Regeneration of normal and fertile plants that express octopine synthase, from tobacco crown galls after deletion of tumour-controlling functions

Crown gall, a neoplasmic transformation of plant cells, is caused by transfer and integration into nuclear plant DNA of T-DNA, a segment of the Ti plasmid of Agrobacterium tumefaciens (1–8). In plant cells, this T-DNA is expressed (9–12) and determines the morphogenetic growth properties of crown gall cells (12–16) and the synthesis of opines (17–21). The T-DNA in octopine crown galls is composed of either one or two fragments derived from a continuous Ti sequence. Thus far, all octopine tumour lines contain the TL fragment (14 kilobases (kb)), whereas TR-DNA (approx. 6 kb) can be present as an additional sequence (22–24). Foreign DNA, inserted in the T region of a Ti plasmid, is co-transferred to the plant genome (17,18,21). However, an important requirement for the use of T-DNA as vectors for plant genetic engineering is the regeneration of normal plants which stably maintain, express and sexually transmit the transferred DNA. We report here the isolation, from tumour tissues induced by mutant Ti plasmids, of morphologically normal plants that produce octopine synthase in all tissues. This enzymatic activity is inherited as a dominant mendelian marker (25). These regenerated plants retained and expressed only the octopine synthase gene of the T-DNA