Application Of Plant Cell Cultures Research Articles

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Liquid–liquid extraction for recovery of paclitaxel from plant cell culture: Solvent evaluation and use of extractants for partitioning and selectivity

A major challenge in the production of metabolites by plant cells is the separation and purification of a desired product from a number of impurities. An important application of plant cell culture is the biosynthesis of the anticancer agent paclitaxel. Liquid-liquid extraction plays a critical role in the recovery of paclitaxel and other valuable plant-derived products from culture broth. In this study, the extraction of paclitaxel and a major unwanted by-product, cephalomannine, from plant cell culture broth into organic solvents is quantified. Potential solvent mixtures show varying affinity and selectivity for paclitaxel over cephalomannine. The partition coefficient of paclitaxel is highest in ethyl acetate and dichloromethane, with measured values of 28 and 25, respectively; however, selectivity coefficients are less than 1 for paclitaxel over cephalomannine for both solvents. Selectivity coefficient increases to 1.7 with extraction in n-hexane, but the partition coefficient decreases to 1.9. Altering the pH of the aqueous phase results in an increase in both recovery and selectivity using n-hexane but does not change the results for other solvents significantly. The addition of extractants trioctylamine (TOA) or tributylphosphate (TBP) to n-hexane gives significantly higher partition coefficients for paclitaxel (8.6 and 23.7, respectively) but no selectivity. Interestingly, when 20% hexafluorobenzene (HFB) is added to n-hexane, the partition coefficient remains approximately constant, but the selectivity coefficient for paclitaxel over cephalomannine improves to 4.5. This significant increase in selectivity early in the purification process has the potential to simplify downstream processing steps and significantly reduce overall purification costs.

Utilization of plant cell cultures for production of biochemicals

The potential biotechnological application of plant cell cultures for the production of various biochemicals and possible advantages of such a process are reviewed. To date, only one commercial process based on the culture of plant cells for the production of a chemical compound (shikonin) is in operation, and this process is briefly described. Problems related to the large-scale culture of plant cells are also discussed, and a comparison between microbial cells and cultured plant cells is used to illustrate some special features of the plant cells that complicate scaling up. Methods available for the establishment of high yielding cell cultures are described. Finally, the potentials of immobilized plant cells for the production of biochemicals are discussed. Various methods available for the immobilization of plant cells are briefly reviewed and some characteristics of such biocatalysts are given. The possible advantages of immobilized plant cells over suspension cells are pointed out.

Impact of subculture cycles and inoculum sizes on suspension cultures of Vitis vinifera

The commercial application of plant cell cultures is often hindered because of the instability of secondary metabolite biosynthesis, where the metabolite yield fluctuates and declines dramatically over the subculture cycles. This study proposed that such instability is due to the fluctuations of culture variables. To validate this hypothesis, the effects of the fluctuations of two culture variables (subculture cycle and inoculum size) on the biomass, anthocyanin biosynthesis, and intracellular carbon, nitrogen and phosphate during 10 continuous subculture cycles were investigated. The subculture cycle was fluctuated for 12 h over a 7-day cycle (6.5, 7, and 7.5 d), and the inoculum size was fluctuated by 20 % on the wet-weight basis of 2.00 g (1.60, 2.00, and 2.40 g). It was found that all the measured culture parameters fluctuated over the 10 subculture cycles. The fluctuation in terms of inoculum sizes had a greater effect on the stability of anthocyanin biosynthesis in suspension cultures of V. vinifera. Among all the subculture conditions investigated, 7-d subculture cycle and 1.60-g inoculum size were the best for maintaining relatively stable anthocyanin production. The anthocyanin yield showed a negative correlation with intracellular sucrose content or intracellular total phosphate content.

Age-related responses of suspension cultured Taxus cuspidata to hydrodynamic shear stress

Hydrodynamic shear sensitivity is considered one of the obstacles for large-scale plant cell culture. Understanding the mechanism of shear sensitivity might assist the industrial application of plant cell culture. Here we investigated the responses of suspension cultured Taxus cuspidata in different culture phases under shear stress using a Couette reactor. It was found that the pH in medium and peroxide hydrogen (H 2O 2) in cells increased more rapidly in the exponential phase than that in lag phase under shear stress. The pH and H 2O 2 concentration in the exponential phase were also higher than those in the lag phase. Inhibition studies showed that there existed a 30–45 min delay in the action time of G-protein, Ca 2+ channel and phospholipase C of T. cuspidata cells in the lag phase than that in the exponential phase. Age-related different membrane fluidity and H +-ATPase activity may partially contribute to the observed responses. These early responses might be indicators for selecting shear-resistant cell lines and for cell damage caused by shear stress.

Regulating the composition of anthocyanins and phenolic acids in a sweetpotato cell culture towards production of polyphenolic complex with enhanced physiological activity

Recent confirmation in the scientific literature of the health-promoting qualities of natural food colorants prompted their increased application in food products. Anthocyanins, a large group of natural pigments, include compounds with diverse molecular structures and different physiological activities. Therefore, selection of specific anthocyanins with enhanced bioactivity for application in functional foods needs to be addressed. Biotechnology offers an opportunity for ongoing production of selected phytochemicals for application in pharmaceutical, cosmetic or food industries. However, application of plant cell culture for commercial production of natural food colorants is still not an achieved goal due to lack of economical viability. One of the ways of improving the efficiency of the system is the production of high-value compounds superior to those accumulated in field-grown plant. This paper presents a review of our earlier works aiming to establish a plant cell culture-based system for the production of anthocyanin–phenolic acid complex with enhanced stability and physiological activity from a high-anthocyanin accumulating cell line generated from the storage root of sweetpotato ( Ipomoea batatas L.), cv. Ayamurasaki. Modifications of the physical and chemical environment of culture growth have been employed to direct the in vitro production of anthocyanins towards the generation of components with enhanced stability and radical scavenging ability. Under the same conditions we have enhanced the accumulation of caffeic acid and chlorogenic (caffeoylquinic) acid and induced accumulation of derivatives: 4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid and 3,4,5-tricaffeoylquinic acid. In vitro generation of polyphenolic complex consisting of selected anthocyanins and phenolic acids with enhanced nutraceutical value has been achieved.

Anthocyanins—More Than Nature′s Colours

Anthocyanins—More Than Nature′s Colours

TOPICAL PAPER: Utilization of Hairy Root Cultures for Production of Secondary Metabolites

AbstractThe possibility of producing useful chemicals by plant cell cultures has been studied intensively for the past 30 years. However, problems associated with low product yields and culture instabilities have restricted wider industrial application of plant cell culture. The employment of hairy root culture technology, developed in the past 10 years, offers new opportunities for in vitro production of plant secondary metabolites. In contrast to cell suspension cultures, hairy root cultures are characterized by high biosynthetic capacity and genetic as well as biochemical stability. In this review, the establishment and cultivation of hairy root cultures as well as their properties and application for production of secondary metabolites are discussed.

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner. The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.

Influence of light irradiation rates and irradiation modes on caffeine production and cell growth in suspension culture of Coffea arabica cells.

The effect of light on caffeine production by Coffea arabica cells was investigated using chemical actinometry for the purpose of industrial application of plant cell cultures. Caffeine production by the Coffea arabica cells was greatly increased by light. About a six-day irradiation period was required to increase the caffeine production in a batch culture. Caffeine production significantly increased at a low light irradiation rate of 0.11 J/L/s. Meanwhile, cell growth was suppressed by increasing the irradiation rate. Maximum production was achieved at a moderate irradiation rate of 0.70 J/L/s in a 20-day batch culture. It was shown there was a light irradiation rate that optimized production. Since long-term continuous light greatly suppressed cell growth, the culture was divided into two stages: the production period in the light and the growth period in the dark. The high production and good growth were maintained for 65 days.

Recent progress in plant cell culture

Commercial application of plant cell cultures for the production of useful metabolites on a large scale has not yet been realized. However, some recently published papers and patents seem very attractive for industrial use because their results and techniques are apparently applicable to a large-scale production of metabolites. The author believes that commercial success using plant tissue cultures will be realized in the near future with advanced techniques if product selection is determined by economic and political principles.In 1982, the 5th International Congress of Plant Tissue and Cell Culture will be held in Japan and this undoubtedly will encourage research activity in our country.