Cytoplasm Transfer of Nicotiana debneyi to N.tabacum by Protoplast Fusion

Abstract

A protoplast fusion method in which nuclei of a cytoplasm donor protoplast are inactivated was evaluated as an alternative method for introducing male sterility into tobacco (Nicotiana tabacum). Prior to the fusion, various doses of X-rays were irradiated to the cytoplasm donor of N.debneyi protoplasts, and 10kR was found to be effective to inactivate nuclear function of N.debneyi without damage of the cytoplasmic function. Following the fusion of the X-irradiated N.debneyi protoplasts and nonirradiated N.tabacum cv. Consolation 402 protoplasts, about 300 calli were subjected to regenerate. Almost all the regenerates exhibited N.tabacum characters concerning overall morphologies except flower, and had normal somatic chromosome number of N.tabacum (2n=48). Out of 318 regenerates 30 possessed male sterility. These male sterile regenerates exhibited floral phenotypic variation and could be classified into four types. One of these types (Type C) showed almost identical flower morphology with that of plants which were obtained from N.debneyi repeatedly backcrossed with N.tabacum The other three types showed intermediate morphologies between the normal and the type C. Their distinct flower morphologies were stably transmitted to the progenies, although some minor deviation from those of the parental regenerates was observed. All the progenies of the male sterile regenerates never segregated any fertile plants, indicating that the transmission of the male sterility was stable. Analysis of the large subunit of Fraction I protein (ribulose 1, 5-bisphosphate carboxylase, EC4, 1, 1, 39) encoded by a chloroplast gene revealed that 15 male sterile lines possessed the N.debneyi type chloroplast gene and 11 lines the N.tabacum type gene. These results may indicate that the cytoplasmic factors were transferred through the protoplast fusion, and that the male sterility is independent from chloroplast gene. The protoplast fusion, in which nuclei of a cytoplasmic donor protoplasts are inactivated by X-irradiation, may provide an alternative and efiicient method for obtaining male sterile tobacco, because male sterile tobacco plants could be obtained within 6-7 months from the protoplast fusion and under appreciable frequency.