油菜에서 細胞質雄性不稔(CMS)係 cytoplast와 耐寒性 雄性可稔系 原形質體間 融合親和性의 genotype間 差異

Varietal difference in fusion-compatibility between CMS-donor-cytoplast and cold-tolerant CMS-receptor-protoplast of Brassica napus

Subprotoplasts containing enuclic(cytoplasts) and nucleic(miniprotoplasts) subprotoplasts were isolated from freshly isolated protoplasts of Brassica napus ‘CMS Jet Neuf’ cotyledons. Protoplasts were separated by centrifuging at 30, 000g for 60min at 12℃ through specific iso-osmotic density gradient containing colloidial silica gel(Percell), CaCl₂ and mannitol. Using this method highly purified cytoplast fractions with less than 20% contamination with nucleased protoplasts were obtained. Cytoplasts prepared by this method were suitable for the use in fusion experiments aimed at transferring enucleated CMS-Donor-Cytoplast and nucleated Cold-Tolerant-CMS-Receptor-Protoplast seperately. Protoplastfusion culture between enucleated CMS-Donor-Cytoplast of ‘CMS Jet Neuf’ and nucleated Cold-Tolerant CMS-Receptor-Protoplast with PEG 6,000 showed the first cell division 9 days after the start of culture and survived for 26 days there after.