CYBRIDIZATION IN NICOTIANA-TABACUM-L USING DOUBLE INACTIVATION OF PARENTAL PROTOPLASTS AND POSTFUSION SELECTION BASED ON NUCLEAR-ENCODED AND CHLOROPLAST-ENCODED MARKER GENES

An effective selection system preceded by double inactivation of paren tal protoplasts was used to transfer Nicotiana suaveolens Leh. cytopla smic male sterility into a commercial tobacco (N. tabacum L.) breeding line. Mesophyll protoplasts from transformed plants of N. tabacum cul tivar WZ2-3-1-1 possessing a neomycin phosphotransferase II gene were used as the nuclear donors, while those isolated from N. suaveolens pl ants carrying a chloroplast mutation for resistance to spectinomycin, induced using nitrosomethyl urea, were the cytoplasm donors in somatic cybridizations. Prior to fusion, nuclear donor protoplasts were inact ivated with iodoacetamide or rhodamine 6G; while those of the cytoplas m donor were inactivated by X-irradiation. The resultant microcalli we re cultured on a shoot regeneration medium containing both kanamycin a nd spectinomycin to select cybrids. Only regenerants that had typical characteristics of the N. tabacum cultivar were selected for transfer to the glasshouse. Four putative cytoplasmic male-sterile (CMS) plants , out of a total of 44 regenerated plants transferred to the glasshous e, were obtained. Intraspecific somatic transfers of the CMS trait bet ween N. tabacum cultivars with distinctly different morphologies using single inactivation and nonselective shoot regeneration medium were d emonstrated. The implications of the results for practical tobacco bre eding as a means of circumventing lengthy backcrossing procedures are discussed.