| In order to produce selectable marker-free (SMF) transgenic rice containing antisense Wx gene, two T-DNA regions, with the HPT selectable marker gene and the antisense Wx gene, respectively, in two binary vectors or a super-binary vector, were constructed and co-introduced into several rice cultivars, both of Japonica and Indica, by Agrobacterium tumeficien(s)-mediated co-transformation system. For comparing the efficiency of co-transformation and elimination of the selectable marker gene between different co-transformation systems, it was carefully analyzed about the co-integration frequency of the selectable marker and antisense Wx gene in primary transgenic lines and, subsequently, the segregation of the two genes among individuals in TI generation. In addition, several factors influencing the efficiency in two-vector co-transformation mediated by Agrobacteriwn were also studied. The results were as follows.1. To study the factors influencing the efficiency in two-vector co-transformation, two binary vectors, containing the HPT marker gene and GUS reporter gene, respectively, were introduced into various Agrobacterium strains. Based on the co-integration frequency of the marker and reporter genes both in hygromycin-resistant calli and regenerated transgenic plants, the results showed that the efficiency of co-transformation was affected both by the different combination and density ratio of the two mixed Agrobacterium strains. It could achieve a higher co-transformation frequency in the combination of two same Agrobacterium strains, EHA105/pCAMBIA1300 and EHA105/pCAMBIA0301, than that in other combinations, by a density ratio of 1 '. 1. Simultaneously, the co-integration was more frequent in the density ratio of 1 '. 2 than that in other ratios when the above combination were used.2. Two binary vectors, containing the HPT selectable marker gene and the antisense Wx gene, respectively, were introduced into the same Agrobacterium strain EHA105, and used as a two-vector co-transformation system. The callus derived from rice immature embryos were infected with the two strains with a mix ratio of 1 : 2, and subsequently screened at the pressure of hygromycin. 92, 23, 25, and 8 independent transgenic lines were regenerated in four rice varieties 9983 (Japonicd), Xieqingzao, Longtepu and Zhenshan 97 (Indica), respectively. The results of PCR analysis showed that 9,3,2, and 1line of four varieties, respectively, were co-integrated by antisense Wx gene, and the frequency of co-transformation was 10.1%. PCR analysis also confirmed that the HPT selectable marker and the antisense Wx gene segregated among the individuals derived from part of the primary TO transgenic rice plants. Therefore, the SMF transgenic plants could be selected in the progeny of transgenic rice by this system.3. A super binary vector pYH592, containing two T-DNA regions harboring the HPT selectable marker gene and the antisense Wx gene, respectively, was used for co-transformation of rice by Agrobacterium. After co-cultivation with the callus and subsequently screening on the medium with hygromycin, 98 and 7 independent transgenic lines were produced in two cultivars 9983 (Japonica) and LongTePu (Indicd), respectively,. PCR analysis showed that there were 43 and 3 lines of these two cultivars, respectively, in which the antisense Wx gene was co-integrated. The frequency of co-integration by this co-transformation system was about 43.8%, which was significantly higher than that by the two-vector co-transformation system. Among the individuals derived from part of the co-transformed rice plants, the segregation of the HPT selectable marker gene and the antisense Wx gene were also confirmed.4. After the antisense Wx gene was introduced into rice by the above two co-transformation system, the amylose content in the mature seeds (Tl generation) of the co-transformed rice plants decreased with a various degree. In very few co-transformed plants derived from indica rice Longtefu, the lowest amylose level in... |
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