The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10 5 protoplasts g −1 fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2 n = 2 x = 24) ‘C-13’ and diploid (2 n = 2 x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L 1–L 3). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci ( STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.