Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

GEORG MELCHERS 0 1 MARIA D. SACRISTAN 0 1 ANTHONY A. HOLDER 0 1 0 Department of Physiology, Carlsberg Laboratory , Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby 1 Max-Planck-lnstitutffir Biologie , Corrensstrasse 45, D-7400 Tt)bingen - Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca 2+ ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants. G. IVIELCHERSet al.: Potato and tomato hybrids i. INTRODUCTION The development of methods to fuse plant protoplasts reproducibly (19,22,43) has made it possible to produce cell hybrids and to regenerate from these in certain instances plants which are somatic hybrids. Most of the somatic hybrids produced so far can also be obtained by sexual hybridization (7,27,29,30,32,36,37,38). Recently somatic hybrids have been produced by fusion of protoplasts from species which cannot be sexually hybridized either in one direction (25), at a certain ploidy level or without the aid of embryo culture (37). In cases of pure gamete incompatibility somatic hybridization is likely to be successful, but in such cases sexual hybridization by in vitro fertilization can also be a way to achieve the hybrid. In cases of zygote incompatibility either alone or in combination with gamete incompatibility, the fusion of protoplasts is unlikely to lead to hybrid plants. As discussed by ZENKTELERand MELCHERS (45) in situ observation of embryo development after sexual crosses cannot give decisive information on the type of incompatibility involved since impaired zygote development can be caused by inhibition from maternal tissues. Such inhibition is absent in callus and embryo cultures. To the best of our knowledge sexual hybrids between potato (Solarium tuberosum, L.) and tomato (Lycopersicon esculentum Mill.) have not been described. The possibility to fuse the protoplasts between these two species is not surprising as incompatibility of cell fusion generally is absent even between animal and plant cells (6). Division and cell cultures from fusion products have been obtained in as diverse combinations as fusions of mouse and human cells or soybean (Glycine max L. Merr.) and Nicotiana glauca Grah. cells (18, 44). Regeneration of plants from hybrid callus cultures is unpredictable. In many plant species regeneration of plants from non-hybrid somatic callus tissue is not possible at present. One might expect that the chances of regenerating plants from somatic fusion products diminishes with increasing taxonomic distance between the partners involved, but the presently known somatic hybrids are too few to permit an evaluation of this hypothesis. Cases are known in which zygote incompatibility is caused by a single allele difference (15,16,26). It is of interest to note that a sexual hybrid between Petunia parodii W.C.S. and Nicotiana tabacum L. has been described (31). Although somatic hybridization between Petunia parodii and P. hybrida readily yielded plants (32) it has so far been impossible to regenerate plants from fusions ofN. tabacum and P. hybrida (45). It has been reported by MELCHERS(28) that protoplasts from cell lines of dihaploid Solanum tuberosum with 24 chromosomes and the diploid Lycopersicon esculentum with 24 chromosomes can be readily fused. This can be directly seen from the protoplasts of Figure 1 which have resulted from a fusion of a colourless potato protoplast and a green tomato protoplast. Since the dihaploid potato cell line has been kept as a submersed callus culture the protoplasts used in the experiments contain only colourless proplastids. The tomato protoplasts originated from mesophyll cells of green-house grown plants and therefore contain light green chloroplasts. The cytoplasms of the fused protoplasts in Figure 1 have not yet mixed and tomato-potato fusions can therefore be recognised as protoplasts with a colourless and a green part. The present communication presents chromosomal counts and an analysis of ribulose-1,5bisphosphate carboxylase (RuBPCase, E.C. 4.1.1.39) of putative somatic hybrid plants regenerated from callus tissue obtained in the above mentioned fusion experiments. Ribulose-l,5-bisphosphate carboxylase from higher plants can be dissociated into two types of subunit, isoelectric focusing (23,35) and peptide mapping (2,21) studies of the subunits from some Nicotiana species have shown that the large subunit (MW 55,000) is maternally inherited and that the small subunit (MW 12,000Abbreviations: RuBPCase = ribulose-l,5-bisphosphatecarboxylase:PVP = polyvinylpyrrolidone:SDS = sodium dodecyl sulphate. G. I~,q-ELCHERSet al."Potato and tomato hybrids 15,000) is inherited in a Mendelian fashion. The large subunit gene has been located on chloroplast DNA (4,10). The small subunit is synthesized on cytoplasmic polysomes (33) and transported into the chloroplast (5,13). Isoelectric focusing of the S-carboxymethylated RuBPCase has been used to provide phenc~ typic markers of chloroplast and nuclear genomes to study nuclear-cytoplasmic relationships in the evolution of plant species (3,8,9,11,39) and in the analysis of sexual interspecific hybrids (35). KUNG et al. (24) have also used this approach to investigate the result of interspecific protoplast fusion within the genus Nicotiana. The present results demonstrate differences in both the large and small subunits of RuBPCase from potato and tomato which have been used to demonstrate the hybrid nature of four plants produced by fusion o (...truncated)