J Integr Plant Biol. ›› 1994, Vol. 36 ›› Issue (7): -.

• Research Articles •    

Polyethylene GlycolInduced Fusion of Selected Pairs of Single Protoplasts

Sun Meng-xiang, Yang Hong-yuan and Zhou Chang   

Abstract: 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 CaCl2 · 2H2O (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 CaCl2 · 2H2O (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 CaC12 · 2H20 (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.

Key words: Protoplast, Fusion, Polyethylene glycol

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