Study On Genetic Transformation Of Sugarcane Mosaic Virus Coat Protein Gene Via Microprojectile Bombardment

Sugarcane mosaic virus(ScMV) is one of the three wide spread diseases in sugarcane planting, which can cause heavy yield lost to sugarcane. As the rising of the global air temperature and growth of susceptible commercial cultivars in large area sugarcane mosaic virus disease add great pressure on sugarcane production. Presently, traditional farming practice and chemical control are not efficient enough to control the disease. Plant breeding has played an important role in resistance to virus by offering varieties with disease resistance, higher yield and better quality.However, sugarcane is a vegetative propagation crop, which has highly heterozygous allopolyploid or aneuploid chromosomes. Its genetic background is very complex. Moreover, the antiviral inheritance of ScMV is controlled by polygenes, and the population of virus resistance breeding is separated very extensively. It is very difficult to aggregate target characters together, such as virus resistance and yield traits. Plant genetic engineering provides a new option in virus-resistant breeding. Genes coding disease-resistant and other growth traits can be cloned and transferred into cultivars to produce new genotypes with novel characters we wanted. Excellent varieties can be obtained without sexual hybridization cycle and a lot of time will be saved.In this study, two sugarcane varieties susceptible to ScMV and regenerative hardly were selected to set up regeneration systems. Four other sugarcane varieties susceptible to ScMV were selected to establish the geneticin(G418) screening systems and bombarded with pNUSCP containing ScMV-CP via microprojectile bombardment respectively. Three PCR-positive lines were obtained and one was proved transgenic sugarcane plants by southern blotting.The results were as follows:1. The calli regeneration systems for FN95-1702 and FN94-0403 were established. The effects of different concentrations and combinations of 6-BA, KT, NAA and active carbon on regeneration of the two sugarcane genotype calli had been studied by experiment of orthogonal design in this paper, and the optimum culture media were selected. The results showed that the optimum compounds and concentrations were MS+1.5 mg/L 6-BA+2.0 mg/L KT+0.2 mg/L NAA+1.0 g/L active carbon and MS+2.5 mg/L 6-BA+1.5 mg/L KT+1.0 g/L active carbon for FN95-1702 and FN94-0403 respectively.2. The screening systems for sugarcane calli with G418 were established. The results showed that the inhibitory effects of G418 on each six sugarcane calli were similar. The range of minimal inhibitory screening concentrations of G418 in the stages of callus, regeneration and root-formation of sugarcane were 20 mg/L~~-35 mg/L, 15 mg/L~35 mg/L and 25 mg/L respectively.3. Eight G418 resistance seedlings were obtained, of which three lines were PCR-positive and one was proved transgenic sugarcane plant by southern blotting.