Feasibility Studies Using β -Glucuronidase as a Gene Fusion Marker in Apple, Peach, and Radish

Abstract

The udiA gene encoding the enzyme β -glucuronidase (GUS) appears promising as a genetic marker for early confirmation of successful plant cell transformation. Two strains of Agrobacterium rhizogenes and eight strains of A. tumefaciens were selected as hosts to carry a binary plasmid (pBI121) containing the marker gene encoding the GUS marker that is controlled by the CaMV35S promoter. Presence of plasmid pBI121 in the bacteria was confirmed by resistance to kanamycin, plasmid re-isolation, and restriction enzyme analysis. When the GUS enzyme was expressed in transformed plant cells, reaction with the histochemical substrate 5-bromo-4-chloro-3-indolylglucuronide (X-gluc) lead to blue pigment development. Expression of GUS in viable bacteria that had not been eliminated from transformed tissue caused problems with the early transformation detection in radish, peach, and apple stem sections by also producing a positive X-gluc color reaction. Putative transformation of apple xylem parenchyma callus was accomplished, as judged by resistance to kanamycin, opine analysis, GUS marker gene expression, and presence of the APH(3')11 enzyme. In this system, elimination of bacterial contamination was accomplished during multiple culture transfers on selective media. To be more useful as a marker, the GUS gene should be coupled with a promoter that will not be expressed by Agrobacterium. Parenchyma callus may serve as a primary screen to provide an efficient way of determining the ideal strain for transformation of deciduous tree fruit genera. In our studies, strain A281 consistently proved to be a vector superior to others tested.