AGROBACTERIUM-MEDIATED TRANSFORMATION OF A WALNUT CULTIVAR

Abstract

A protocol for Agrobacterium-mediated transformation has been developed for shoots explants of the Paradox (Junglans bindsii x Junglans regia) walnut rootstock 'Vlach', which is one of the most used rootstocks for walnut. The effect of different kanamycin concentrations on regeneration was evaluated and complete inhibition from control explants was observed when 70 mg/L kanamycin was applied. Also, the influence of antibiotic on the growth of the regenerated buds, micropropagated shoots and rooting was tested. Limiting concentration of kanamycin for growth of buds and shoots was 100 mg/L, where all of them died. Rooting decreased from 58% rooted shoots without selection to 1.7% when 30 mg/L of kanamycin was applied. Kanamycin at different concentrations from 65 to 150 mg/L was assayed in transformation experiments. After inoculation of explants with the strain EHA105/p35SGusint, buds that were able to regenerate in selective medium were rescued and transferred to medium with 75 mg/L kanamycin for development of elongated shoots. Transformation efficiency, based on PCR analysis of individual putative transformed shoots, ranged from 0.7 to 2.7% when antibiotic concentration was between 75 to 65 mg/L. To our knowledge, this is the first report of transformation of a commercial walnut genotype.