Currently employed transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these selectable markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce selectable marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a selectable marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two selectable marker genes and successfully obtained progeny plants in which the second selectable marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.

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Alpha-linolenic acid (ALA, 18:3[?]9,12,15) and γ-linolenic acid (GLA, 18:3[?]6,9,12) are important trienoic fatty acids which are beneficial for human health in their own right, or as precursors for the biosynthesis of long chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2[?]9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and [?]6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with a FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fibre quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils. Hosted fileGao_et_al.doc available at https://authorea.com/users/296166/articles/425022-engineeringtrienoic-fatty-acids-into-cottonseed-oil-improves-low-temperature-seed-germinationplant-photosynthesis-and-cotton-fibre-quality

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