Molecular And Cytological Identification Of Transgenic Rice Offspring Co-expressing RCH10 And AGLU1 Genes And Studies Related To Their Resistance To The Sheath Blight Disease

The sheath blight disease caused by the fungal pathogen Rhizoctonia solani K(u|¨)hn is one of the most destructive diseases of rice in China.Due to the lack of genetic source for effective resistance to the sheath blight disease,genetic engineening becomes important for providing an alternative approach to obtain broad and stable resistance.A previous work done in Zhejiang University introduced the rice chitinase gene RCH10 and the alfalfaβ-1,3-glucanase gene AGLU1 into rice cv. Taipei 309 by Agrobacterium-mediated transformation and obtained transgenic rice lines of YH-19 and YH-24 resistant to the sheath blight disease.In this study,we analysed the genetic stability of the transgenes in the T₆ generation of the YH-19 and YH-24 transgenic rice lines and the resistance of T₆ plants to the sheath blight disease. PCR analysis revealed that all the tested T₆ plants kept the NPTâ…¡marker gene where as 15%of the tested T₆ plants lost RCH10 gene or AGLU1 gene;an individual tested plant even lost both RCH10 and AGLU1.Northern blot analysis of RCH10 and AGLU1 transcripts and histochemical staining for GUS reporter gene revealed the expression of transgenes in the T₆ plants which kept the double transgenes.These T6 plants keeping the transgenes showed a remarkably higher resistance than that of the wide type rice plants to the inoculation of R.solani.In accordance with the resistance difference,these T₆ plants keeping the RCH10 and AGLU1 transgenes had much higher enzyme activities for both chitinase and glucanase than those in the wide type plants.Moreover,immunogold labeling for chitinase and glucanase showed that leaf cells of these T₆ plants contained a much higher amount of chitinase and glucanase than those in the wide type plants and that RCH10 and AGLU1 accumulated in the chloroplasts in the transgenic plants.Furthermore,we established a method to distinguish between R.solani mycelia on the leaf surface and those in leaf sheaths by differential fluorescence display observed by combining of light microscopy and fluorescence microscopy after staining of mycelia with trypan blue or chiorazole black E.In conclusion,this study identified the resistance of T₆ plants of YH-19 and YH-24 transgenic rice lines containing RCH10 and AGLU1 to the sheath blight disease and developed a differential fluorecence display method to distinguish fungal mycelia inside and outside of the leaf sheath.It provides new materials and methods for further studies on the interaction between rice plants and R. solani and to know the resistance mechnisms for transgenic plants to the sheath blight disease.