The culture of manually isolated heterokaryons of Nicotiana tabacum and Nicotiana rustica

Abstract

Protoplasts derived from leaf mesophyll tissue of Nicotiana tabacum were fused with cell suspension-derived protoplasts of N. rustica. Heterokaryons were isolated using a micromanipulator and were cultured. Nuclear fusion was observed to occur in many of these heterokaryons after culturing them for a few days. Hybrid cell division was also observed. Fluorescein isothiocyanate (FITC) staining of N. rustica protoplasts prior to fusion did not interfere with subsequent hybrid cell division, and the FITC fluorescence was observed to persist beyond the first division stage of hybrid cells. From a total of thirty heterokaryons which were placed in a nurse culture of protoplasts of albino Petunia hybrida, thirteen green colonies were subsequently obtained of which six have regenerated somatic hybrid plants. Plants were characterised for their hybrid nature by analysis of vegetative and floral morphology, isoelectric focusing pattern of leaf esterases and Fraction 1 protein. All the six plants are nuclear hybrids. Chloroplast segregation appears to have occurred in these plants, with five having the Fraction 1 protein large subunit of N. rustica, and one having the large subunit of N. tabacum. Some of the plants possess sexual fertility.