灭蚊真菌——大链壶菌原生质体形成和再生的研究

采用1％纤维素酶与1％真菌脱壁酶混合液作脱壁酶,0.6mol/L山梨醇为渗透压稳定剂,从摇床培养的12—14小时菌龄的大链壶菌(Lagenidium giganteum)菌丝体获得原生质体。酶解3—5小时后,产量可达1.4—2.0×10~6/mL。并在双层培养基上初步实现了原生质体再生。

PROTOPLAST FORMATION AND REGENERATION OF THE MOSQUITO FUNGAL PATHOGEN LAGENIDIUM GIGANTEUM (OOMYCETES: LAGENIDIALES)

The facultative parasitic fungus Lagenidium giganteum is one of the most promising biological control agents of mosquitoes because of its good attributes, such as specificity for mosquito larvae, safety to non-target organisms, ability to recycle under suitable conditions, simple and rapid means of mass production. But its weak tolerance to salinity and organic pollution limits its application partially. Protoplast fusion and DNA vectored transformation are some of new techniques to modify fungi for different purposes and there have been many successful cases in other fungi. It is necessary and possible to use these techniques in the improvement of L. giganteum genetically and thus, to develop suitable procedures of making protoplast and having it regenerated is one of the basic steps. A fast and reliable method of obtaining protoplasts from L. giganteum mycelia is reported here. A Protoplast yield of 1.4-2.0 × 106/ml can be obtained in 3-5hours under observed optimal conditions, which are described as follows: Using 12-14 hour old PYG liquid cultures of L. giganteum; no mercaptoethanol is needed; 1 % cellulase plus lywallzyme as combined enzyme system; using 0.02 mol/L pH 6.0 phosphate buffer; petri dishes of 9cm in diameter as incubating container with 4ml of mycelia and enzyme mixture; 0.6mol/L sorbitol as osmotic stabilizer; pH range 5.0-6.0; temperature range 30-35℃. Three forms of protoplast release were observed. They can be released from (1) the swelling of hyphal tips, (2) broken lateral wall of mycelia at subapical or middle regions, (3) directly from fragments of broken mycelia. The diameter of protoplasts ranged from 5 to 30μm, mostly 15μm. The conditions for protoplast regeneration were also investigated. When incubated on two layer plates of 2% agar (bottom) plus 0.6% agaros (cover), the protoplasts regenerated and formed colonies successfully.