A simple method for cryopreservation of shoot tips of Arabidopsis genotypes.

　　BACKGROUND: The use of the model plant Arabidopsis could be a valuable tool to elucidate the basic mechanisms involved in plant cryopreservation. OBJECTIVE: A simple and powerful protocol, independent of Arabidopsis genotypes, was established using a PVS2 protocol. MATERIALS AND METHODS: Two PVS2 (a and b), one PVS3 droplet-vitrification and one DMSO droplet-freezing protocol were tested with alternating temperatures during the growing phase of donor plants. RESULTS: PVS2 protocols, including cold acclimation of donor plants, resulted in highest recovery. The PVS2a protocol was successfully applied to a collection of different Arabidopsis genotypes with an average recovery of 94%. In addition, Differential Scanning Calorimetry confirmed the occurrence of glass transitions in the PVS3 and PVS2 protocols. CONCLUSION: The PVS2a protocol is suitable to screen the large collection of Arabidopsis mutants and transgenic lines with the aim to identify cellular functions associated with cryopreservation tolerance.

Melatonin-loaded alginate beads improve cryopreservation of yam (Dioscorea alata and D. cayenensis).

BACKGROUND: The cryopreservation of yam is constrained with many challenges. OBJECTIVE: This study tested the effects of melatonin on shoot tips of D. alata and D. cayenensis accessions exposed to water and liquid nitrogen (LN) stresses. MATERIALS AND METHODS: Sucrose pretreatment (0.3 M) was applied for 48 h before cryopreservation. Shoot tips were encapsulated in beads loaded with 0.75 M sucrose, with and without melatonin and desiccated over sterile dry silica gel for 0 - 9 h. RESULTS: The beads moisture content declined from 100% to ~ 13% after 9 h. The 3 h desiccation period without melatonin produced a significantly higher regeneration compared to 6 h and 9 h. Shoot tips with melatonin had significantly higher regeneration after 3 - 6 h desiccation compared to 9 h and the regeneration of all accessions after 6 h was >80%. Regeneration following 6 h desiccation and LN was significantly greater for melatonin-treated shoot tips compared to non-treated ones. CONCLUSION: The results indicate that melatonin significantly increased regeneration from 15% to 35%.

Cryopreservation for plant genebanks - a matter between high expectations and cautious reservation.

Cryopreservation is the best method of storing germplasm efficiently and safely, particularly for the maintenance of vegetatively propagated material. In IPK cryopreservation is used for potato, garlic, mint and yam. IPK collaborates with other cryobanks and research groups (ECPGR, COST, EURALLIVEG) and finds considerable differences in the adoption of cryopreservation between crops and their host institutes, depending on crop, local and historical circumstances. A better understanding of the long-term benefits of cryopreservation and its further integration into general genebank management is therefore needed. Recommended approaches include: comparative validation of methods between different laboratories, detailed comparisons of crop-based methods, economical analyses, efficient integration strategies of cryobanks by genebanks; including safe duplication of cryopreserved resources for the limitation of risk of loss Importantly, there has been recent progress in the development of quality management systems. Cryopreservation is, however, characterized by high expectations. Therefore, to ensure its sustainable and practicable use, basic knowledge of storage protocols must be combined with increased awareness of the rationales required to validate, implement and apply cryobanking technologies in working genebanks.

Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol.

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25 degree C or in alternating temperature regimes (25/15C or 25/-1C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25/-1C regime than in plants cultivated at 20C or 25C or at 25/15C regime for all nine tested accessions. The mean re-growth levels increased from 36 percent at 20C to 69percent at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89 percent. A pre-culture at alternating temperatures of 25/15C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions.

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.