Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids

We report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR+, NR+, KmR, and NR-KmR). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred. © 1990 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after "gamma"-fusion

Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt60,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition. Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via "gamma"-fusion. © 1991 Springer-Verlag.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Translocation events demonstrated by molecular, in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts of Nicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts of Petunia hybrida. Molecular, cytogenetic and in situ hybridization analyses performed on the asymmetric somatic hybrid P1, previously identified as having a clearly metacentric chromosome besides a nearly complete Nicotiana chromosome complement, are reported. Meiotic analysis and in situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 of N. plumbaginifolia. Southern hybridization with an rDNA probe showed that only Nicotiana-specific rDNA was present. In situ hybridization experiments, using total genomic DNA of P. hybrida as a probe, demonstrated that the translocated fragment represented Petunia DNA. © 1992 Chapman & Hall.SCOPUS: ar.jinfo:eu-repo/semantics/publishe