An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants

Abstract

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.