Gene transfer into Indian cultivars of safflower (Carthamus tinctorius L.) using Agrobacterium tumefaciens

摘要

Direct and callus-mediated shoot regeneration following co-cultivation with Agrobacterium tumefaciens was obtained in two Indian cultivars of safflower (Carthamus tinctorius L.), A-1 and A-300. The procedure yielded 23 and 34 transformation events per 100 co-cultivated explants with direct and callus-mediated shoot recovery, respectively. The use of uid A gene in pKIWI105 that lacks a bacterial ribosome binding site precluded uid A expression in residual Agrobacterium cells. High levels of GUS activities were detected in selected putative transgenic calli and in shoot regenerants by histochemical assay. Western blot analyses using GUS antiserum and the NPT II expression assays confirmed the expression of marker genes in the putative transformants. Transgene integration was examined by PCR and dot blot hybridization of the transformants. Compared to controls, the efficiency of regeneration was markedly decreased subsequent to co-cultivation. Extended periods of callus-mediated regeneration led to hyperhydricity and vitrification of the shoots. Shoots regenerated from explants directly had, however, a normal appearance. The rooting response of regenerated shoots was poor and remains a continuing obstacle for safflower plant regeneration and transformation.