Isolation and Reculturing of Protoplasts from Taxus Suspension Cultures

Paclitaxel (Taxol^®, Bristol-Myers Squibb) is an anti-cancer agent originally isolated from the bark of the Pacific yew, Taxus brevifolia.  Plant cell culture is currently being used to industrially produce paclitaxel because it is a renewable alternative to natural harvest, but suspension cultures are limited by the slow growth rate and variable product yields. Aggregates in suspension culture lead to the development of microenvironments, which can result in cellular heterogeneity.  To study the properties of Taxus suspension cultures on a single cell level, protoplasts can be isolated from the aggregates by incubating the cells in cell wall digesting enzymes. A procedure was developed for the reculturing of protoplasts isolated from Taxus suspension cultures. Digestion and purification conditions were chosen to minimize cell death and lysis due to changes in the osmolarity of the media.  Reculturing conditions, such as seeding density and media composition, were adjusted in order to maximize the growth rate and viability of the protoplasts over time.  To study the cells throughout the growth period, hemocytometry and fluorescence microscopy with image analysis were used to determine the culture density and viability. Initial results indicate that samples with higher seeding densities remain viable.  Further optimization of this reculturing procedure will allow for the transformation of protoplasts using polyethylene glycol (PEG)-mediated transformation for gene upregulation studies and the flow cytometric analysis of paclitaxel accumulation and distribution in the Taxus cultures.