Figure 3.

T-DNA integration assays of rat5 and Ws plants. (A) Transient and stable GUS expression in Ws and rat5. Sterile root segments of Ws and rat5 plants were infected with the nontumorigenic Agrobacterium strain GV3101 containing the binary vector pBISN1 (21). Two days after cocultivation, the roots were transferred to callus-inducing medium (CIM) containing timentin. Three days after infection, half of the segments were stained with X-gluc to determine the efficiency of transient GUS expression. The other group of segments was allowed to form calli on CIM. After 4 weeks, these calli were stained with X-gluc to determine the efficiency of stable GUS expression. (B) T-DNA integration in rat5 and Ws plants. Suspension cells were derived from the calli generated from Ws and rat5 root segments infected with the nontumorigenic Agrobacterium strain GV3101 containing the binary vector pBISN1. The suspension cell lines were grown for 3 weeks (without selection for transformation) in the presence of timentin or cefotaxime to kill Agrobacterium. Genomic DNA was isolated from these cells, subjected to electrophoresis through a 0.6% agarose gel, blotted onto a nylon membrane, and hybridized with a gusA gene probe. After autoradiography, the membrane was stripped and rehybridized with a phenylalanine ammonia-lyase (PAL) gene probe to determine equal loading of DNA in each lane.