Introduction of antifungal genes in sunflower via Agrobacterium
Laura Mabel Radonic
Julián Marcos Zimmermann#
Marisa López Bilbao*
Financial support: National Agency of Scientific and Technological Promotion (ANPCyT), PICTO Nº 08-13164 and 08-12925, Argentina.
Keywords: antifungal genes, double expression cassettes, polyprotein cassettes, sunflower, transgenic plants.
#Present Address: Chacra Experimental Agrícola, PO box nº 8 (A4531WAA), Colonia Santa Rosa, Salta, Argentina. E-mail: firstname.lastname@example.org
There is evidence that overexpression of transgenes codifying antifungal proteins may confer protection to pathogen attack, and that this protection is increased due to the synergic effect of the expression of two or more genes. On the other hand it is well known that sunflower is a recalcitrant specie, highly difficult to be genetically transformed. In this context, the final aim of this project was to obtain sunflower plants expressing at least two antifungal genes, avoiding sequential transformation. The antifungal genes used encode for two enzymes that degrade the fungal wall (glucanase and chitinase), an osmotin and a ribosome inhibitor protein. Two types of transformation vectors were used: a more traditional system with a double cassette and a novel system producing a unique polyprotein with antifungal proteins released in equimolecular quantities. The polyprotein vector system generated hyperhydric shoots with necrotic areas and abnormal growth at the end of the tissue culture procedure, making impossible the use of this interesting vector in sunflower. Transformation assays carried out with the pHGC39 vector (including glucanase and chitinase genes) vector produced 0.83% efficiency, corresponding to 13 rooted shoots in kanamycin (Km) from a total of 1568 agroinfected shoots. T0 rooted shoots resulted positive by PCR analysis and were transferred to greenhouse to obtain their offspring. In addition, we corroborate the transformation protocol using Km as selective marker, previously described (Radonic et al. 2006) with a reporter gene, but in this opportunity with antifungal genes.