Production of male sterile interspecific somatic hybrids between transgenic N-tabacum (bar) and N-rotundifolia (npt II) and their identification by AFLP analysis

A protoplast fusion experiment was carried out aiming to obtain somatic hyb rid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt I I). The bialaphos resistance marker (bar) was introduced into A tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosp hinothricin acetyltransferase. A rotundifolia (npt II) was recovered after direct gene transformation of protoplasts by the pGP6 plasmid carrying the npt II gene for neomycin phosphotransferase. Both plasmids possessed 35S Ca MV promoters. Hybrid selection was based on dual bialaphoskanamycin resista nce. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, whic h confirmed their hybrid nature. IV. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids whi ch possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolia-like hybrids had nuclear DNA contents near that of N. tabacum (9.40 +/- 0.24 pg) or N. rotundifolia (5.29 +/- 0.36 pg), respectively, and were highly asymm etric. Other hybrids combined traits from the two species at various levels - N. tabacum habit or branched, similar to IV rotundifolia. Their leaves v aried in shape. The flowers of the hybrid plants were of N. tabacum or IV. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male ste rile. The nuclear DNA content varied from 8.90 +/- 0.30 to 19.57 +/- 0.33 p g. The data from the morphological and cytological analysis provided eviden ce that parental chromosome elimination in the hybrid clones was spontaneou s and not species-specific and that diploidization of the tobacco genome mi ght have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.