The generation and characterization of Nicotiana cybrids

In this investigation the `donor-recipient' fusion method was used to investigate intraspecific transfer of organelles from donor to recipient Nicotiana species. Three mutant lines of Nicotiana tabacum were used in three independent fusion studies. Each of these lines was chosen for its ability to facilitate selection of cybrids by the identification of plastome and/or chondriome-controlled traits and included a streptomycin-resistant line, SR-1, a cytoplasmic albino line, GWG-6, and an alloplasmic male sterile line, CMS, carrying the cytoplasm of N. bigelovii in a N. tabacum nuclear background. In the first fusion between GWG-6 and SR-1 (6kRad), morphological analysis of 111 cybrid shoots from 12 calli suggested the existence of three distinct groups, all expressing the `recipient's' nuclear morphology. Only one of the calli analysed retained a mixture of chloroplasts and in only two other cases was more than one chloroplast-type plant differentiated from a single callus. These results indicated that the mixture of two chloroplast types is almost exclusively sorted into one or other parental type by the time the mature leaves are formed. In the second fusion between CMS and SR-1 (6kRad), deliberate selection for SR-1 parental chloroplasts enabled this investigation to concentrate on the cytoplasmic consequences at the mitochondrial level. Of the 21 calli obtained, plants from 14 were established in the greenhouse and characterized with respect to morphology, chromosome number, tentoxin sensitivity and chloroplast and mitochondrial DNA restriction profiles. As expected, all lines expressed SR-1 chloroplasts and, with two exceptions, CMS nuclear morphology. Eleven of the lines exhibited normal anther morphology and, with two exceptions, were self fertile or gave rise to self fertile plants. The two remaining lines were self sterile and bore the `feathery' anthers of the CMS parent. The MtDNA of seven second generation plants was apparently identical to the original donor's (SR-1) and the MtDNA of two other plants (with `feathery' anthers) was apparently identical to the original recipient's (CMS). Only two plants (self fertile) gave restriction profiles which differed from those of either parent. These results were indicative of a correlation between anther morphology and mitochondrial type and also demonstrated an independent assortment of chloroplasts and mitochondria from the fusion products. The third fusion between CMS (6kRad) and GWG-6 was very slow-growing. Morphological analysis of shoots from 12 calli confirmed the chloroplast segregation patterns observed in the first fusion study. The role of somatic cybridization in general and the relevance of irradiation for facilitating limited gene transfer is also discussed.