A cryopreservation protocol based on air dehydration of explants placed on aluminium cryo-plates, termed D cryo-plate, was successfully developed for in vitro mat rush (Juncus decipiens Nakai) lateral buds. The buds of line 'Hiroshima 4gou(1)' with basal stems were dissected from multiple shoots and precultured overnight at 25°C on solid MS medium containing 0.3 M sucrose. Precultured buds were placed on aluminium cryo-plates, each one with 10 elliptical wells (2.5 mm long, 1.5 mm wide and 0.75 mm deep) and embedded in calcium alginate gel with 0.4 M sucrose and 1 M glycerol. Osmoprotection was performed by immersing the cryo-plates with buds for 30 min at 25°C in loading solution (2 M glycerol+1.0 M sucrose). Buds were dehydrated to 26% moisture content (fresh weight) by placing the cryo-plates for 2.5 h either in the air current of a laminar flow cabinet or in Petri dishes containing silica gel. Cooling was performed by placing the cryo-plates in uncapped cryotubes, which were immersed in liquid nitrogen. For rewarming, cryo-plates were immersed in liquid medium with 1.0 M sucrose for 15 min at room temperature. Under these conditions, regrowth of cryopreserved buds of line, 'Hiroshima 4gou (1)' was 93% after four weeks culture. The average regrowth of 20 mat rush lines was 88%. The D cryo-plate procedure will facilitate cryostorage of mat rush germplasm.
Cryopreservation using an aluminium cryo-plate was successfully applied to in vitro-grown strawberry (Fragaria £ ananassa Duch.) shoot tips. The shoots were cold-hardened at 58C for 3 weeks with an 8-h photoperiod. The shoot tips (1.5 -2.0 mm £ 0.5-1.0 mm) were dissected from the shoot and pre-cultured at 58C for 2 d on Murashige and Skoog medium containing 2 M glycerol and 0.3 M sucrose. The pre-cultured shoot tips were placed on the aluminium cryo-plate containing ten wells embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates in a loading solution (2 M glycerol and 0.8 M sucrose) for 30 min at 258C. Dehydration was performed by immersing the cryo-plates in plant vitrification solution 2 for 50 min at 258C. Then, the cryo-plate with shoot tips was transferred into an uncapped cryotube that was held on a cryo-cane and directly immersed into liquid nitrogen (LN). After storage in LN, shoot tips attached to the cryo-plate were directly immersed into 2 ml of a 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 15 strawberry cultivars reached 81%. This new method has many advantages and will facilitate the cryostorage of strawberry germplasm.
We modified the D cryo-plate protocol by paper mounting to the cryo-plate with alginate gel and shoot tips, and cryopreserved shoot tips of 13 potato genotypes (potato genetic resources in Mexico) using the revised and optimized D cryo-plate protocol. There were no significant differences in regrowth of cryopreserved shoot tips by addition of the paper mounting step to the base D cryo-plate protocol, besides a reduction in dropping shoot tips in steps during the whole procedure. Some steps of revised D cryo-plate protocol were optimized or reconfirmed, and the effect of optimizations such as cold-hardening, loading solution treatment, and postrewarming treatment on the regeneration of shoot tips was studied. This optimized protocol was successfully applied to 'B-71-240-2' and 12 additional potato genotypes, resulting in 70.0%-93.3% regrowth with an average of 82.8% and stable storage for 1 year. When introducing new cryopreservation techniques, modification and optimization of the method are required to adjust to each laboratory's circumstances. This optimized D cryo-plate method will facilitate cryobanking of potato and other plant genetic resources in Mexico for long-term preservation in a genebank.
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