Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice / 生物工程学报

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.

Marker-assisted selection and pyramiding for three blast resistance genes, Pi-d(t)1, Pi-b, Pi-ta2, in rice / 生物工程学报

G46B is a promising holding line used for three-lines breeding strategy in hybrid rice, but it is susceptible to blast disease caused by Pyricularia grisea. To improve its blast resistance, three rice varieties, Digu, BL-1, and Pi-4, with blast resistance genes, Pi-d(t), Pi-b, and Pi-ta2, respectively, were used to be crossed with G46B, and 15 plants with these three blast resistance genes, Pi-d(t)1, Pi-b, and Pi-ta2, were selected from their F2 and B1C1 populations via a marker-aided crossing procedure. Among them, four plants were heterozygotes in the three resistance genes, with the genotype of Pi-d(t)1 pi-d(t)/Pi-b pi-b/ Pi-ta2 pi-ta2; ten plants were heterozygotes in two of the three resistance genes, of which six with the genotype of Pi-d(t)1 Pi-d(t)1/Pi-b pi-b/Pi-ta2 pi-ta2, three with the genotype of Pi-d(t)1 pi-d(t)1/Pi-b pi-b/Pi-ta2 Pi-ta2, and one with the genotype of Pi-d(t)1pi-d(t)1/Pi-b Pi-b/Pi-ta2 pi-ta2; and only one plant was homozygote in two of the three resistance genes with the genotype of Pi-d(t)1 Pi-d(t)/Pi-b pi-b/Pi-ta2 Pi-ta2. These results demonstrate the capacity of maker-assisted selection (MAS) in gene pyramiding for rice blast resistance and its enhancement for the efficiency in rice resistance breeding.

Genome analysis of transgenic homozygous line "Minghui 63-Xa21" / 生物工程学报

By using rice SSRP, RAPD and AFLP molecular markers, the genome of rice transgenic line "Minghui 63-Xa21" was analyzed. 32 SSRP primers, 42 RAPD primers and 8 AFLP primers could produce obvious PCR bands in the analysis of at least 12 individual plants selected randomly from "Minghui 63-Xa21" T3 generation. Totally 550 PCR bands, equivalent to 550 genomic sites, were detected. Different individual plants of the transgenic homozygous line displayed almost the same PCR pattern. Compared with the control "Minghui 63", no difference was found in their PCR patterns. This indicated that the introduction of Xa21 into the genome of "Minghui 63" did not change these 550 genome sites and their heredity. Very few variant PCR bands were observed in some individual plants from both "Minghui 63-Xa21" and "Minghui 63". However, the variant percentage was equivalent between the transgenic line and the non-transgenic control line.