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Breeding of edible mushroom using cell fusion technique

利用细胞融合技术育种食用菌

基本信息

批准号：
59560157
负责人：
MIURA Kiyoshi
金额：
$ 1.41万
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：
1984
资助国家：
日本
起止时间：
1984 至 1986
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-59560157/
关键词：
Electrofusion Protoplast Plating efficiency Auxotrophic mutant 栄養要求性株

项目摘要

Optimum result in obtaining protoplast suitable for an electrofusion technique and having a high viability as well,was observed when hydrolysis was conducted for 3 hours at 30゜C in an enzyme solution on mycelia cultured for 3 to 5 days in soyonion medium. The solution contained 2% of cellulase, 0.6% zymolyase, and 6 units of chitinase, respectively. The yield of protoplast was over 10/ml, the average plating efficiency being approximately 20%. Investigation of the ultrastructure of the protoplast showed that those dikaryon protoplasts with a dense concentration of cytoplasm, had a higher plating efficiency. In terms of fusion conditions,the best result was obtained by adding 0.5mM magnesium chloride and 0.1mM calcium chloride to a 0.5M mannitolsolution, with the pulse length and voltage set at 40 sec, and 4kV/cm, respectively. And this gave a fusion rate of approximately 60%. However, further investigations have yet to be conducted with regard to the decrease in aggregation of protoplasts in a high frequency electric field caused by the addition of electrolyte, and on influences of increased high frequency voltage on the hyphae regeneration, etc. Study of isozyme analysis exhibited that a specific isozyme pattern could be observed for esterase and malate dehydrogenase, thus constituting an efficient selection index for the screening of somatic hybridoma. The development of strains having markers, such as auxotrophic mutants is also under consideration. Screening tests are now being carried out although as yet no new somatic hybridoma have been obtained.

当在 soyonion 培养基中培养 3 至 5 天的菌丝体在酶溶液中于 30°C 下进行水解 3 小时时，观察到获得适合电融合技术并具有高活力的原生质体的最佳结果。该溶液分别含有2％的纤维素酶、0.6％的酶解酶和6单位的几丁质酶。原生质体产量超过10个/ml，平均铺板效率约为20%。对原生质体超微结构的研究表明，细胞质浓度较高的双核原生质体具有较高的铺板效率。融合条件方面,在0.5M甘露醇溶液中添加0.5mM氯化镁和0.1mM氯化钙,脉冲长度和电压分别设置为40秒和4kV/cm时获得最佳融合效果。这使得融合率约为 60%。但对于添加电解质引起的高频电场中原生质体聚集的减少、高频电压升高对菌丝再生的影响等还有待进一步研究。同工酶分析研究表明，酯酶和苹果酸脱氢酶可观察到特定的同工酶模式，从而构成体细胞筛选的有效选择指标。杂交瘤。具有标记的菌株（例如营养缺陷型突变体）的开发也在考虑之中。尽管尚未获得新的体细胞杂交瘤，但目前正在进行筛选试验。