Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally 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 conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.