Transmission genetics of nicotiana hybrids produced by protoplast fusion 2. genetic constitution of nicotiana tabacum and nicotiana plumbaginifolia hybrid cell lines obtained using selective and nonselective screening methods

Gleba-Yu-Yu ; Kanevsky, I.F.; Cherep, N.N.

Plant Cell Tissue and Organ Culture 4(1): 19-28


ISSN/ISBN: 0167-6857
Accession: 006829775

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Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of N. plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of A. tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All 8 cell lines selected on the screening medium were identified as nuclear hybrids, while only 3 of the 8 evaluated clones obtained by mechanical isolation and cloning were nuclear hydrids; the rest of them were nuclear segregants of tobacco (1) or N. plumbaginifolia (4) type. Independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products that can be revealed only by using nonselective methods for hybrid screening is presented. The value of microisolation for the recovery of cytoplasmic hybrids is demonstrated.