CONICET | Buscador de Institutos y Recursos Humanos

Since the first transgenic plants were obtained using Agrobacterium tuimefaciens system many other important species have been transformed. However monocots, including the major cereals crops were not amenable to Agrobacterium manipulation. Particularly, cereals have been very recalcitrant to genetic manipulation in vitro. Only after the development of direct methods for plant transformation, the stable transformation of rice and maize was informed. Wheat transformation has been achieved in several laboratories including our own. In this work we describe a rapid and efficient method to obtain fertile stable transgenic wheat plants, employing a gunpowder device built in our laboratory following the Sanford concept. Immature embryos of spring wheat used to obtain embryogenic calli. Calli were bombarded with microparticles coated with one of five plasmid constructions. We have used as selectable marker either bar and hpt genes, which confer resistance against the herbicide glufosinate and the antibiotic hygromycin, respectively. Also we employed the reporter nidÁ gene that encodes for the B-glucuronidase enzyme. Bombarded calli were cultured in the dark on selective pressure. After a short period of selection, calli were transferred to regeneration medium with a photoperiod of 14 h (10 W.m2). Resistant plants were rusticated in a Percival growth chamber. Screening to detect Ro transgenic plants were made by PCR assay. Results were confirmed by slot and Southern blots ofgenomic DNA. Transgenic plant were recovered in about 30 weeks. About a 37% of them were fertile and produced seeds. In sorne cases, embryo rescue of transgenic plants was carried out to accelerate the next generation while others seeds were allowed to mature. In five experiments where the bar gene was used as a selectable rnarker, we have obtained 27 Ro transgenic plants with an efficiency of 0.5 - 2.6 % of plant per calli bombarded.

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