Influence of culture vessels on mesophyll protoplast yield of Pelargonium ssp

Somatic hybridization is a multistage process with many factors of influence. It requires a high yield of stable protoplasts. Plant protoplasts can be isolated from different plant tissues. Leaves of in vitro cultivated plants are one of the preferential sources. Using the same isolation protocol for four Pelargonium genotypes (Pelargonium 'Pink Capitatum', P. graveolens, P. × hortorum 'Antik Violet', wild type 585) the yield of leaf protoplasts from in vitro plants was very inconstant between the experiments, from high yield up to no protoplasts. To investigate the influence of culture conditions of the starting plants on the protoplast yield, plants were cultivated in Magenta vessels®, baby jars and 300 mL flasks. After 15 weeks, apex necrosis was observed in all vessels but with different strength: in Magenta® vessels it was lower, whereas plants cultivated in 300 mL flasks showed stronger symptoms. At the same time, these plants had a number of adventitious shoots with small leaves. Independently of the kind of culture vessel, 'Pink Capitatum' protoplasts could be isolated and they developed further in protoplast culture medium (PPM 1). The protoplast yield ranged between 1.6-5.0 × 10(5) protoplasts g-1 fresh weight (both from Magenta® vessel). Wild type 585 protoplasts were received also from all three plant culture conditions. However, getting protoplasts from Magenta® vessel plants involved some problems, the protoplast viability was low and a further development, at least a cell wall formation was not observed. Protoplasts of P. × hortorum could be isolated from all three variants. High number of protoplasts (1.8 × 10(6) g-1 fresh weight) was received from 300 mL flasks plants. Despite the high yield these protoplasts did not develop further. P. × hortorum protoplasts from Magenta® vessels showed cell divisions. A strong influence of culture vessels was found for P. graveolens protoplasts. Plants from 300 mL vessels gave stable protoplasts. These protoplasts showed a striking cell division activity. Plants from other vessels gave only once (from baby jar) viable protoplasts. The results show a considerable effect of plant culture conditions on Pelargonium protoplasts. Unfortunately, there is no vessel which would be optimal for all.