Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of "clean" GM wheat containing only the foreign genes of agronomic importance.

Expressing an (E)-ß-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid.

(E)-ß-Farnesene (EßF) synthase catalyses the production of EßF, which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies. Many plants possess EßF synthase genes and can release EßF to repel aphids. In order to effectively recruit the plant-derived EßF synthase genes for aphid control, by using chloroplast transit peptide (CTP) of the small subunit of Rubisco (rbcS) from wheat (Triticum aestivum L.), we targeted AaßFS1, an EßF synthase gene from sweet wormwood (Artemisia annua L.), to the chloroplast of tobacco to generate CTP + AaßFS1 transgenic lines. The CTP + AaßFS1 transgenic tobacco plants could emit EßF at a level up to 19.25 ng/day per g fresh tissues, 4-12 fold higher than the AaßFS1 transgenic lines without chloroplast targeting. Furthermore, aphid/parasitoid behavioral bioassays demonstrated that the CTP + AaßFS1 transgenic tobacco showed enhanced repellence to green peach aphid (Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae, thus affecting aphid infestation at two trophic levels. These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EßF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.