Application of imaging tools to plant cell culture: Relationship between plant cell aggregation and flavonoid production

Abstract

Image analysis tools were developed to measure biomass concentration, aggregate size and distribution, and pigmentation from anthocyanin-producing cell suspension cultures of ohelo (Vaccinium pahalae). The ex situ imaging system could image cell aggregates from 30 gm to 2 mm in diameter. The image analysis algorithm was based on extracted geometric features and morphological methods for biomass volume estimates, and hue, saturation, and intensity color characteristics for pigmentation estimates. Detailed information available from sampled cell culture images was validated by comparison to standard destructive manual measurements. Image analysis measurements revealed that pigment accumulation was negatively correlated with aggregate size. Although a substantial proportion of small aggregates remained colorless, the highlypigmented small aggregates, 18 to 238 gm in breadth, contributed over 70% of the culture anthocyanin production (mg L 1), despite their minor contribution to the overall biomass. The relative frequency of pigmented aggregates was higher in large-size aggregate classes; however, the pigmented sectors were mostly confined to only the periphery of the aggregates. As a result, large aggregate classes had only a minor contribution to overall culture anthocyanin yield.

Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures

“…Colored products such as anthocyanins have been measured directly via microscopy, and indicate that smaller aggregates favor higher metabolite production 19 . Another approach is to alter the starting aggregate size distribution, which has shown that smaller aggregates 20,21 or cultured single cells 22 result in higher metabolite production.…”

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

“…The predominance of aggregates formed by a small number of cells is a positive factor with regard to the production of secondary metabolites, especially anthocyanic pigments. Some authors have observed a reduction in anthocyanin production in enlarged cell aggregates (Madhusudhan and Ravishankar 1996;Pèpin et al 1999). Because anthocyanin biosynthesis is also strongly influenced by light intensity, which affects the activity of key enzymes involved in their biosynthetic pathway, such as phenylalanine ammonia-lyase and chalcone synthase (Zhang et al 2002), it is possible that increased aggregate size can cause a lack of light at the core cells.…”

Characterization of aggregate size in Taxus suspension cell culture

“…We assumed that the polar axis was an average of the major and minor axes, which essentially minimizes the irregularity of each aggregate by assuming the roundest shape possible. Several reports have used a similar averaging method (Edahiro and Seki 2006; Keβler et al 1999), while others have used the minor axis as the polar axis (Pepin et al 1999). Calculating volume using either the major or minor axis as the polar axis will shift the equivalent spherical diameter size distribution.…”

“…The effect of aggregate size on metabolic activity has been studied extensively, but no definitive trend regarding aggregate size and secondary metabolite production has emerged across species and cell culture systems. Larger aggregates have been shown to have a positive effect on secondary metabolite production (Edahiro and Seki 2006; Hulst et al 1989), a positive effect up to a critical size (Zhao et al 2003; Madhusudhan and Ravishankar 1996), or a negative effect (Pepin et al 1999; Bolta et al 2003). Additionally, aggregate size has been shown to affect culture growth rates (Forni et al 1999), rheological properties of the culture broth (Rodriguez-Monroy et al 2004), and is the most likely cause of heterogeneity in single cell populations (Naill and Roberts 2005a; Naill and Roberts 2005b).…”

“…Image analysis techniques consist of sample dilution and plating, followed by microscopic image acquisition and subsequent analysis using a variety of software programs (Pepin et al 1999; Rodriguez-Monroy et al 2004). While this method can allow for better resolution than filtration, the processing steps increase the likelihood of altering the aggregate size due to breakage.…”