Somatic hybridization between Nicotiana rustica and N. tabacum. II. Protoplast fusion and selection and regeneration of hybrid plants

Abstract

Protoplasts derived from cell suspensions of chlorophyll-deficient Nicotiana rustica L. cv. chlorotica and albino N. tabacum L. were fused by a polyethylene glycol (PEG) treatment. As early as 17 days after fusion bright green cell colonies appeared as a result of genetic complementation for chlorophyll synthesis in the hybrid cells only. For optimal hybrid production a low calcium level (1.05 mM) was essential in the PEG solution. Dimethyl sulfoxide (DMSO) at optimal concentration (2% v/v) when used with PEG increased the frequency of hybrid production over PEG alone. Temperature was not a critical factor in PEG-induced fusion except that where DMSO was used in conjunction with PEG, temperatures of 25 °C or greater were optimal. Over 65% of the hybrid cell colonies could be induced to undergo shoot morphogenesis on osmoticum-free (Nagata and Takebe) medium with 1.0 mg/Lbenzyladenine + 0.05 mg/L naphthaleneacetic acid and 1% sucrose. Under these conditions glucose (0.175–0.35 M) depressed morphogenesis. A number of physiological and environmental conditions were tested to elicit root formation in hybrid shoots but these failed to enhance the frequency of rooting obtained with hormone-free medium. Somatic hybrid plants of N. rustica + N. tabacum were produced thus by-passing sexual incompatibility mechanisms which partially isolate these species.