Interspecific chloroplast recombination in a Nicotiana somatic hybrid

Abstract

Genetic recombination between chloroplasts of two flowering plant species, Nwotiana tabacum and Nicotana plumbaginifolia, after somatic cell fusion is described. The parental lines differed in three cytoplasmic genetic markers. The N. tabacum mutant SR1-A15 was streptomycin-resistant, defective in chloroplast greening, and lincomycin-sensitive. The N. plumbaginifolia mutant LR400 was streptomycinsensitive, normal green, and lincomycin-resistant. Streptomycin-resistant clones in cell culture are identified by their ability to form a green callus on a selective medium. Streptomycin resistance in the SR1-A15 mutant could not be expressed due to defective chloroplasts. Protoplasts of the two species were fused, and calli grown from the fused population were screened for the expression of streptomycin resistance from the SR1-A15 line as the result of interspecific chloroplast recombination. A somatic hybrid, ptl4, expressed a new combination of the cytoplasmic genetic markers. In the ptl4 chloroplast genome three N. tabacum and four N. plumbaginifolia parent specific restriction sites have been identified, indicating that the ptl4 chloroplast genome contains at least six recombination sites.

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

“…These results have also demonstrated that in higher plants the high number of plastid DNA (ptDNA) copies in a plastid, and plastids in a cell, does not hamper the production of a stable and uniform plastome population of new genetic composition, provided segregation is directed by a suitably stringent selection pressure. Chloroplast gene transfer by recombination, however, seems to be accompanied by numerous, random recombination events scattered along the entire plastome (Fejes et a/., 1990;Medgyesy et al, 1985a). In experiments aimed at introducing defined changes, the use of cloned DNA sequences, and a transformation approach, is therefore, unavoidable.…”

“…Genetic transformation gives the most useful results when the wild-type information can be stably exchanged with the desired new DNA sequences via homologous recombination. Although plastid DNA recombination is a frequent phenomenon in the green alga Chlamydomonas (Lemieux etal., 1981), higher plants with stably exchanged plastid genes have only been produced through stringent cell selection for genetic complementation after protoplast fusion (Medgyesy et a/., 1985a). In distant species combinations, where the entire plastome is non-functional with an alien nucleus, a limited plastid gene transfer by recombination has been able to overcome this incompatibility, offering a genetic tool to identify genes crucial in plastid-nucleus co-operation (Thanh and Medgyesy, 1989).…”

“…The frequency of spontaneous antibiotic-resistant plastid mutants in protoplast-derived cell cultures of N. plumbaginifolia is 1-2 x lop4 under standard culture conditions (Cseplo and Maliga, 1984;Medgyesy et a/., 1985a). Therefore a test for an unselected marker (streptomycin resistance), after the selection for spectinomycin resistance, or an ab ovo selection for double resistance was used to distinguish plastid transformants in our experiments.…”

“…Seedlings and leaf calli were tested for their resistance to the appropriate antibiotics as described by Maliga et a/. (1982) and Medgyesy et a/. (1985a), respectively.…”

Cell-to-cell movement of mitochondria in plants

“…Absence of recombination between the plastid genomes suggested movement of intact organelles rather than naked, fragmented DNA (11,12). Indeed, chloroplasts rarely fuse (22), precluding recombination that enables new combinations of chloroplast genes.…”