Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum
Debasis Patnaik, Dalia Vishnudasan and Paramjit Khurana*
Centre for Plant Molecular Biology, Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India
Plant regeneration studies in cereals have been undertaken in immature embryos,scutellum and also in immature inflorescence tissue. The wheat mature embryos can also be employed for callusing and regeneration, as they are available throughout the year and have presently been employed for transformation studies. An efficient and reproducible method for Agrobacterium-mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat(Triticum durum) is reported. Presence of acetosyringone at 200 µM concentration in the bacterial growth medium, inoculation medium and cocultivation medium was essential for achieving a 1.5– 2.0 fold increase in transient expression of the introduced gus gene. Successful generation of T. aestivum and T. durum transgenic plants at a transformation frequency ranging from 1.28 to 1.77% has beenachieved following 2–3 days co-cultivation using mature embryos and also mature embryo-derived calluses with binary Agrobacterium strain LBA4404 (pBI101 :: Act1) and LBA4404 (p35SGUSINT) respectively. Paromomycin and phosphinothricin served as effective selection agents as they did not adversely affect plantlet regeneration. Successful integration as well as inheritance of the transgene was confirmed bySouthern hybridization and PCR amplification in T0 as well as T1 generation. Optimization of this method facilitated the introduction of bar gene as a selectable marker conferring herbicide resistance as well as potato proteinase inhibitor gene (pin2) for insect resistance into wheat. Keywords: Agrobacterium, embryos, transformation, Triticum aestivum, Triticum durum, wheat. GENETIC transformationof crop plants by Agrobacterium-mediated co-cultivation is an efficient and costeffective method for gene delivery. Monocotyledonous plants, including important cereals were earlier thought to be recalcitrant to Agrobacterium-mediated gene transfer1,2, but the scenario has changed in the last few years. Consistent efforts by researchers on cereal crop plants have resulted in the development ofprotocols for efficient gene delivery via Agrobacterium into rice3,4, maize5, barley6 and wheat7.
*For correspondence. (e-mail: email@example.com) CURRENT SCIENCE, VOL. 91, NO. 3, 10 AUGUST 2006
One of the key points in these protocols has been the use of actively dividing cells/tissues such as immature embryos and immature embryo-derived calluses that were further co-cultivated withAgrobacterium in the presence of potent inducers of virulence genes8. Mooney and coworkers9 were the first to demonstrate the wound-independent in vitro attachment of Agrobacterium to wheat embryos. Subsequently, Chen and Dale10 reported a higher frequency of infection by incubation of exposed apical meristems of dry wheat seeds with Agrobacterium. Previous work from this laboratory also reportedthe transient expression of gus gene in meristematic leaf bases, calluses, mature seeds and mesocotyl punctured seedlings following co-cultivation with different strains and vectors of Agrobacterium tumefaciens11. Stable Agrobacterium-mediated transformation of wheat and transmission of the transgenes to subsequent generations have now been reported by many workers7,12–15. Nonetheless, thewidescale application of this methodology in diverse genotypes is still restricted. The present study thus focuses on the use of excised mature embryos and mature embryo-derived calluses as primary explants for Agrobacterium-mediated transformation of bread wheat (Triticum aestivum) and also the macaroni wheat (Triticum durum). The optimized protocol was subsequently used for the introduction of potato...