摘要
用微吸管选取单对原生质体，在含聚乙二醇（ＰＥＧ）的微滴中诱导融合。此法克服了常规的ＰＥＧ群体融合方法中的盲目性，能排除一方亲本原生质体自相融合和多个原生质体的融合，以及未融合的原生质体的混杂，保证融合产物来自选定的成对原生质体，从而使ＰＥＧ融合技术精确化。
A new method for polyethylene glycol (PEG) induced fusion between single pairs of selected protoplasts was developed.The protoplasts were prepared from tobacco leaves.Under an inverted microscope two defined protoplasts were selected with a hand made micropipette and transferred into a droplet of fusion solution containing 25% PEG (M.W.6000), 0 1 mol/L mannitol and 0 01 mol/L CaCl 2·2H 2O (pH 5 6).Slightly moving the pipette caused the protoplasts to contact and adhere to each other.the fusion pairs were then transferred to a solution containing 10% PEG, 0 35 mol/L sucrose and 0 01 mol/L CaCl 2·2H 2O (pH 5 6) for approximately 10 min, followed by subsequent washing with a solution containing 0 45 mol/L sucrose and 0 04 mol/L CaCl 2·2H 2O (pH 7—9).Compared with conventional fusion methods adopted to protoplast population, the present method can avoid either blind fusion of protoplasts belonging to one partner and fusion among multiple protoplasts, or the presence of unfused protoplasts, thus ensure the fusion to be precisely at the level of a selected pair of single protoplasts.Moreover, it is simple and convenient enough to show its potentiality for wide application in somatic hybridization and particularly in the case of small quantity of parental protoplasts such as in vitro intergametic fusion studies.
基金
国家自然科学基金
