CELL COMPETENCE FOR AGROBACTERIUM-MEDIATED DNA TRANSFER IN PISUM-SATIVUM L

Distribution and properties of pea (Pisum sativum L.) cells, competent for Agrobacterium-mediated transformation were analysed by in situ hi stochemical detection of GUS (beta-glucuronidase) activity, 4 d after inoculation with engineered Agrobacterium tumefaciens. The vector syst em consisted of the hypervirulent disarmed strain EHA101 and the binar y plasmid pIBGUS, carrying an intron-containing, 35S-promotor driven g usA (or uidA) gene and two selectable marker genes. Cells competent fo r transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantif ication of factors influencing number and distribution of competent ce lls. In etiolated seedlings, competence for transformation decreased w ith the distance of the epicotyl explant from the shoot apex and was s pecifically induced by the exogenous application of auxins. Transient expression of gusA after Agrobacterium-mediated DNA transfer was drama tically reduced upon application of cell-cycle and DNA replication inh ibitors aphidicolin, colchicine and nalidixic acid. GUS expression aft er direct DNA transfer of double-stranded plasmid DNA (via PEG into pr otoplasts or via particle bombardment of epicotyl segments) was indepe ndent of cell-division/DNA replication. A GUS-positive mutant of EHA10 1 was constructed to allow in situ analysis of attaching bacteria with in the plant tissue. Attachment and invasion was inhibited by well-dev eloped cuticula but was restored after chloroform treatment of the tis sue surface. Moreover, no correlation was found between distribution o f attaching bacteria and the pattern of transformation-competent cells .