Self-fertile cybrids Nicotiana tabacum (+Hyoscyamus aureus) with a nucleo-plastome incompatibility

Abstract

Cytoplasmic hybrids (cybrids) in a novel inter-generic combination, Nicotiana tabacum (+ Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and gamma-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated cybrids N. tabacum (+ H. nigrum). In contrast to the cybrids N. tabacum (+ H. nigrum), cybrids N. tabacum (+ H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+ H. nigrum) plants. Therefore, the cybrids N. tabacum (+ H. aureus) present a self-propagating system of Nicotiana (+ Hyoscyamus) nucleo-plastome incompatibility in its pure form.

Intergeneric somatic hybridization and its application to crop genetic improvement

“…The somatic hybrids have mainly two kinds of morphology, intermediate between the fusion parents, identical or similar to one fusion parent. The former is primarily seen in symmetric fusions and the latter in asymmetric fusions (Yan et al, 1999;Sigareva and Earle, 1999b;Varotto et al, 2001;Zubko et al, 2002;Hu et al, 2002a;Xia et al, 2003). However, plants with intermediate morphology have also been derived from asymmetric fusion, and regeneration of somatic plants with morphology identical to one of the fusion parents has been reported in many symmetric fusions (Kushnir et al, 1987;Hansen and Earle, 1997;Kisaka et al, 1997;Liu et al, , 2000Szarka et al, 2002).…”

“…Somatic hybrids between gamma-ray-irradiated Zizania latifolia and rice (Oryza sativa) had chromosome number equal to rice, but Southern analysis using both total genomic DNA and moderate-copy Z. latifolia-abundant DNA sequences as probes detected signal from the donor, indicating that possibly only chromosome segments from the donor have been integrated into the hybrids . In addition, creation of cybrids in many asymmetric fusions provides convincing evidence that exposure of the donors to irradiation prior to fusion could cause complete loss of donor chromosomes, in which only cytoplasm from the donor is transferred to the somatic hybrids (Glimelius and Bonnett, 1986;Kushir et al, 1987;Vardi et al, 1987Vardi et al, , 1989Hinnisdaels et al, 1991;Perl et al, 1991;Varotto et al, 2001;Zubko et al, 2002). Compared to symmetric fusion, asymmetric fusion strategies with irradiation of donor protoplasts lead to regeneration of normal plants, as demonstrated in fusions between Arabidopsis and Brassica (Hoffmann and Adachi, 1981), Lycopersicon hybrid and Solanum melongena (Guri et al, 1991;Liu et al, 1995a, b).…”

“…Treatment of the donor protoplasts with bromodeoxyuridine prior to UV and gamma irradiation could promote formation of highly asymmetric hybrids (Trick and Bates, 1996). In most of the asymmetric fusions, protoplasts that have been purified or are being isolated were irradiated before fusion (Somers et al, 1986;Vlahova et al, 1997;Kaimori et al, 1998;Zubko et al, 2002). But callus, leaf, cell suspension cultures or in vitro plantlets have also been used for irradiation prior to protoplast isolation (Tempelaar et al, 1991;Wolters et al, 1991;Hansen and Earle, 1997).…”

“…A number of selection strategies have been used for the selection of hybrids, of which utilization of biochemical mutants and antibiotics or herbicide resistance are used frequently. As far as biochemical mutants are concerned, mainly three kinds have been used, cytoplasmic chlorophyll deficiency mutant (Toki et al, 1990;Dragoeva et al, 1999), nitratereductase deficiency mutant (Kushnir et al, 1991) and albino mutant (Schoenmakers et al, 1993;Zubko et al, 2002). Difference in resistance to specific antibiotic(s), amino acid analogues or herbicides between fusion parents could also expedite hybrid selection when the fusion products are cultured in a medium supplemented with the abovementioned chemicals Vlahova et al, 1997;Kulawiec et al, 2003).…”

“…The above discussion indicates that it is necessary to minimize the introduction of genomes from the fusion parents into the hybrids, which is mainly done by breaking or fragmentating chromosomes using irradiation with X or gamma rays (Dudits et al, 1980;Zubko et al, 2002). Dudits et al (1980) produced the first intergeneric asymmetric hybrid between X-ray irradiated parsley (Petroselium hortense) protoplasts and tobacco protoplasts.…”