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

1.冷冻处理对芦笋试管苗生根的影响未冷冻和-20-ﾟ℃以上冷冻对贮藏根状白根形成的促进作用不明显，而液氮冷冻(超低温冷冻)显著促进了白根的形成。芦笋试管苗液氮冷冻解冻培养后茎段的组织学观察。存活的细胞含有经Paragon染色的细胞质，可与冰冻的死亡细胞区分开来。添加DMSO或糖的冷冻液对液氮冷冻的芦笋茎段(有节；来自培养物)存活和生长的影响。在冻存液中单独加入DMSO时，DMSO浓度以12%为宜，DMSO浓度为8~16%时，存活率高达70%。添加山梨醇使成活率更高，合理的DMSO浓度更宽(4-12%)。山梨醇的适宜浓度为0.3m.4。游离氨基酸和糖的种类和含量的差异。在所有组织中均检测到17种氨基酸。所有游离氨基酸在节内的含量均高于节间。葡萄糖、果糖和蔗糖在节部的含量高于节间。马铃薯试管培养的植株再生。冷冻在液氮中的节茎切段。‘Norin-Ichigo’的小茎段(单节)在0.4M蔗糖培养基上预培养，用含有5%DMSO和0.4M山梨醇的冷冻液处理，成活率为25%，并在几乎所有的茎段上形成了芽和根。

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.