the medium used in protoplast fusion experiments is

In cases where a species is polymorphic for CPDNA and at least one of the molecular variants is diagnostic for a second species, interspecific hybridization is a plausible explanation. That intraspecific variation in cpDNA is potentially indicative of hybridization is founded on the expectation that slowly evolving loci or genomes will produce greater molecular variation between than within species. Rieseberg (1995) suspected that a review of the literature at that time would reveal over 100 cases of intraspecific variation in CPDNA that could be attributed to hybridization and introgression. Rleseberg and Wendel (1993) list 37 cases of proposed hybridization in plants that include intraspecific variation in cpDNA, 24 (65%) of which they considered to be more » probable instances of introgression. However, documentation of intraspecific variation in cpDNA has become increasingly common and has been attributed in many cases to ''chloroplast capture'' following genetic exchange across species boundaries. This finding has been attributed to relatively low rates of sequence evolution and has been maintained as justification for the lack of intraspecific sampling in studies examining, relationships at the species level and above. « lessĮarly studies of evolutionary change in chloroplast DNA indicated limited variability within species.

Reasons for the rapid sorting out of the chloroplast types are discussed. In all, 46 F/sub 2/ progeny and 8 different F/sub 3/s were analyzed and each of these, with few if any exceptions, showed the same single subunit type as the F/sub 1/ and F/sub 2/ parent hybrid plants. A single plant retained a mixture of L and G chloroplast DNAs this later yielded six F/sub 2/ progeny from one branch, all of which were G type, and three asexual progeny from another branch, all of which had the L-type pattern. From one hybrid callus, two plants were regenerated, of which one had exclusively L-type more » large subunit and the other had exclusively G. Fourteen of the hybrids displayed the large subunit electrofocusing pattern characteristic of only one parent (eight L and six G). All the hybrids showed the combined small subunits of both parents, thus confirming the hybridity of each of the fusion products. langsdorffii (L), were analyzed for fraction I protein (ribulose-1,5-bisphosphate carboxylase/oxygenase) which consists of large subunit polypeptides coded by chloroplast DNA and small subunit polypeptides coded by nuclear DNA. Sixteen different mature interspecific parasexual hybrids, produced by fusing leaf protoplasts of Nicotiana glauca (G) and N. The unusual chromosome numbers found in the parasexual hybrids may have been due primarily to triple fusions (giving 60-66 chromosomes) followed by losses during callus growth, accompanied by selection for a particular range of aneuploidy (2n = 56 to 64) favorable for development of plantlets from calli. Seeds or progeny have been obtained from 16 of the hybrids. At meiosis mostly bivalents were formed and pollen fertility was high, averaging 84 percent.

The parasexual hybrids were examined cytologically and, instead of the amphiploid number 42, they were found to have a range of from 56 to 64 chromosomes, which accounted for the different characteristics observed. Each of the plants was shown to be more » a parasexual hybrid in that it formed tumors and the corolla, leaf, and plant habit were similar to, but somewhat different from, the amphiploid produced by cross pollination. Mature flowering plants were differentiated from 19 different calli, and more than one from three of these, making 23 regenerated plants in all. A total of 174 calli that grew on the hormoneless medium were obtained. The mixed population of protoplasts was grown first on an enriched medium (M3 of Kao, et al.) and was then transferred to a medium lacking phytohormones, which selects against the parental types. langsdorffi were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol.

The medium used in protoplast fusion experiments is