Fertile somatic hybrids between transgenicNicotiana tabacum and transgenicN. debneyi selected by dual-antibiotic resistance

Abstract

A simple, yet effective selection system was used to produce fertile somatic hybrids betweenNicotiana tabacum andN. debneyi. This approach utilized transgenic antibiotic-resistantN. tabacum andN. Debneyi as donor plants for mesophyll protoplast fusions. Thirteen somatic hybrid plants were regenerated from calli capable of growth on medium containing both antibiotics. The majority of the hybrids displayed a range of leaf and floral morphologies and growth habits that were intermediate to those of the parental species, and had chromosome numbers varying from amphidiploid (2n = 96) to hypoaneuploid (2n = 60). Isoenzyme and RFLP analysis demonstrated the presence and expression of nuclear genes from both parents in all of the hybrids. Most plants are fully fertile. Thus, these plants differ from the malesterile tobacco 'cybrids' and alloplasmic lines produced by transferring theN. debneyi cytoplasm to tobacco. A nonrandom pattern of cytoplasmic segregation in the fusion products occurred with a bias towards the presence ofN. debneyi cp and mtDNA. Evidence for the presence of rearranged or recombinant cp and mtDNA in some of the hybrids was obtained. The somatic hybrids were successfully backcrossed to theN. tabacum parent and are now being tested for immunity to black root rot, a trait specific toN. debneyi, but not existent in theN. tabacum parental line.

Morphological and cytological characteristics of somatic hybrids of Nicotiana tabacum L. (+) N. megalosiphon Heurk. et Müll

“…Cytological analysis of Nicotiana somatic hybrids has revealed the recovery of plants with various ploidy levels and DNA rearrangements such as recombinations and translocations (De Vries et al, 1987;Hinnisdaels et al, 1991aHinnisdaels et al, , 1992. Considering the reported data, the chromosome number can vary from amphidiploid to aneuploid with limited elimination of one of the parental species chromosomes (Sproule et al, 1991), the presence of only one or two donor species chromosomes (Piastuch and Bates, 1990), or entire elimination of the donor genome and recovery of cybrids (Zelcer et al, 1978). Also, polyploidization of the recipient genome can result in double, triple and even hexaploid recipient genomes (Famelaer et al, 1989).…”

Production of male sterile interspecific somatic hybrids between transgenic N. tabacum (Bar) and N. rotundifolia (NPT II) and their identification by aflp analysis

“…It is generally considered that the more distant the parental species are, the more unstable hybrids are obtained. The cytological analysis of Nicotiana somatic hybrids revealed plants with various ploidy levels from amphidiploid to aneuploid, and DNA rearrangements such as recombinations and translocations (De Vries et al, 1987;Famelaer et al, 1989;Piastuch and Bates, 1990;Hinnisdaels et al, 1991bHinnisdaels et al, , 1992Sproule et al, 1991;Ilcheva et al, 2000). These events may be a result of chromosome interactions between the donor and the recipient, in vitro cultivation conditions (spontaneous polyploidization), protoplast fusion conditions (multiple fusions) or the effect of irradiation on donor species protoplasts in induced asymmetric hybridizations.…”

Morphological, cytological and molecular characterization of a novel symmetric somatic hybrid betweenN. tabacumandN. glauca

“…Successful regeneration of plants from cell fusion has been reported from about 50 interspecific and intergeneric protoplast combinations. Most somatic hybrids produced between N. tabacum and wild species in the genus Nicotiana were asymmetric, while only a few symmetric somatic hybrids have been obtained (Carlson et al, 1972;Douglas et al, 1981;Gleba et al, 1984;Bates, 1990;Sproule et al, 1991;Hagimori et al, 1993;Donaldson et al, 1995;Ilcheva et al, 2000). Using cell fusion, we have discovered a rare symmetric somatic hybrid between Nicotiana tabacum and N. glauca, which, to date, is the first symmetric interspecific hybrid of tobacco bred by cell fusion, as identified by random amplified polymorphic DNA (RAPD), SCAR and morphological characterization.…”