resources of selected horticultural woody plant species. The advantages of employing DB in preservation are widely known; the most relevance is no requirements of aseptic cultures, high processing throughput and involvement of a relatively low skilled technical support; but the method has also shortcomings, such as seasonality of processing and lack of procedural modifications that might support preservation of all accessions in a collection. The US, NLGRP cryopreserved DB of 2168 unique Malus (Mill.) accessions (among 51 species) with a ≥40% post cryo viability. The method worked well for all accessions in 20 species, for ≥90% of accessions in six species and at a various percent (0-89%) in the remaining 25 species. For species with the largest number of processed accessions, the M. domestica, Borkh., M. hybrid and M. sieversii (Ledeb.) M. Roem. (1355, 326 and 128 cryopreserved accessions, respectively), the percent of accessions responding favorable to the DB method was high (96, 96 and 82%). The results indicated variability in Malus DB response to liquid nitrogen exposure; similar results were recorded in the Pyrus L. collections. Procedural refinements of the DB cryopreservation method could increase the method applicability to a much higher number of Malus species and its use in other collections of horticultural woody plants like Prunus L. and Pyrus L.

The USDA-ARS National Plant Germplasm System (NPGS) maintains a citrus genetic resource collection of 1522 accessions of Citrus cultivars and crop wild relatives. Of these, 540 are maintained as pathogen-free duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. An additional 815 accessions of citrus and citrus relatives are maintained in greenhouses or orchards in Riverside until sanitation for pathogens can be completed. This paper describes progress in backing up the 540 pathogen-tested accessions using cryopreservation techniques and methods to accelerate processing and viability testing. Vegetative budwood was harvested from trees in the screenhouse and sent to NLGRP. At least 170 shoot tips from each accession were excised from surface-sterilized budwood, and then cryopreserved using a PVS2 droplet-vitrification technique. Viability was assessed by micrografting 10 thawed shoot tips onto in vitro grown 'Carrizo' seedling rootstock. The remaining shoot tips are held in long-term liquid nitrogen storage. A total of 451 of the pathogen-tested citrus accessions were cryoprocessed: 354 accessions had regrowth levels of 40% or greater, 17 had 0% regrowth, 47 had viability levels between 10 and 30%, and 33 had endogenous contaminants. Technical staff familiar with shoot-tip cryoprocessing could be fully trained for specific applications of citrus in about 2 months and thereafter processed a single accession of at least 170 shoot tips in about 16 h. This large-scale effort has revealed that shoot tip cryopreservation can be successfully scaled-up to secure the NPGS Citrus collection.

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to identify a method whereby clonally propagated sugarcane accessions could be successfully introduced into tissue culture, multiplied, and then cryopreserved as shoot tips for long-term preservation at the National Laboratory for Genetic Resources Preservation in Fort Collins, Colorado. For many accessions, 70% isopropyl alcohol and 20% commercial bleach treatments, followed by three rinses of sterile water were sufficient to remove microbial contaminants during the introduction process. However, in some cases, cefotaxime was particularly effective for removing bacterial contamination. We found that antioxidant treatments of glutathione, glycine betaine, and ascorbic acid did not improve regrowth after liquid nitrogen exposure using either PVS2 or PVS3 as cryoprotectants in droplet vitrification cryopreservation methods. Exposure durations of PVS2 and PVS3 were optimized, with and without exposure to liquid nitrogen (LN), and shoot tip regrowth levels ranged from 0 to 37% after LN exposure. Published methods for encapsulation-dehydration and V-plate vitrification cryopreservation procedures were tested to determine if acceptable results could be obtained. Using these methods, shoot tip regrowth levels ranged from 0 to 50% after LN exposure. We conclude that the sugarcane cryopreservation methods that we tested are not yet ready for implementation in the NPGS.