AGROBACTERIUM-TUMEFACIENS-MEDIATED EXPRESSION OF GUSA IN MAIZE TISSUES

To develop a system for Agrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of beta-glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity in A. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A 188 were inoculated with A. tumefaciens. Four days after inoculation, transient expression of GUS activity was found in mesocotyl segments originating from the intercalary meristem region. This GUS activity was specific to the vascular cylinder and was not found in the internal cortical or epidermal layers, nor was it found in mature mesocotyl tissue (segments 5 mm below the coleoptilar node). Transient GUS activity was also detected in leaf and coleoptile tissues of shoot segments, but not in the shoot apex per se or in leaves younger than the first leaf. Maize tissues inoculated with A. tumefaciens strains that harbour gusA-containing binary vectors but no Ti-plasmid did not show GUS activity, supporting evidence from previous work that vir gene activity was essential for the observed GUS activity. A. tumefaciens strains containing different types of Ti-plasmids were also tested. A strain harbouring an agropine-type Ti-plasmid was the most effective for expressing GUS activity in mesocotyl segments, whereas a strain harboring a nopaline-type Ti-plasmid was most effective for expression of GUS activity in the apical meristem-containing segment. These results indicate that different interactions occurred between the different A. tumefaciens strains and the susceptible plant tissues. Maize genotype specificity for GUS activity in mesocotyl tissues was observed; variations in the cocultivation medium had a profound effect on the frequency of expression of GUS activity.