Cold treatment of seeds, obtained from crosses between cultivars of T. gesneriana L., affects the developmental stage of embryos, which in turn influences the frequency of callus induction and the development of different callus types. Cold-treated, mature embryos and basal segments of in vitro-derived bulblets, were suitable explants for the initiation of regenerative callus on medium with 2,4-dichlorophenoxyacetic acid. The bulblets were initiated on flower-stalk segments from cold-stored bulbs of T. gesneriana 'Christmas Marvel.' Histological analyses of regenerative callus revealed the regeneration of bulb-like structures. The influences of culture medium, culture conditions, growth regulators and acetylsalicylic acid, an inhibitor of ethylene, on the initiation and establishment of regenerative callus cultures are discussed.
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 β-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 β-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.
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