Abstract: This paper reports an enzymatic maceration-osmotic shock method for isolation of tobacco embryo sac and its component cell protoplasts, and also a new method for fusion between single pairs of selected mesophyll protoplasts using polyethylene glycol (PEG) as on inducing agent. An integration of these two methods has led to the successful fusion of female gametoplasts with other kinds of protoplasts. The female gametoplasts described here, in a broad sense, include the egg cell (E), central cell (C) and synergid (S). One of the female gametoplasts was selected and fused with another female, male (generative cell, G) or somatic (mesophyll, M)protoplast. Various combinations were involved: E+S, E+C, E +G, E+M, C+C, C+S, C+G, C+M, S+S, S+G, S+M, etc. Briefly, the authors were able to choose any desired combination to realize single-pair fusion by the new PEG method. For the purpose of culturing such fusion products that were limited in number, the authors had done some preliminary experimets using mesophyll protoplasts as feeder cells. Two methods were adopted: the microdrop culture, and the millicell culture with feeder cells. The mesophyll protoplasts were precultured for 2—3 days in large for population expansion before they were used as feeder cells. One or several protoplasts were cultured in a microdrop or a millicell and were induced formation of small cell clusters. This result indicated that the culture methods might also be suitable for culturing the products from fusion of female gametoplasts and other protoplasts in this plant species.

Key words: Egg cell, Synergid, Central cell, Protoplast fusion, Nicotiana tabacum