Looking for a way forward for the cryopreservation of orchid diversity.

The family Orchidaceae, with over 25,000 species, includes five subfamilies and nearly 700 genera. Loss of plants in the wild has resulted from clearing of forests and excessive collection for various purposes. Moreover, the requirement of symbiotic association during seed germination under natural conditions adds a certain level of difficulty in retaining the orchid resources in the wild. Cryopreservation is an important arena in conservation science due to its potential of storing genetic resources without altering the genetic makeup. Cryopreserved orchids are a very small percentage of the species, and are also not representative of most genera. Finding effective protocols for the various explant types is of prime importance in conserving orchid diversity. Seed is the most commonly stored and directly useful explant, and direct plunging in liquid nitrogen or PVS2 vitrification appear to be suitable for most tested species. The myriad of other species should be screened as they become available, with special emphasis on seed maturity and moisture content. Studies of protocorms and protocorm-like bodies mostly employ desiccation, PVS2 vitrification or encapsulation-dehydration. Pollinia are generally stored successfully following desiccation or slow cooling. There are too few examples of shoot tip cryopreservation to make a determination, however vitrification techniques are likely the most useful for a range of genera. A systematic and coordinated effort is needed to screen all available species in as many taxa as possible, initially with seed, protocorms and pollinia. It is a charge to the orchid research community to organize this effort and fill in the required data for the large number of untested taxa. In addition, providing stored samples to established orchid cryo collections would greatly increase preservation of these endangered treasures.

Cryopreservation of shoot tips of recalcitrant and tropical species: Advances and strategies.

There is a pressing need for practical and successful conservation efforts to establish long-term germplasm collections of recalcitrant and tropical species, given the challenge and threat that these plants are facing. Cryopreservation is the only way of conserving some of these species, especially those with temperature or desiccation sensitive (recalcitrant) seeds. This review covers reports on cryopreservation studies of shoot tips (apical and axillary) of tropical and subtropical plants. Since many of these species have recalcitrant seeds, the cryopreservation successes, failures and problems involved with these seeds are also discussed. The methodologies, important factors and steps involved in successful cryopreservation protocols are analyzed. Finally strategies are suggested to develop a successful cryopreservation protocol for new plant species, in particular those with tropical recalcitrant seeds.

CRYOTHERAPY AS A METHOD FOR REDUCING THE VIRUS INFECTION OF APPLES (Malus sp.).

BACKGROUND: There is an urgent need in Kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. An in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries. METHODS: Malus sieversii Ledeb. M. Roem. and Malus domestica Borkh. accessions were initiated in vitro for virus identification and elimination. Reverse transcription and multiplex PCR were used to test for five viruses. PVS2 vitrification was used as a tool for cryotherapy. RESULTS: Four viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) were detected in 17 accessions. Tomato ringspot virus (ToRSV) was not detected. ACLSV affected 53.8% of the accessions, ASPV 30.8%, ASGV 5.1%, and ApMV was found only in 'Aport Alexander'. Cryotherapy produced virus-free shoot tips for seven of nine cultivars tested. Six cultivars had 60-100% elimination of ACLSV. CONCLUSIONS: An in vitro collection of 59 accessions was established. Virus elimination using cryotherapy produced virus-free shoots for seven of nine cultivars and is a promising technique for developing a virus-free apple collection.

Transcriptomic profiling revealed the regulatory mechanism of Arabidopsis seedlings response to oxidative stress from cryopreservation.

KEY MESSAGE: Elevated antioxidant status and positive abiotic stress response in dehydration enhance cell resistance to cryoinjury, and controlling oxidative damage via reactive oxygen species homeostasis maintenance leads to high survival. Cryoprotectants are important for cell survival in cryopreservation, but high concentrations can also cause oxidative stress. Adding vitamin C to the cryoprotectant doubled the survival ratio in Arabidopsis thaliana 60-h seedlings (seedlings after 60-h germination) cryopreservation. In this study, the metabolites and transcriptional profiling of 60-h seedlings were analyzed in both the control cryopreservation procedure (CCP) and an improved cryopreservation procedure (ICP) to reveal the mechanism of plant cell response to oxidative stress from cryopreservation. Reactive oxygen species (ROS) and peroxidation levels reached a peak after rapid cooling-warming in CCP, which were higher than that in ICP. In addition, gene regulation was significantly increased in CCP and decreased in ICP during rapid cooling-warming. Before cryogenic treatment, the number of specifically regulated genes was nearly 10 times higher in ICP dehydration than CCP dehydration. Among these genes, DREBs/CBFs were beneficial to cope with cryoinjury, and calcium-binding protein, OXI1, WRKY and MYB family members as key factors in ROS signal transduction activated the ROS-producing and ROS-scavenging networks including AsA-GSH and GPX cycles involved in scavenging H2O2. Finally, elevated antioxidant status and oxidative stress response in the improved dehydration enhanced seedling resistance to cryogenic treatment, maintained ROS homeostasis and improved cell recovery after cryopreservation.

ROS-induced oxidative stress and apoptosis-like event directly affect the cell viability of cryopreserved embryogenic callus in Agapanthus praecox.

KEY MESSAGE: Oxidative stress and apoptosis-like programmed cell death, induced in part by H 2 O 2 , are two key factors that damage cells during plant cryopreservation. Their inhibition can improve cell viability. We hypothesized that oxidative stress and apoptosis-like event induced by ROS seriously impact plant cell viability during cryopreservation. This study documented changes in cell morphology and ultrastructure, and detected dynamic changes in ROS components (O 2 (·-) , H2O2 and OH·), antioxidant systems, and programmed cell death (PCD) events during embryonic callus cryopreservation of Agapanthus praecox. Plasmolysis, organelle ultrastructure changes, and increases in malondialdehyde (a membrane lipid peroxidation product) suggested that oxidative damage and PCD events occurred at several early cryopreservation steps. PCD events including autophagy, apoptosis-like, and necrosis also occurred at later stages of cryopreservation, and most were apoptosis. H2O2 is the most important ROS molecule mediating oxidative damage and affecting cell viability, and catalase and AsA-GSH cycle are involved in scavenging the intracellular H2O2 and protecting the cells against stress damage in the whole process. Gene expression studies verified changes of antioxidant system and PCD-related genes at the main steps of the cryopreservation process that correlated with improved cell viability. Reducing oxidative stress or inhibition of apoptosis-like event by deactivating proteases improved cryopreserved cell viability from 49.14 to 86.85 % and 89.91 %, respectively. These results verify our model of ROS-induced oxidative stress and apoptosis-like event in plant cryopreservation. This study provided a novel insight into cell stress response mechanisms in cryopreservation.

Cryopreservation affects ROS-induced oxidative stress and antioxidant response in Arabidopsis seedlings.

Plant recovery status after cryopreservation by vitrification had a negative relationship to the oxidative stress induced by reactive oxygen species (ROS). Arabidopsis thaliana seedlings germinated for 48 h or 72 h with different survival tolerances were examined at five steps of cryopreservation, to determine the role of ROS (O2(-), H2O2 and OH) and antioxidant systems (SOD, POD, CAT, AsA and GSH) in cryo-injury. In addition, the effects of the steps on membrane lipid peroxidation were studied using malondialdehyde (MDA) as an indicator. The results indicated that H2O2-induced oxidative stress at the steps of dehydration and rapid warming was the main cause of cryo-injury of 48-h seedlings (high survival rate) and 72-h seedlings (no survival). The H2O2 was mainly generated in cotyledons, shoot tips and roots of seedlings as indicated by Amplex Red staining. Low survival of 72-h seedlings was associated with severe membrane lipid peroxidation, which was caused by increased OH generation activity and decreased SOD activity. The antioxidant-related gene expression by qRT-PCR and physiological assays suggested that the antioxidant system of 48-h seedlings were activated by ROS, and they mounted a defense against oxidative stress. A high level of ROS led to the weakening of the antioxidant system of 72-h seedlings. Correlation analysis indicated that enhanced antioxidant enzymes activities contributed to the high survival rate of 48-h seedlings, which could reflect by cryopreservation of antioxidant mutant seedlings. This model system indicated that elevated CAT activity and AsA content were determinants of cryogenic stress tolerance, whose manipulation could improve the recovery of seedlings after cryopreservation.

Cryopreservation of native Kazakhstan apricot (Prunus armeniaca l) seeds and embryonic axes.

BACKGROUND: Preserving the genetic diversity of Central Asia includes conserving wild apricots found in the foothills of several mountain ranges. These include primitive and genetically diverse populations with important characteristics for crop improvement. Apricot seeds have a short storage life, so cryopreservation of the seeds of wild populations is important for conserving the genetic diversity. OBJECTIVE: This study was to determine a suitable protocol for long-term storage. METHODS: This study tested a range of protocols using embryos and embryonic axes for storage of an important population of wild apricots and to determine if seed size and the distribution of moisture in the seed play a role in successful cryopreservation. RESULTS: Germination of scarified whole seed from trees in the Jungar population of Prunus armeniaca varied from 63 to 90 percent after 1 h in liquid nitrogen (LN) and was generally better at 7 % moisture content (MC) than at the original 14 percent MC. Embryos (4 percent MC) from stratified seed had only 33 % germination after LN exposure. Isolated embryonic axes from non-stratified seed germinated at 86 to 100 % following drying to 4 % or 7 % MC. Examination of three seed sizes determined that the MC of whole seed, embryos and isolated axes varied with the seed size and shape. MC of whole seeds and embryos decreased as size decreased, however, the axis MC did not. MC of medium-size seed was more evenly distributed between the axis and endosperm than in the larger or smaller samples. Cryopreservation of axes from medium-sized seed was good at any moisture content and a 1-h drying time was significantly better than 90 min. for axes of all seed sizes. Cryopreservation of axes using vitrification protocols initially designed for shoot tips produced germination similar to or lower than seed and axis drying techniques. CONCLUSION: We recommend storing apricot germplasm as unstratified seed dried to 7 % MC or as isolated embryonic axes.

Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus americana L.).

Climate change and global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are exposed to multiple stresses during the cryopreservation process. The current study was designed to evaluate the potential of melatonin to improve survival through the process of cryopreservation. Shoot tips of in vitro-grown plantlets and dormant winter buds of American elm were successfully cryopreserved in liquid nitrogen (LN) at -196°C under controlled environmental conditions following melatonin treatment and cold acclimation with either vitrification or encapsulation­vitrification protocols. Explants had optimal regrowth following cryopreservation when treated with the plant vitrification solution#2 (PVS2) for 10 min. Supplementation of both preculture and regrowth media with melatonin significantly enhanced regrowth of frozen shoots compared with the untreated control (P < 0.05). Approximately 80­100% of shoot explants grew under optimized conditions using melatonin-enriched media. Shoot tips of dormant winter buds consistently produced nearly 100% regrowth with both techniques. The main steps of the optimized protocol are14-day cold-acclimated cultures exposed to preculture medium with 0.1­0.5 lM melatonin for 24 hr, application of PVS2 for 10 min, rapid cooling in LN, rapid rewarming, removal of cryoprotectants, and recovery on a medium supplemented with 0.1­0.5 lM melatonin. Our results demonstrate the usefulness of the antioxidant melatonin for long-term storage of naturally resistant elm germplasm.

Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation.

Cryopreservation can be a safe and cost-effective tool for the long-term storage of plant germplasm. In Arabidopsis, the ability to recover from cryogenic treatment was lost as growth progressed. Growth could be restored in 48-h seedlings, whereas 72-h seedlings died after cryogenic treatment. Why seedling age and survival are negatively correlated is an interesting issue. A comparative transcriptomics was performed to screen differentially expressed genes between 48- and 72-h seedlings after exposure to cryoprotectant. Among differentially expressed genes, oxidative stress response genes played important roles in cryoprotectant treatment, and peroxidation was a key factor related to cell survival. Seedlings underwent more peroxidation at 72-h than at 48-h. A comprehensive analysis indicated that peroxidation injured membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Furthermore, the apoptosis-like events were found in cryogenic treatment of Arabidopsis seedlings. 48- and 72-h seedlings underwent different degrees of membrane lipid peroxidation during cryoprotectant treatment, and reducing the injury of oxidative stress was an important factor to successful cryopreservation. This study provided a novel insight of genetic regulatory mechanisms in cryopreservation, and established an excellent model to test and evaluate the effect of exogenous antioxidants and conventional cryoprotectants in plant cryopreservation.

Micropropagation of pear (Pyrus sp.).

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 µM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.

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.

Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation.

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more alpha- and gamma-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.

Cold acclimation improves regrowth of cryopreserved apple shoot tips.

Cryopreservation is important for safeguarding the genetic resources of apple germplasm in Kazakhstan, the center of origin for apples. In this study, conducted with five apple genotypes [Malus domestica Borkh. and Malus sieversii (Ledeb.) M. Roem] we determined cold hardiness and the effect of cold acclimation on shoot tip recovery following cryopreservation using two techniques. Apple shoot tips were cold acclimated (CA) for 0 to 6 weeks and cryopreserved using PVS2 vitrification and encapsulation dehydration (ED). Cold hardiness was indicated by the temperature at which 50 percent of the shoot tips were lethally injured (LT50). For non-acclimated shoots, LT50 ranged from -6.7 degree C to -9.3 degree C. These LT50 values resembled the natural cold hardiness of field grown plants and resulted in 10-12 percent regrowth after cryopreservation. Acclimated plantlets had LT50 values of -12 degree C to -15 degree C after 1 to 3 weeks CA, and after 3 weeks CA, cryopreservation resulted in 65 percent regrowth. There were no significant differences between the two techniques for regrowth of shoot tips after each cold acclimation period. Overall, 2 to 5 weeks CA produced high regrowth for each of the five cultivars tested. Three weeks of alternating temperature CA can be recommended as a standard protocol for Malus germplasm cryopreservation. These conditions resulted in moderate (60 percent) to high (80 percent) recovery for all five genotypes tested with both cryopreservation methods used.

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.).

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.

A comparative study of three cryopreservation protocols for effective storage of in vitro-grown mint (Mentha Spp.).

This study was designed to determine the response of diverse mint genotypes to three commonly used cryopreservation techniques. Four mints [Mentha x piperita nothosubsp. citrata (Ehrh.) Briq.; M. canadensis L.; M. australis R. Br, and M. cunninghamii Benth] were cryopreserved using three protocols: controlled rate cooling (CC), encapsulation dehydration (ED) and PVS2 vitrification (VIT). Regrowth of mint species following controlled rate cooling (93 percent) was significantly (P < 0.0001) better than encapsulation dehydration (71 percent) and vitrification (73 percent). All four genotypes responded well to the controlled rate cooling protocol but there was some variability with the other two protocols. Genotype specific response to the individual protocols showed that there were significant differences in the recovery of Mentha x piperita nothosubsp. citrata and M. australis with CC > VIT > ED. There were also significant differences in the recovery of M. cunninghamii and M. canadensis, with CC and ED significantly better than VIT. Regrowth of the shoot tips of these mints ranged from 60 percent to 95 percent for all but one treatment. The overall results of this study compare favorably to other techniques. These improved results may be due to a combination of favorable growth conditions, cold acclimation and recovery medium. Controlled rate cooling was the most successful technique for the storage of these diverse mint genotypes; however recovery of shoot tips from VIT and ED was high and these techniques could also be used for cryogenic storage of mint germplasm.

Cryopreservation of shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification.

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.

Evaluation of critical points in technology transfer of cryopreservation protocols to international plant conservation laboratories.

Cryopreservation of plant tissues in liquid nitrogen is now used for long-term conservation of vegetatively-propagated crops. Development of standard techniques for cryopreservation is important to the international plant-conservation community for successful implementation of storage protocols in diverse and internationally dispersed laboratories. Evaluation of the critical points of each preservation technique will greatly assist in developing and validating internationally-used cryopreservation protocols. The goals of this project were to assess critical points of two major cryopreservation techniques (PVS2 vitrification and encapsulation dehydration) during their transfer to international laboratories; analyze post-storage viability for each technique and location; and develop recommendations based on the assessments and data from the participating laboratories. Investigators from Germany, Kazakhstan, Poland and UK participated in a 2-week training workshop in cryopreservation methods after which the techniques were tested in the home laboratories of the participants. After one-year site visits by the technology trainers identified critical points in the protocols. Critical points were identified as 1) Cryogenic (cryoprotection, LN exposure, rewarming); 2) Non-cryogenic (plant health status, pre- and post-storage culture); 3) Operational (skills transfer, training, interpretation of procedures); 4) Facility (growth room, ambient conditions, media preparation, equipment). The most critical factors in all laboratories were culture health, operator skills and experience, and clarification of the technical details of the procedures. Final results showed that correction of critical factors improved the post-storage recovery in all the laboratories.

Cold storage and cryopreservation of hops (Humulus l.) shoot cultures through application of standard protocols.

The USDA-ARS National Clonal Germplasm Repository (NCGR) stores the global diversity of Humulus for the US Plant Germplasm System as trellised plants in a field genebank. In vitro storage and cryopreservation are now considered excellent ways to provide medium and long-term storage for plant collections. Developing a new cryopreservation or cold storage protocol for every accession or genus of large multi-crop collections can be a very time consuming and long-term activity. We propose that standard cold storage and cryopreservation techniques used for other temperate crop genera would be successful for additional crops with few modifications. This study was initiated to determine if a large collection of hops germplasm could be successfully stored with techniques developed for unrelated genera. In this study we characterized the response of diverse Humulus genotypes to in vitro storage under low light at 4 degree C following techniques used for strawberry and mint plants, and cryopreservation in liquid nitrogen by slow cooling with a pear protocol. The average storage time without transfer for the 70 genotypes evaluated was 14 +/- 3.5 months with a range of 6 to 26 months. Mean recovery of cryopreserved shoot tips of accessions with 1-wk cold acclimation was 41 +/- 18 percent and increased to 54 +/- 13 percent with 2-wk cold acclimation. This demonstrates that application of a well-tested standard technique can provide a quick start for storing additional germplasm collections.