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 hybrid plants of transgenic Nicotiana tabacum (bar) (+) N. rotundifolia (npt II). The bialaphos resistance marker (bar) was introduced into N. tabacum via Agrobacterium tumefaciens using vector pGV1500 carrying the bar gene phosphinothricin acetyltransferase. N. 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 CaMV promotors. Hybrid selection was based on dual bialaphos— kanamycin resistance. Amplified fragment length polymorphism (AFLP) analysis of regenerated plants showed the presence of species-specific bands for both parents, which confirmed their hybrid nature. N. tabacum (bar) (+) N. rotundifolia (npt II) hybrids exhibited a great diversity in morphology. Fertile hybrids which possessed N. tabacum or N. rotundifolia morphology were recovered. Flow cytometric analysis revealed that the N. tabacum- and N. rotundifolio-like hybrids had nuclear DNA contents near that of N. tabacum (9.40±0.24pg) or N. rotundifolia (5.29±0.36 pg), respectively, and were highly asymmetric. Other hybrids combined traits from the two species at various levels—N. tabacum habit or branched, similar to N. rotundifolia. Their leaves varied in shape. The flowers of the hybrid plants were of N. tabacum or N. rotundifolia type, or had N. rotundifolia dimensions, pink with N. tabacum corolla or white with curly fused petals. All were self-sterile or male sterile. The nuclear DNA content varied from 8.90±0.30 to 19.57±0.33 pg. The data from the morphological and cytological analysis provided vidence that parental chromosome elimination in the hybrid clones was spontaneous and not species-specific and that diploidization of the tobacco genome might have occurred in some clones during in vitro culture. This reflects the genomic incompatibility between the two species.