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

园艺作物遗传资源超低温冻融冻融培养系统的建立

基本信息

批准号：
02660020
负责人：
HARADA Takashi
金额：
$ 0.96万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

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.

1.冷冻处理对芦笋茎段生根的影响试管苗培养。不冻和-20 ℃以上冻藏对白根形成的促进作用不明显，而液氮冻藏（超低温冻藏）对白根形成的促进作用显著.芦笋试管苗茎段液氮冻融培养的组织学观察。存活细胞的胞浆被Paragon染色液深染，可与冰冻死亡细胞区分开来.冷冻溶液添加DMSO或糖对冷冻在液氮中的芦笋茎段（带节;来自培养物）存活和生长的影响。当冻存液中仅含DMSO时，DMSO浓度以12%为宜，当DMSO浓度为8-16%时，成活率可达70%。添加山梨醇使存活率更高，DMSO的合理浓度范围更宽（4-12%）。山梨醇的适宜浓度为0.3M。游离氨基酸和糖的种类和含量差异。在所有组织中检测到17种氨基酸。所有游离氨基酸在节中的含量均高于节间。对葡萄糖、果糖和蔗糖进行了分析，发现结部比结间部含有更丰富的葡萄糖、果糖和蔗糖。5.马铃薯离体培养的植株再生-液氮冷冻下切下的茎节段。在含0.4M蔗糖的培养基上预培养，并用含5%DMSO和0.4M山梨醇的冷冻液处理的“Norin-ichigo”的小芽段（有一个节）显示出25%的存活率，并且几乎所有的芽段都形成了芽和根。