Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia.

After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only TL-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both TL- and TR-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the TR-DNA markers were found. Not each of the TL-, or TR-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the TR-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the TR-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained TR-DNA. It also confirmed the absence of the TL-DNA, demonstrating the independent integration of the TR-region of the octopine-type Ti plasmid pTi Ach5.