Molecular Testing And Disease Resistance Analysis Of T1 And T2 Generations Of Red Clover Expressing Coat Protein Genes Of Alfalfa Mosaic Virus (AMV) And White Clover Mosaic Virus (WCMV)

Red clover (Trifolium pratense L.) virus disease is one of the most serious diseases apart from root eelworm, especially AMV (Alfalfa Mosaic Virus, AMV) and WCMV (White Clover Mosaic Virus, WCMV). Chemical control not only cause high cost, but also pollute enrironmrnt and had influence on animal products. For red colver, the fundental way to control virus disease is AMV and WCMV transgenic breeding. Some methods can be used to achieve multi-tolerance of transgenic plants: construct binary vectors or multiple vectors; transfer the other resistant gene to the transgenic plants with a foreign gene; make cross between two different transgenic plants having different transgenes.In this study, transgenic red clover plants with the AMV and the WCMV coat protein gene respectively were crossed and T1,T2 generation were obtained. The expression of the transgenes in the offsping and their disease tolerance were screened. The results showed as follows:1) Total DNA of T1,T2 transgenic red clover generation and non-transformed control was extracted by CTAB method and Plant Genomic DNA Purification Kit (TIANGEN) respectively. The results of agarose gel electrophoresis of 34 DNA samples of T1 generation, 30 DNA samples of T2 generation, and 1 DNA sample of control non-transgenic plant obtained showed clear single high molecular weight bands in all the samples. Analysis of RNA from the 30 transgenic red clover plants of T2 generation, in agarose gels showed that the 28S, 18S and 5S rRNA were all intact.2) The PCR was performed for the DNA from the leaflets of 34 putative transgenic red clover plants of T1 generation and a non-transformed control plant to detect the presence of virus coat protein genes and selectable marker gene. Of the putative transgenics, 5 were positive for both AMV and WCMV; 6 were WCMV positive; 1 was AMV positive; 3 were nptâ…¡positive.3) The PCR was performed for the DNA from the leaflets of 30 putative transgenic red clover plants of T2 generation and a non-transformed control plant. All of the putative transgenics were nptII positive, 10 were AMV and WCMV positive, 13 were WCMV positive, 3 were AMV positive.4) RT-PCR analyses were performed to detect AMV, WCMV and nptII transgene transcription among the T2 generation transgenic progenies. DNA fragments of expected sizes were obtained in most of the T2 generation plants. Among them, 9 expressed both AMV and WCMV CP transcripts; 3 expressed AMV CP transcript, 6 expressed WCMV CP trasncript and 3 expressed the nptâ…¡gene.5) Twenty-one transgenic red clover plants of T2 generation that expressed the viral coat protein genes, which indicated that foreign genes can be transferred to proheny by artificial hybridization and expressed, and foreign gene pyramiding can be achieved by artificial crossing.6) The T2 positive plants were conducted AMV and WCMV tolerance analysis. Compared with the control, T2 generation of transgenic red clover ahd significantly higher disease tolerance. The bioassay also showed that the disease spots of the transgenic plants were remarkably lower than that of the control.