DEVELOPMENT OF BREEDING TECHNIQUES OF BOLETACEAE USING BIOTECHNOLOGY

1)建立了3种牛肝菌属（Boletaceae）菌根真菌：灰盖乳牛肝菌（Suillus grevillei）、褐盖乳牛肝菌（Suillus luteus）和洞穴牛肝菌（Boletinus cavipes）的原生质体制备技术。我们发现，一般来说，琼脂不利于这些真菌的原生质体再生，结冷胶更好。3）我们研究了各种物质如碳水化合物，以Ohta培养基为基本培养基，研究了氨基酸及相关化合物对三种真菌生长的影响。4）共收集牛肝菌科野生菌株26株。利用线粒体DNA的限制性片段长度多态性（RFLP）分析了11个来自日本不同地区的灰盖乳牛肝菌（Suillus grevillei）野生菌株的遗传多样性，发现11个菌株可分为3个类型。对褐乳牛肝菌原生质体再生菌株的形态学和氧化酶活性进行了研究，发现了许多突变。7）对褐乳牛肝菌进行了细胞学研究。9）以密花Pinua densiflora无菌苗为材料，用灰盖乳牛肝菌（Suillus grevillei）和褐盖乳牛肝菌（Suillus luteus）的液体培养物成功地产生了菌根。从华丽牛肝菌（Boletus ornatipes）子实体中提取得到一种新的大环内酯类酚类化合物，并对其化学结构进行了测定，命名为“ornatipolide”。

1) We developed techniques for preparation of protoplasts of three mycorrhizal fungi of Boletaceae ; Suillus grevillei, Suillus luteus and Boletinus cavipes.2) We developed techniques for regeneration of protoplasts of the three fungi. We found that, in general, agar is not good for protoplast regeneration of these fungi, and gellan gum is better.3) We studied the effects of various substances such as carbohydrates, amino acids and related compounds on the growth of the three fungi using Ohta medium as the basal medium.4) We collected 26 wild strains of Boletaceae.5) We studied the phylogeny of 11 wild strains of Suillus grevillei collected from various region of Japan using RFLP of mitochondrial DNA and found that 11 strains could be grouped into 3 types.6) We studied morphology and oxidase activities of regenerated strains of protoplasts of Suillus luteus and found many mutations appeared.7) We studied the cytology of Suillus luteus.8) We conclude that it is possible to breed these three fungi using protoplast techniques.9) We succeeded to produce mycorrhiza by inoculating sterilly grown seedling of Pinua densiflora with liquid cultures of Suillus grevillei and Suillus luteus.10) We extracted a novel macrolide phenolic compound from fruiting bodies of Boletus ornatipes and determined the chemical structure of the compound and named it as "ornatipolide".