Agrobacterium-mediated transformation of cotyledonary node of Vicia faba L.

Abstract

Broomrape represents one of the most intractable parasitic weed problems for faba bean in Mediterranean region. It causes enormous yield losses, and few control measures are effective. Recently, a novel strategy based on parasite-induced expression of a selective sarcotoxin IA polypeptide in transgenic tobacco was used to control broomrape. Generation of transgenic plants with broomrape resistance requires an efficient genetic transformation system. The present study explored the possibility of faba bean plant regeneration via direct organogenesis from cotyledonary node explants to improve transgenic efficiency. We studied the effect of some factors (genotype, Agrobacterium strains, Acetosyringone and thiols products). Cotyledonary nodes of two cultivars’ Lobab’ and ‘Aquadulce’ were inoculated and co-cultivated with two A. tumefaciens strains, EHA101 and AGL1, both carrying the pBIBhyg vector containing sarcotoxin IA and hptII genes. The results revealed a significant effect of genotype, genotype x acetosyringone interaction and addition of thiol compounds in co-cultivation medium on frequency of hygromycin resistant shoots of faba bean. Thus, the highest frequency of hygromycin resistant shoots was 38.3% with ‘Aguadulce’ inoculated by AGL1 and co-cultivated in medium with cysteine and without acetosyringone. The integration of sarcotoxin IA gene construct in transgenic plants was confirmed by PCR analysis.