Establishment of freezing-thawing-culturing system for super low temperature-freeze-preservation of genetic resources of horticultural crops.

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1. Effect of freezing treatments on rooting of shoot segments of asparagus(Asparagus officinalis L.)plantlets cultured in vitro. Enhancement of storage root-like white root formation was not recognized with no freezing and above -20-ﾟC, freezing, whereas remarkably occurred with liquid nitrogen freezing(super low-temperature freezing).2. Histological observation of shoot segments of in vitro-cultured asparagus plantlets after liquid nitrogen freezing, thawing and culture. Surviving cells contained cytoplasm deeply stained by Paragon staining solution, and could be discriminated from a frozen, dead cell.3. Effects of freezing solution-added DMSO or sugar on survival and growth of asparagus shoot segments(with a node ; from cultures)frozen in liquid nitrogen. When freezing solution contained DMSO alone, a reasonable DMSO concentration was 12% ; a high survival rate of 70% was obtained at 8-16% of DMSO. Addition of sorbitol made the survival rate more higher, and reasonable DMSO concentrations more wider(4-12%). A suitable concentration of sorbitol was 0.3M.4. Differences in kinds and contents of free amino acids and sugars. Seventeen kinds of amino acids were detected in all tissues. All of the free amino acids existed more quantitatively in nodes than in internodes. Glucose, fructose and sucrose were analyzed and contained more abundantly in a nodal part than in a internodal part.5. Plantlet regeneration of in vitro-growing potato cultures-excised nodal shoot segments frozen in liquid nitrogen. Small shoot segments(with one node)of 'Norin-ichigo', which were precultured on a 0.4M sucrose-containing medium, and treated with a freezing solution containing 5% DMSO and 0.4M sorbitol, showed a survival rate of 25%, and formed shoots and roots on almost of all the segments.