Enhancing transgenic pea (Pisum sativum L.) resistance against fungal diseases through stacking of two antifungal genes (chitinase and glucanase)

Abstract

One way of enhancing and broadening resistance of plants to different biotic and abiotic stresses is to combine transgenes expressing several genes into a single line. This can be done using different strategies such as crossing, single vector with multiple genes, co-transformation, sequential transformation and IRES elements. In the present study conventional crossing method was used. Parental transgenic lines transformed via Agrobacterium tumefasciens-mediated gene transformation with pGreenII binary vector harbouring a bar gene as selectable marker in combination with the family 19 chitinase gene from Streptomyces olivaceoviridis for one line and 1,3-β-glucanase from barley (Hordeum vulgare) for the other line were used for crossing. Both chitinase and glucanase genes were cloned into pGreenII vector under the control of the constitutive double 35S-promoter from cauliflower mosaic virus. Progenies expressing the two genes were characterised at the molecular level using PCR, RT-PCR and Southern blot analysis, as well as segregation and stability studies of the respective expression levels. Leaf paint assay was used as functional test for herbicide resistant gene. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated. The synergistic effect of crossed plants was tested using in vitro assay which shows higher inhibition of spore germination.