Interspecific T-DNA transfer through plant protoplast fusion

Summary An attempt was made to transfer the T-DNA of Agrobacterium tumefaciens, previously introduced into plant cells, via protoplast fusion from one species into another. For the experiments two cell lines were used: firstly, a Nicotiana paniculata cell line transformed with the Agrobacterium strain B6S3. This cell line exhibits both hormone independent growth and synthesis of octopine as a result of the incorporated T-DNA from Agrobacterium. These two markers are dominant. The second cell line was the nitrate reductase deficient cnx-68 cell line of N. tabacum which contains an intracellular calcium oxalate druse. These two markers are recessive. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone free agar medium supplemented with 50 mM KClO₃. This compound is toxic for cells possessing nitrate reductase activity. From about 1.1×10⁷ cultivated protoplasts 18 cell lines survived the selection treatment. Of these seven exhibited the two dominant and the two recessive markers, whereas the others showed either only one or none of the recessive or only one of the dominant markers. In dot-blot experiments using species specific DNA clones of the donor and the recipient plant species it was confirmed that besides the T-DNA other nuclear genomic DNA of the donor species had also been transferred in various amounts. The possible consequences of these results for plant breeding programmes are discussed.