The chemical diversity of plant-derived natural products allows them to function in a multitude of ways including flavor enhancers, agricultural chemicals, and importantly, human medicinals. Supply of pharmaceutically active natural products is often a challenge due to the slow growing nature of some species, low yields found in nature, and unpredictable variability in accumulation. Several production options are available including natural harvestation, total chemical synthesis, semisynthesis from isolated precursors, and expression of plant pathways in microbial systems. However, for some medicinal natural products, such as the anticancer agent paclitaxel, where low yields in nature, chemical complexity and lack of knowledge of the complete biosynthetic pathway, preclude many of these options, plant cell culture technology is an attractive alternative for supply. Plant cell suspension cultures are amenable to scale-up, environmental optimization, and metabolic engineering. This review focuses on some of the key challenges in utilizing and commercializing plant cell culture suspension technology, with a focus on pharmaceutically active natural products. Recent research has been directed toward application of traditional strategies such as reactor design, cell immobilization, and enzyme elicitation as well as emerging strategies such as characterizing cellular heterogeneity and variability through flow cytometric techniques, metabolic engineering, and system-wide analysis.

Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol® (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regards to paclitaxel accumulation, cell cycle participation and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.

Objectives To characterize culture heterogeneity through investigation at genomic levels  To assess the metabolic stability of plant species and understand the progression of cell cycle and DNA content distribution and its control in greater detail through evaluation of DNA content / Genome size and protein content  To help establish relationships between primary and secondary metabolism to determine the position of the cell within the cell cycle to predict its further development trajectories

Plant cell cultures of Taxus provide the most reliable production methods for the anti-cancer drug paclitaxel. In order to comprehend the inherent culture heterogeneity and production variability in cell cultures, it is essential that the cellular metabolism is studied at the genomic level. Genomic stability in plant cell cultures is crucial as it affects cell growth and division, metabolite accumulation and protein synthesis. A rapid and efficient method to prepare nuclei suspensions from aggregated cell cultures of Taxus was employed. Methods were subsequently developed to simultaneously stain them for DNA and protein content using Propidium Iodide and Fluorescein Isothiocyanate respectively. Flow cytometry was used to analyze and quantify the DNA content and genome size of Taxus using known reference species as standards. Furthermore, their genomic stability was evaluated by correlating DNA content and genome size with cell size and complexity, protein content, and elicitation effects using multiparameter flow cytometry. These techniques to evaluate and correlate various culture characteristics can be very useful in designing superior bio processes for enhanced production.