Somatic cybridization between Nicotiana tabacum and N. repanda based on a single inactivation procedure of nuclear donor parental protoplasts

In the present study, mesophyll protoplasts isolated from N. tabacum var. K326 were inactivated by Rhodamine-6G (5 μg/ml, 15 min), and induced by PEG (6000) to fuse with those from N. repanda. All 31 plants regenerated from fusion-derived protoplast cultures showed uniform characteristics identical to K326 except flower morphology and male sterility. Morphological, cytological, biochemical investigation and RAPD analysis for nuclear genomes, as well as PCR amplification using primers for mitochondria atpA gene sequence of N. repanda revealed that they were cybrids with nuclei of K326 and cytoplasm carrying mitochondrial factors from N. repanda. The cybrids were further sexually backcrossed with K326 and other tobacco cultivars for three successive seasons. No revertants were observed among the progenies showing a strict maternal inheritance of the male-sterile phenotype. The methodology described in this paper is a simple and effective method for interspecific somatic cybridization in terms of rapid creation of tobacco cytoplasmic male-sterility (CMS) source with mitochondrial factors from wild species. It also seems to have a wide applicability, as there is no requirement for fusion partners. In addition, the creation of the new cytoplasmic male-sterile source is of much value for tobacco breeding programs in China.