ABSTRACT
This study deals with internodal development in vegetative plants of Nicotiana tabacum cv Samsun NN and its reflection in changes of the cellular competence for regeneration. During elongation of the internodes, the cells of the epidermis, subepidermis, and cortex exclusively expanded and increased their DNA content cell type specifically, generally from 2C to 4C. Cells with the 8C DNA content were found mainly among the cortex cells of mature internodes. The frequency of shoot regeneration (directly from subepidermal and epidermal cells together) on thin cell layer explants increased to an optimum along with elongation of the internodes and decreased in mature internodes along with aging. The frequencies of diploid shoots among the regenerants from elongating and mature internodes were high (88 and 75% on the average, respectively), indicating that most cells that had achieved the 4C DNA content generally retained the G2 phase of the diploid cell cycle. Shoots regenerated from explants of young plant material mainly had a vitrified appearance. The occurrence of this type of malformed growth was already determined by the physiological state of the cells in the internode and did not interfere with their acquisition of competence. Vitrification was unrelated to the degree of polysomaty of the internodal tissue. Using the occurrence of tetraploid root regenerants (from intermediate cortex-derived callus), up to a frequency of 50%, we show that the position in the plant where a majority of the 4C cortex cells switched to the G1 phase of the tetraploid cell cycle was at the transition from the elongation phase to the mature phase.
ABSTRACT
Several hybrid callus lines were produced through somatic hybridization between the diploid transformed Solanum tuberosum plant clone 413 (2n = 2x = 24) and a diploid wild-type plant clone of Nicotiana plumbaginifolia (2n = 2x = 20). The hybrid callus lines with subdiploid numbers of potato chromosomes were studied for karyotypic evolution as well as for segregation of the transformation marker characters (i.e. hormone autotrophy, opine synthesis, kanamycin resistance and ß-glucuronidase activity). Initially, these hybrids (cultured in kanamycin-containing medium) expressed all of the transformation characters. Six callus lines were selected for the establishment of cell suspension cultures; two of these were also used to initiate sublines, one from single cells of a suspension culture, and the other from callus-derived protoplasts. The cell suspension cultures and the sublines were cultured in kanamycin-free medium. After prolonged culture, karyotypic analysis of the various cell suspension lines revealed independent evolution of both parental genomes. Out of the six suspension lines, four showed a considerably reduced number of potato chromosomes as compared to the original hybrid callus lines, whereas the karyotypes of the individual sublines generally reflected the karyotypic diversity of the original cultures. The fate of the marker characters in various suspension cultures and sublines revealed independent segregation of the markers of TL-DNA (hormone autotrophy) and vector T-DNA (kanamycin resistance and ß-glucuronidase activity). Loss of the TR-DNA marker (opine synthesis) was observed only in combination with the simultaneous loss of the TL-DNA marker and the vector T-DNA markers. The results on segregation patterns of marker characters are discussed in the light of specific chromosome loss in the hybrid lines and gene linkage relationships.
ABSTRACT
Electrofusion was carried out between mesophyll protoplasts from the transformed diploid S. tuberosum clone 413 (2n=2x=24) which contains various genetic markers (hormone autotrophy, opine synthesis, kanamycin resistance, ß-glucuronidase activity) and mesophyll protoplasts of a diploid wild-type clone of N. plumbaginifolia (2n=2x=20). Hybrid calli were obtained after continuous culture on selection medium containing kanamycin. Parental chromosome numbers, determined at 2 months after fusion, revealed hybrid-specific differences between the individual calli. On the basis of these differences three categories of hybrids were distinguished. Category I hybrids contained between 8 and 24 potato chromosomes and more than 20 N. plumbaginifolia chromosomes; category II hybrids had between 1 and 20 N. plumbaginifolia chromosomes and more than 24 potato chromosomes; category III hybrids contained diploid or subdiploid numbers of chromosomes from both parents. The hybrids were evenly distributed over the three categories. After a 1-year culture of 24 representative hybrid callus lines on selection medium the karyotype of 10 hybrids remained stable, whereas 8 hybrids showed polyploidization of the genome of one parent, together with no or minor changes of the chromosome numbers of the other parent. Six hybrids showed slight changes in the hybrid karyotype. The elimination of chromosomes of a particular parent was not correlated to their metaphase location. The processes of spontaneous biparental chromosome elimination leading to the production of asymmetric hybrids of different categories are discussed.
