Establishment Of Protoplast Transformation System Of Sclerotinia Sclerotiorum With Agrobacterium Tumefaciens And Biology Of Mutants With Abnormal Sclerotia

Sclerotinia sclerotiorum [Sclerotinia sclerotiorum (Lib) de Bary] is a soil-borne disease caused by a variety of important plant pathogenic fungi, whose host have many species, that it was reported that as many as 75 families, more than 450 species, which can cause many important plant Sclerotinia disease, such as rapeseed, soybean, sunflower and other oil crops, lettuce, celery, radishes and other vegetables, beans and other crops.It can cause a serious threat to yield and quality of crop. The pathogenic fungus is difficult to control because of whose survival ability of sclerotia. Now this disease was controlled mainly by chemical pharmaceutical and biological control.Sclerotia plays an important role in the life history of S. sclerotiorum, in 2006, The whole genome of S. sclerotiorum was sequenced, giving a new era to study the formation and development of sclerotia on molecular biology level of this pathogenic fungus. However, the molecular biology research of this fungus is dropping behind since the lack of efficient transformation system. At present, S. sclerotiorum can be transformed with polyethylene glycol-mediated protoplast and Agrobacterium-mediated bi-nucleate ascosporous, but all these approaches are consuming of time and troublesome. Agrobacterium mediated transformation system of S. sclerotiorum with hyphae is batter than above system, although it can produce a lot of transformants which were not stable. In order to overcome these disadvantages, an Agrobacterium mediated transformation system of S. sclerotiorum with protoplast was established, so far as now we have produced nearly 500 transformants, and on this basis, some studies about mutant with abnormal sclerotia as follows:First the preparation of protoplasts of S sclerotiorum, whom was next co-cultured with Agrobacterium strain EHA105 containing the plasmid pTFCM in the AS condition, after 2 days, the co-cultures were selected on PDA containing 500?g/ml cefotaxime and 30?g/ml hygromycin. Now more than 500 transformants have been successfully obtained. To identify these putative transformants, one pair of specific primers was designed based on the sequence of HPH, and were used for PCR amplification from the genomic DNA of putative transformants, and the results showed that an expected DNA segment could be amplified from all tested putative transformants. The transformants Sunf-M-N250 and Sunf-M-N156 with abnormal sclerotia was purified by protoplast regeneration to obtain renewable strains Sunf-M-N250-R10 and Sunf-M-N156-R10 having stable genetic traits, and some biology research were started about these two renewable strains as follows:Sunf-M-N250-R10 does not produce sclerotia on PDA medium,while doing on carrot medium, the mycelium of whose colony was thick, this strain have pathogenicity to rape, Arabidopsis thaliana and Brassica with hyphae growing rapid; For Sunf-M-N156-R10, few sclerotia were producted on PDA medium while many were produced on carrot medium, the hyphae grow slower than control, this strain have pathogenicity to rape, Arabidopsis thaliana and Brassica with hyphae growing rapid.