Genetic engineering of the forage legume lotus corniculatus using Agrobacterium: mediated transformation systems

Abstract

Gene transfer vectors based on the Agrobacterium tumefaciens Ti plasmid were used to develop a successful disarmed Agrobacterium tumefaciens-mediated transformation method for Lotus comiculatus. A binary vector construct, pJIT73, was used during the development of the Agrobacterium tumefaciens transformation system due to its selectable (Aph IV, nos- neo) and scorable markers. The effects of the antibiotics geneticin (G-418) and hygromycin B were studied. Use of kill curves and selection delay experiments allowed potentially suitable selection pressure parameters to be proposed. Using such selection during transformation experiments led to further optimisation of this stage of transformation. The influence of plant hormones on the regeneration of Lotus comiculatus explants was investigated and a modification of an established protocol using leaf explants was introduced as an attempt to reduce the overall time of regeneration. Various explants were used but leaf pieces were chosen as the most suitable explant on which to focus research. So, through alteration of various stages, including length of cocultivation and subsequent decontamination within the transformation process, a successful method was developed. Experiments indicated the optimum Agrobacterium tumefaciens strain to be used with Lotus comiculatus was the disarmed Ach5 type, LBA4404(pAL4404). Transgenic Lotus comiculatus plants were produced which expressed the scorable marker β-Glucuronidase gene (GUS) and the selectable marker for hygromycin B resistance, AphIV. Gene transfer was confirmed by Southern blotting. The new Agrobacterium tumefaciens-mediated vector system was used to introduce the cowpea trypsin inhibitor gene (CpTi) into Lotus comiculatus. However, although there was evidence for transformed callus development, no shoots were induced. By the use of previously established Agrobacterium rhizogenes-mediated system, an attempt was made to introduce the pea lectin gene (psl) into Lotus corniculatus. Hairy root regenerants were produced but genetic transfer was unconfirmed and attempted investigation of the plant - Rhizobium symbiosis involving Lotus corniculatus was not fulfilled.