Agrobacterium-mediated Transformation Of Dianthus Chinensis And Regeneration Of Transgenic Plants Containing The Maize Chitinase Gene

Rainbow pink (Dianthus chinensis) is a native plant in China, and is one of the importmant members of ground cover plant. But it is susceptible to Septori asp. when cultivated in wet and warm environment. Chitinase was induced by the infection of fungal as a defense response on plants. Although plants do not produce chitin, most fungi contain chitin as a major component of their cell walls. Chitinase can inhibit the growth of fungal pathogen. The application of chitinase plays an important role in genetic engineering of plant resistance to diseases recently. Many transgenic plants demonstrated significantly higher levels of resistance to diseases than control. In this study, a transformation system via Agrobacterium tumefaciences was firstly established, and we want to obtain Chitinase gene modified rainbow pink which can resist fungi by using genetic engineering technology. The results are as following:1. Regeneration systems of leavesThe second and thirth pairs of leaves were used for explant preparation. The leaves were cut into 0.5cm or so in length and precultured on MS1(MS+1.0mg/L2,4-D+0.1mg/LBA) for 3⁴d with their abaxial surface on the medium. Subsequently, explants were transferred to MS2(MS +1.0mg/LBA +0.3mg/LNAA). Shoots were formed at a frequency of 68.3%.2. A.tumefaciences-mediated transformation system of leavesIt was studied that several factors affected genetic transformation of rainbow pink(D.chinensis) by pBH2I/LBA4404. The optimized condition was that: leaves segments from the second and thirth pairs were precultured on MS1 for 3⁴d with their abaxial surface on the medium. The A.tumefaceins cells which OD₆₀₀ comes up to 0.5 or so were collected by centrifugation and resuspended in same volume of liquid MS containing 100 μmol/L AS. The leaves segments were transferred into A.tumefaceins suspension as described above for 6⁸min. After the A.tumefaceins cells on the surface of leaves segments were removed, the explants were then placed on a filter paper...