Conditioned culture for protoplasts isolated from chrysanthemum: an efficient approach

An efficient approach was described for the culture of protoplasts isolated from chrysanthemum, and factors affecting the plating efficiency, defined as the number of protoplasts developing microcolonies divided by the number of cultured protoplasts, were investigated here. A yield of 4.86+/-0.22x10(5) mesophyll protoplasts per gram fresh weight were achieved by an enzymolysis procedure. The viability of fresh isolated protoplasts was 64.5+/-8.2% as confirmed by Evans blue staining. Sustained cell division and microcolony formation from the protoplasts were supported by modified Murashige and Skoog medium, complemented with 1:4 diluted conditioned medium harvested from three different callus suspension cultures. Results showed that the plating efficiency was obviously influenced by various concentrations of agarose and antibiotic, and significantly increased by conditioned medium harvested from chrysanthemum suspensions, while the other two species had a positive effect generally. Results also indicated that the growth-stimulative effect was reduced when the conditioned medium was stored for 6-14 days prior to use, suggesting the unstable characteristic of conditioning factors.