フジマメとダイズの培養細胞由来のプロトプラスト培養および細胞融合について

Abstract

The effective isolation and culture methods of protoplasts derived from cell culture of soybean (Glycine max L. Merr. ‘aotyouhin’) and hyacinth bean (Dolichos lablab L. ‘waseakabana’) were investigated. Somatic hybrids from the protoplasts have also been obtained by polyethylene glycol (PEG) method.1. Protoplasts were isolated from 10 day-old hyacinth bean cell cultures suspended in liquid medium and 12 day-old soybean callus on agar medium. The enzyme mixture consisted of 3% Macerozyme R 10, 1% Pectolyase Y 23, 0.5% Pectinase•Sigma, 1% Cellulase Onozuka RS, 1% Driselase, 7mM CaCl2, 0.7mM KH2PO4, 5mM 2-(N-Morpholino) ethan sulfonic acid (MES), 0.2M mannitol and 0.2M sorbitol. Isolation of viable protoplasts required three-hour incubation at 31°C with shaking at 60rpm.2. Weak osmotic pressure was essential for inducing the division of protoplasts isolated from hyacinth bean cells. The addition of dimetylsulfoxide (DMSO) to the media was effective for regeneration of colonies.3. Fusion between hyacinth bean and soybean protoplasts was induced by the modified Menczel and Wolfe′s method (12). The mixture ratio 2: 3 of hyacinth bean and soybean protoplasts was necessary for obtaining hybrid cells.4. Approximately 400 putative somatic hybrid cells formed colonies on a suitable medium for hyacinth bean within 60 days after the fusion. Ninety percent of those putative hybrids were confirmed as real fusion products by Southern hybridization experiment using ribosomal DNA of rice as a probe.