NONRANDOM CHLOROPLAST SEGREGATION IN NICOTIANA-TABACUM (+) N-RUSTICA SOMATIC HYBRIDS SELECTED BY DUAL NUCLEAR-ENCODED RESISTANCE

Nicotiana tabacum (+) N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant Nicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistant N. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessed N. rustica chloroplasts and only one had N. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar to N. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns of coxII hybridization identical with N. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.