High-efficiency colony formation and whole plant regeneration from mesophyll protoplasts of Pelargonium x hortorum 'Panache Sud'

This study aimed to establish a plant regeneration system from protoplasts of Pelargonium X hortorum, efficient enough to be used for further direct gene transfer experiments. A rapid and efficient system that allowed high efficiency colony formation (40%) and whole plant regeneration (83%), as well as rooting within 4 months, was established using mesophyll protoplasts of the cultivar 'Panache Sud'. Protoplast culture in liquid medium was found to be better than culture on solid medium both for cell division and colony formation. The optimum density for high colony formation (31-40%) from viable cultivated protoplasts was 3-5 X 10(4) protoplasts ml(-1). Reducing the osmotic pressure and increasing the macronutrient and sucrose contents of the culture medium after the first week of culture facilitated the rapid development of colonies. The transfer of microcalli to mannitol-free callus-induction medium produced green calli in all cases. The highest frequency of bud and shoot regeneration from protoplast-derived calli (83%; 6.6 per callus) was obtained at a density of 3 X 10(4), on medium containing 0.2 mg I-1 indole-3-acetic acid (IAA), 1.0 mg I-1 zeatin and 0.1 mg I-1 thidiazuron (TDZ). The best results were obtained when the medium was gelled with Gelrite (R) and cultures were maintained under low light (12 mu mol s(-1) m(-2)). Sixty-five percent of protoplast-derived calli underwent bud and shoot regeneration and 2.6 rootable plantlets were obtained per callus after 3 weeks on elongation medium. All acclimatised plants grew normally and gave fertile flowers. However, flow cytometry on 42 plants showed that 40 of these were tetraploids, and only two were diploids, like the mother plant. This protocol can now be used in transformation experiments and applied to other genotypes to improve regeneration.