Characterization of factors involved in the tissue growth recovery and the sustainability of the GUS activity in Hevea brasiliensis friable calli transformed by Agrobacterium tumefaciens

The influence of various parameters on the efficiency of Agrobacterium tumefaciens-mediated gene transfer and on the growth of inoculated Hevea brasiliensis friable calli was investigated. Transient GUS activity was more dependent on the duration of the co-cultivation step than the inoculum concentration. Washing of the inoculated tissues in a solution supplemented with tetracycline reduced GUS activity, probably reflecting the lower number of transformation events occurring after co-cultivation. Replacement of cefotaxime by ticarcillin in the decontamination medium delayed tissue browning and allowed part of the inoculated tissue to proliferate. Growth recovery of the whole inoculated tissue was stimulated when higher concentrations of growth regulators were introduced in the culture medium following co-cultivation. However stability of the GUS activity was adversely affected by these modifications in growth regulator concentration and timing of application. The transfer of smaller pieces of non-washed inoculated tissues onto the decontamination medium further improved growth recovery and allowed maintenance of the GUS activity.