INTERSPECIFIC HYBRIDIZATION BY PROTOPLAST FUSION IN NICOTIANA - CONFIRMATION AND EXTENSION

Protoplasts of N. glauca and N. langsdorffii were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. The mixed population of protoplasts was grown 1st on an enriched medium (M3 of Kao, et al.) and was then transferred to a medium lacking phytohormones, which selects against the parental types. A total of 174 calli that grew on the hormoneless medium were obtained. Mature flowering plants were differentiated from 19 different calli, and more than 1 from 3 of these, making 23 regenerated plants in all. Each of the plants was shown to be a parasexual hybrid in that it formed tumors; and the corolla, leaf, and plant habit were similar to, but somewhat different from, the amphiploid produced by cross pollination. The parasexual hybrids were examined cytologically and, instead of the amphiploid number 42, they were found to have a range from 56-64 chromosomes, which accounted for the different characteristics observed. At meiosis mostly bivalents were formed and pollen fertility was high, averaging 84%. Seeds or progeny were obtained from 16 of the hybrids. The unusual chromosome numbers found in the parasexual hybrids were probably due primarily to triple fusions (giving 60-66 chromosomes) followed by losses during callus growth, accompanied by selection for a particular range of aneuploidy (2n = 56-64) favorable for development of plantlets from calli.