Theor Appl Genet (1985) 70:590-594 9 Springer-Verlag 1985 A light sensitive recipient for the effective transfer of chloroplast and mitochondriai traits by protoplast fusion in Nicotiana P. Medgyesy, R. Golling* and F. Nagy Institute of Plant Physiology, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary Accepted February 2, 1985 Communicated by R. Hagemann Summary. A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wild- type donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with meso- phyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. Key words: Cytoplasm transfer - Light sensitive plastids - Male sterility - Nicotiana - Protoplast fusion Introduction Protoplast fusion provides a new approach in trans- ferring cytoplasmic organelles between plant species as compared to the conventional breeding methods (Galun and Aviv 1983). * On leave from: Department of Genetics, Section of Bioscien- ces, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic An increase in the ratio of cybrids (new nucleus-cytoplasm combinations) in the fusion derived clones was achieved by irradiating the cytoplasm donor protoplasts to inactivate their nuclei (Zelcer et al. 1978). On the other hand, selection for the cytoplasm to be transferred, using a selectable cytoplasmic mutation, made it easy to isolate fusion-derived cell lines with the desired cytoplasm (Medgyesy etal. 1980). Using a com- bination of these methods, cytoplasms of N. tabacum, N. megalosyt)hon and ?4. sylvestris have effectively been trans- ferred into N. plumbaginifolia, used as a model species in our laboratory (Menczel etal. 1982, 1983; Csrplo etal. 1984). These transfer experiments were based on selection for strep- tomycin or lincomycin resistant plastids derived from the lethally irradiated donor protoplasts. The small number of mutants of such types (Maliga 1984), however, limits the range of available cytoplasm donors. In this paper we report on the use of a cytoplasmic light sensitive N. plumbaginifolia line as a recipient, which made possible the efficient transfer of chloro- plasts from a wild-type donor. The selection was based on the greening ability of wild-type plastids from the irradiated donor protoplasts in the heterokaryon derived clones. In the experiment of Menczel et al. (1983) the selec- tion for streptomycin resistance (a chloroplast trait) resulted in the co-transfer of male sterility (a mito- chondrial trait) of the cytoplasm donor. In our experi- ment the co-transfer of chloroplasts and a mitochon- drial trait (alloplasmic male sterility) has also been ob- served. The role of the various selection systems in this phenomenon is discussed. Materials and methods Plant material Nicotiana gossei (2n = 36) plants were maintained on RM salts (Murashige and Skoog 1962) with 2% sucrose, solidified with 0.6% Bacto-Agar (RM medium), under culture room condi-