Plant regeneration from protoplasts of micropropagated Pelargonium x hortorum ‘Alain’: effect of some environmental and medium factors on protoplast system efficiency

Abstract In order to further develop somatic hybridisations within the genus Pelargonium , a procedure of protoplast isolation, culture, and plant regeneration from protoplast-derived calli was set up. Micropropagated plants of Pelargonium x hortorum ‘Alain’ were used as donor plants. Among the explants tested (leaf, petiole, stem, root), young leaves were the best source for protoplast isolation (2×10 7 protoplasts per g FW), culture, and plant regeneration. These results were obtained, when enzymatic digestion was carried out under light during 6 h in a medium containing Murashige and Skoog mineral salts, 0.4% (w/v) cellulase Onozuka RS, pectinase Sigma 0.2% (w/v) and 0.5 M mannitol. The highest frequency of division (37%) and colony formation (30%) was observed when protoplasts were plated at 1–5×10 4 protoplasts per ml, under light, in liquid or solid medium containing either sucrose or mannitol as osmoticum. Shoot regeneration (12% of protoplast-derived calli) was obtained on a culture medium containing 0.2 mg l −1 IAA, and a combination of cytokinins (1 mg l −1 zeatin and 1 mg l −1 BAP l −1 ). Plants acclimatised in the greenhouse grew normally.