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  • 1.
    Waara, Sylvia
    Department of Physiological Botany, University of Uppsala, Uppsala, Sweden.
    Production and characterization of intraspecific somatic hybrids of potato (Solanum tuberosum L.)1991Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    A culture protocol has been developed for mesophyll protoplasts isolated from various d¡haploid clones of potato. A large number of call¡ was obtained after serial dilution of the cultures w¡th a suitable amount and type of medium at different stages of cell colony development.

    A polyelhylene glycol fusion procedure was developed that yielded up to 12 % heterokaryons. Using this fusion protocol hybrid cells have been manually lsolated, cultured and regenerated into plants. Fus¡on products were identified 2-3 days after fuslon by the dual fluorescence emission from the chloroplasts in mesophyll protoplasts (red) and the fluorescein diacetate stain in l¡ght and norflurazon bleached protoplasts (yellow-green). This fusion and select¡on strategy leads to the recovery of almost 100 % hybr¡d plants as established by isozyme analysls.

    Cytolog¡cal analysis of protoplast-derived plants and somatic hybrid plants revealed genetic changes as a consequence of protoplast culture and protoplast fus¡on. Twenty-three tetraplo¡d somatic hybrid plants were obta¡ned from 6 different calli and 9 of these were euploid.

    Morphological assessments of somatic hybr¡d plants of different ploidy levels demonstrated that tetraploid as well as several hexaploid somatic hybrids showed an increased vigour as compared to the parental plants. Most tetraploid somat¡c hybrids had a similar appearance although not all euploid plants were identical. Loss of vigour was evident in all mixoploid and octoplo¡d plants. These were stunted, weak and had an abnormal leaf morphology.

    The plastid type in hybrid plants was determined by RFLP analysis. All analysed hybrids had a cpDNA restrict¡on fragment pattern ¡dent¡cal to one of the parents wh¡ch contained either S. tuberosum or S. stoloniferum cpDNA. A slight preference for S. tuberosum plastids was observed in hybild plants. No influence on tho assortment of chloroplast by the norflurazon bleaching could be detected.

  • 2.
    Waara, Sylvia
    et al.
    Department of Plant Physiology, Uppsala University, Uppsala, Sweden.
    Tegelström, H.
    Department of Plant Physiology, Uppsala University, Uppsala, Sweden.
    Wallin, Anita
    Department of Plant Physiology, Uppsala University, Uppsala, Sweden.
    Eriksson, Tage
    Department of Plant Physiology, Uppsala University, Uppsala, Sweden.
    Somatic hybridization between anther-derived dihaploid clones of potato (Solanum tuberosum L.) and the identification of hybrid plants by isozyme analysis1989In: Theoretical and Applied Genetics, ISSN 0040-5752, E-ISSN 1432-2242, Vol. 77, no 1, p. 49-56Article in journal (Refereed)
    Abstract [en]

    Green mesophyll protoplasts of the dihaploid potato line 198∶2 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 67∶9 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%-4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.

  • 3.
    Waara, Sylvia
    et al.
    Department of Physiological Botany, Uppsala University, Uppsala, Sweden.
    Wallin, Anita
    Department of Physiological Botany, Uppsala University, Uppsala, Sweden.
    Ottosson, Agneta
    Department of Physiological Botany, Uppsala University, Uppsala, Sweden.
    Eriksson, Tage
    Department of Physiological Botany, Uppsala University, Uppsala, Sweden.
    Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants1991In: Plant Cell Tissue and Organ Culture, ISSN 0167-6857, E-ISSN 1573-5044, Vol. 27, no 3, p. 257-265Article in journal (Refereed)
    Abstract [en]

    A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina). 

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