ANALYSIS OF CONDITIONS FOR AGROBACTERIUM-MEDIATED TRANSFORMATION OF TOBACCO CELLS IN SUSPENSION

We have developed an Agrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring beta-glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in the uidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterial vir-region was monitored using a heterologous gene construct composed of a virB promoter fragment from pTiC58 fused to the chloramphenicol acetyltransferase (CAT) gene of Tn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterial vir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number of Agrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivating Agrobacterium strain also carried the plasmid used to monitor vir induction, the frequency of transformation was reduced by as much as 97%.