INTERSPECIES PROTOPLAST FUSION IN LARIX - COMPARISON OF ELECTRIC AND CHEMICAL METHODS

Larix was chosen for the study on interspecies protoplast fusion due t o its ability to regenerate plants from protoplasts derived from embry ogenic cultures. L. laricina line L2 was used in fusion experiments wi th either L. X eurolepis line L6 or L. X leptoeuropaea line L5. A meth od of unambiguous labeling of parental protoplasts prior to fusion ws developed using vital fluorescent dyes. Of a number of dyes tested, on ly rhodamine B hexyl ester chloride (R6) and 3,3'-dihexyloxcarbocyanin e iodide (DiOC(6)) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internall y labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental cond itions. The fusion products could he identified by dual fluorescence s everal h after the onset of fusion. Heterofusion frequencies of approx imately 18% and 6% in the presence of PEG and an electric field, respe ctively, were attained. Postfusion cultures between L. X laricina prot oplasts and protoplasts of L. X leptoeuropaea gave rise to cell coloni es and between L. laricina and L. X eurolepis, to mature somatic embry os.