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  • 1
    ISSN: 1432-2242
    Keywords: Key words Rice ; Oryza sativa ; Porteresia coarctata ; Somatic hybridisation ; Flow cytometry ; Random amplification of polymorphic DNA (RAPD) ; Genome in situ hybridisation (GISH) ; Halophyte ; Chromosomes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Somatic hybrid plants were obtained following the electrofusion of rice (Oryza sativa L. cv ’Taipei 309’, 2n = 2x = 24) cell suspension–derived protoplasts with non-dividing leaf protoplasts of Porteresia coarctata (2n = 4x = 48), a saline-tolerant wild species. Fusion-treated protoplasts were plated on the surface of cellulose nitrate filter membranes, overlaying Lolium multiflorum nurse cells. The nurse cells were embedded in KPR medium containing 0.5 mg l−1 2,4–dichlorophenoxyacetic acid and semi-solidified with SeaPlaque agarose. Putative somatic hybrid cell colonies were selected on the basis of their growth, whereby faster growing colonies were transferred preferentially to MS-based medium with 2.0 mg l−1 kinetin, 0.5 mg l−1α-naphthaleneacetic acid, 30 g l−1 sucrose and 4.0 g l−1 SeaKem agarose to induce shoot regeneration. One hundred and nineteen regenerated plants were micropropagated clonally on MS-based medium containing 2.0 mg l−1 6–benzylaminopurine, 50 g l−1 sucrose and 4.0 g l−1 SeaKem agarose, prior to DNA extraction of plant samples. Putative somatic hybrids were initially identified by RAPD analysis, and 8 plant lines were selected for further investigation by flow cytometric ploidy determination and cytology. Plants of one line had an allohexaploid chromosome complement (2n = 6x = 72) and, following examination of its vegetative clones by GISH, were confirmed as somatic hybrids containing full chromosome complements of both O. sativa and P. coarctata.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1615-6102
    Keywords: Electrofusion ; Flow cytometry ; Heterokaryon selection ; Plant protoplasts ; Somatic hybridization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Protoplasts from a range of tissue sources for 21 species within the plant familiesCompositae, Cruciferae, Leguminosae, andSolanaceae were fused electrically to produce heterokaryons. Protoplasts were labelled by application of green fluorescein fluorescence to one parental protoplast population, and the red fluorescence of either naturally occurring chlorophyll or of rhodamine exogenously applied to the other parental type. Heterokaryon populations were identified, characterized and isolated using an EPICS 541 flow cytometer. Following culture, sorted heterokaryons from 11 of the 14 combinations investigated underwent division; in 5 of these combinations the heterokaryons developed into cell colonies, 4 produced callus and 2 gave rise to somatic hybrid shoots.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1615-6102
    Keywords: Electroporation ; Flow cytometry ; Macromolecules ; Protoplasts ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.
    Type of Medium: Electronic Resource
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