Optimization of protoplast-to-plant system for Pelargonium x hortorum 'Alain' and genetic stability of the regenerated plants

A system for establishing cultures and inducing plant regeneration from mesophyll protoplasts of Pelargonium x hortorum 'Alain' has been developed. Among several enzyme combinations, cellulase onozuka RS (0.4%) with pectinase (0.2%) proved to be optimal for protoplast division (37%) and colony formation (31%). Environmental conditions affected plant tissues differently, as leaf anatomy was different if obtained from jar or from test tube culture. Water loss was higher in test tubes than in jars. Moreover, yield and viability of protoplasts from mother plants propagated in jars were significantly higher than for those grown in test tubes. The concentrations of ammonium nitrate and calcium chloride in the culture medium appeared as a crucial factor for successful protoplast cultures. Generally, cell division decreased as ammonium and calcium concentrations increased up to 5.15 and 15 mM, respectively. Flow cytometry analysis showed a low variability (8%) in the relative DNA content between mother plants and protoplast-derived plants. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.