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

The 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.     

Electronic Measurement of Plant Cell Number and Size in Suspension Culture

The rapid and accurate determination of cell number and sizeis very desirable for obtaining data on growth kinetics andchanges in the physiological state of a culture. The CoulterCounter has been widely applied to bacterial and animal cellsuspension cultures to determine electronically cell numberand cell size. The application of such techniques to plant cellsuspension cultures is discussed in the following paper anddemonstrated for cultures of Pauls Scarlet Rose (Rosa sp.) andsoybean (Glycine max L.). A new formula for coincidence correctionsis suggested.     

First principle-based models in plant suspension cell cultures: a review

In this work, the development and application of published models for describing the behavior of plant cell cultures is reviewed. The structure of each type of model is analyzed and the new tendencies for the modeling of biotechnological processes that can be applied in plant cell cultures are presented. This review is a tool for clarifying the main features that characterize each type of model in the field of plant cell cultures and can be used as a support on the selection of the more suitable model type, taking into account the purpose of specific research.     

Flow cytometry of plant cells with applications in large-scale bioprocessing

In recent years, there has been a significant upsurge in the application of flow cytometry to plant cells and plant cell cultures. As well as a range of uses in plant biology, flow cytometry offers many advantages for monitoring plant cell cultures used in large-scale bioprocessing operations. This review summarizes the current status of the field, concentrating on methods for DNA measurement and multiparameter cell cycle analysis. Techniques for screening and selection of elite cell lines with high productivity of secondary metabolites are also addressed.     

Comparative potency of different plant growth retardants in cell cultures and intact plants

A comparison of the efficiency of a broad range of plant growth retardants on cell division growth of 13 cell suspension cultures is presented. The results show that (1) the new plant bioregulator tetcyclacis (NDA) is the compound with the highest activity in inhibiting cell division of all cultures tested, and (2) cell cultures react species-specifically to various compounds. Significant correlations between the results from suspension cultures and intact seedlings of the same plant species demonstrate the usefulness of cell cultures for identifying substances with a growth-regulating potency. Futhermore, the usefulness of cell cultures for establishing structure-activity relationships was shown with structural analogues of chlormequat and mepiquat chloride.     

Comparative potency of different plant growth retardants in cell cultures and intact plants

A comparison of the efficiency of a broad range of plant growth retardants on cell division growth of 13 cell suspension cultures is presented. The results show that (1) the new plant bioregulator tetcyclacis (NDA) is the compound with the highest activity in inhibiting cell division of all cultures tested, and (2) cell cultures react species-specifically to various compounds. Significant correlations between the results from suspension cultures and intact seedlings of the same plant species demonstrate the usefulness of cell cultures for identifying substances with a growth-regulating potency. Futhermore, the usefulness of cell cultures for establishing structure-activity relationships was shown with structural analogues of chlormequat and mepiquat chloride.     

刺激剂(elicitor)在植物细胞培养次生代谢物生产中的应用

刺激剂（ｅｌｉｃｉｔｏｒ）在植物细胞培养中被用来作为提高次生代谢物产量的手段。文中概括介绍了微生物、寡聚糖、蛋白质、第二信使及其他物质作为刺激剂在植物细胞培养中的应用及其研究成果。     

动植物细胞或组织生物反应器悬浮培养过程中的通气方式

通气在动植物细胞或组织生物反应器培养过程中起着至关重要的作用,而同时通气过程所产生的机械损伤力亦可对细胞造成直接的伤害,因此,通气方式是动植物细胞或组织生物反应器培养过程设计与工程放大的关键技术之一。本文综述了动植物细胞或组织生物反应器悬浮培养过程中三种主要通气(异养培养时又称供氧)方式的结构特点,及其对气液传质、生物量、代谢产物量和细胞损伤的影响,以及改进的新型通气方式和几种通气方式的融合并用。     

Novel chemically synthesized salicylate derivative as an effective elicitor for inducing the biosynthesis of plant secondary metabolites

Trifluoroethyl salicylate (TFESA), a novel salicylate derivative, was chemically synthesized and evaluated by bioassay as a potential elicitor for inducing the biosynthesis of plant secondary metabolites. A cell line of Taxus chinensis, which stably produced a high level of bioactive taxuyunnanine C (Tc), was taken as a model plant cell system. The application of TFESA to the cell cultures could significantly induce Tc biosynthesis, although the cell growth was slightly inhibited. More interestingly, Tc production was enhanced more in the presence of TFESA compared with a structure-similar well-known elicitor, salicylic acid (SA). For example, addition of 100 microM TFESA on day 7 led to a high Tc content of 21.9 +/- 0.1 mg g(-1) (at day 21), whereas the Tc content was 14.0 +/- 0.2 and 16.7 +/- 0.3 mg g(-1) for the control and that with addition of 100 microM SA, respectively. The results indicate that the newly synthesized TFESA can act as a powerful elicitor for secondary metabolism induction in plant cell cultures.     

Metabolism of nicotinic acid in plant cell suspension cultures: II; Isolation, characterization and enzymology of nicotinic acid N-alpha-arabinoside (author's transl)]

A very hydrophilic compound was isolated from parsley cell suspension cultures in high yield after application of nicotinic acid. Using chemical, chromatographic and spectroscopic procedures the structure of this new plant constituent has been elucidated as nicotinic acid N-alpha-L-arabinopyranoside. This structure has been proved by chemical synthesis. An arabinosyltransferase was isolated from parsley cell suspension cultures and purified about 19-fold. The enzyme converted nicotinic acid N-alpha-arabinoside with UDP to nicotinic acid and UDP-arabinose. pH-Optimum (pH 7.0-8.0), Km value for nicotinic acid N-alpha-L-arabinoside (2.2 X 10(-4) mol/l) and mol. wt. (app. 70 000) of the transferase were measured. Function and biosynthesis of the arabinoside in cell cultures are discussed.     

Production and engineering of terpenoids in plant cell culture

Terpenoids are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition. Their chemical diversity has led to the discovery of over 40,000 different structures, with several classes serving as important pharmaceutical agents, including the anticancer agents paclitaxel (Taxol) and terpenoid-derived indole alkaloids. Many terpenoid compounds are found in low yield from natural sources, so plant cell cultures have been investigated as an alternate production strategy. Metabolic engineering of whole plants and plant cell cultures is an effective tool to both increase terpenoid yield and alter terpenoid distribution for desired properties such as enhanced flavor, fragrance or color. Recent advances in defining terpenoid metabolic pathways, particularly in secondary metabolism, enhanced knowledge concerning regulation of terpenoid accumulation, and application of emerging plant systems biology approaches, have enabled metabolic engineering of terpenoid production. This paper reviews the current state of knowledge of terpenoid metabolism, with a special focus on production of important pharmaceutically active secondary metabolic terpenoids in plant cell cultures. Strategies for defining pathways and uncovering rate-influencing steps in global metabolism, and applying this information for successful terpenoid metabolic engineering, are emphasized.     

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative “chemical factory”. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.     

Production of anthocyanins by plant cell cultures

Anthocyanins, responsible for the various attractive colors in plants, are becoming important alternative to many synthetic colorants due to increased public concerns over the safety of artificial food colors. Production of anthocyanins by plant cell cultures has been suggested as a feasible technology that has attracted considerable industrial and academic interests in the past two decades. This paper is to provide an overview of the present status and the future prospects in the commercial development of plant cell cultures for production of anthocyanins. The focus is on the strategies for enhancement of anthocyanin biosynthesis to achieve an economically viable technology for commercial applications. Through strain improvement, optimization of media and culture conditions, and intelligent process strategies such as elicitation and two-stage system, significant enhancement in productivity has been achieved in a number of cultures. However the yield of anthocyanins obtained so far is still far away from the full potential of anthocyanin synthesis by plant cell cultures. Further improvements require the insights on the regulation of anthocyanin synthesis, accumulation, storage and breakdown that will eventually lead to genetic manipulation of anthocyanin biosynthesis. Many studies have elucidated the metabolic pathway of anthocyanin biosynthesis. Preliminary studies on the regulation of anthocyanin biosynthesis on the levels of genes and enzymes are reviewed, showing that it is feasible to clone genes from secondary metabolism with an improved yield of anthocyanins. There is currently no commercial-scale trial for production of anthocyanin by plant cell cultures, but an intelligent integration of those existing strategies could provide a technology for industrial application competitive to the current production methods.     

Hairy Roots, their Multiple Applications and Recent Patents

In the last years, hairy root (HR) cultures are gaining attention in the biotechnology industry. This particular plant cell culture derives from explants infected with Agrobacterium rhizogenes. They constitute a relatively new approach to in vitro plant biotechnology and modern HR cultures are far away from the valuables findings performed by Philip R. White in the 1930?s, who obtained indefinite growth of excised root tips. HR cultures are characterized by genetic and biochemical stability and high growth rate without expensive exogenous hormones source. HR cultures have allowed a deep study of plant metabolic pathways and the production of valuable secondary metabolites and enzymes, with therapeutic or industrial application. Furthermore, the potential of HR cultures is increasing continuously since different biotechnological strategies such as genetic engineering, elicitation and metabolic traps are currently being explored for discovery of new metabolites and pathways, as well as for increasing metabolites biosynthesis and/or secretion. Advances in design of proper bioreactors for HR growth are being of great interest, since scale up of metabolite production will allow the integration of this technology to industrial processes. Another application of HR cultures is related to their capabilities to biotransform and to degrade different xenobiotics. In this context, removal assays using this plant model system are useful tools for phytoremediation assays, previous to the application in the field. This review highlights the more recent application of HRs and those new patents which show their multiple utilities.     

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications

A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties.     

Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid

To clarify the causal factors for ploidy variation in plant cell culture, we attempted to alter ploidy distribution in cell cultures of a tetraploid cultivar of Doritaenopsis by changing the plant growth regulators (PGRs) in the culture medium. The original suspension cultured cells, which had been maintained in medium containing 0.1 mg l⁻¹ 1-naphthaleneacetic acid and 1 mg l⁻¹ benzyladenine, were transferred onto various gellan gum solidified media with a single application of PGRs, and the ploidy distributions of the cells were examined using flow cytometry analysis during 3 weeks of culture. Among the PGRs tested, 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid caused a drastic reduction in the 4C-cell proportion in cell cultures with an increased cell proportion of 8C or higher C-values. In the case of 2,4-D application, a reduction of cell viability was observed. A decreasing proportion was also observed in the 8C-cell population accumulated by 2,4-D treatment, following transfer back to the medium containing the standard PGR composition. These results suggest that the exogenous application of 2,4-D arrested the cell cycle at G2 phase in the Doritaenopsis cells, and the removal of 2,4-D might induce further endoreduplication or recover the mitotic cycle of the G2-arrested cells.     

Elicitation: A biotechnological tool for enhanced production of secondary metabolites in hairy root cultures

Elicitation is a possible aid to overcome various difficulties associated with the large‐scale production of most commercially important bioactive secondary metabolites from wild and cultivated plants, undifferentiated or differentiated cultures. Secondary metabolite accumulation in vitro or their efflux in culture medium has been elicited in the undifferentiated or differentiated tissue cultures of several plant species by the application of a low concentration of biotic and abiotic elicitors in the last three decades. Hairy root cultures are preferred for the application of elicitation due to their genetic and biosynthetic stability, high growth rate in growth regulator‐free media, and production consistence in response to elicitor treatment. Elicitors act as signal, recognized by elicitor‐specific receptors on the plant cell membrane and stimulate defense responses during elicitation resulting in increased synthesis and accumulation of secondary metabolites. Optimization of various parameters, such as elicitor type, concentration, duration of exposure, and treatment schedule is essential for the effectiveness of the elicitation strategies. Combined application of different elicitors, integration of precursor feeding, or replenishment of medium or in situ product recovery from the roots/liquid medium with the elicitor treatment have showed improved accumulation of secondary metabolites due to their synergistic effect. This is a comprehensive review about the progress in the elicitation approach to hairy root cultures from 2010 to 2019 and the information provided is valuable and will be of interest for scientists working in this area of plant biotechnology.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Plant cryopreservation: a continuing requirement for food and ecosystem security

This issue of In Vitro Cellular and Developmental Biology—Plant is dedicated to current developments in liquid-nitrogen cryopreservation methods and their use in plant biology and germplasm preservation. The development of cryopreservation for storage of plant cells, tissues, and organs began in the 1960s and continues to this day. Long-term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains an important requirement for many scientists, organizations, and companies. The continued development of cryopreservation techniques and their application to new plants is the subject of this issue.     

An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond

Epigenetic mechanisms are highly dynamic events that modulate gene expression. As more accurate and powerful tools for epigenetic analysis become available for application in a broader range of plant species, analysis of the epigenetic landscape of plant cell cultures may turn out to be crucial for understanding variant phenotypes. In vitro plant cell and tissue culture methodologies are important for many ongoing plant propagation and breeding programmes as well as for cutting-edge research in several plant model species. Although it has long been known that in vitro conditions induce variation at several levels, most studies using such conditions rely on the assumption that in vitro cultured plant cells/tissues mostly conform genotypically and phenotypically. However, when large-scale clonal propagation is the aim, there has been a concern in confirming true-to-typeness using molecular markers for evaluating stability. While in most reports genetic variation has been found to occur at relatively modest frequencies, variation in DNA methylation patterns seems to be much more frequent and in some cases it has been directly implicated in phenotypic variation. Recent advances in the field of epigenetics have uncovered highly dynamic mechanisms of chromatin remodelling occurring during cell dedifferentiation and differentiation processes on which in vitro adventitious plant regeneration systems are based. Here, an overview of recent findings related to developmental switches occurring during in vitro culture is presented. Additionally, an update on the detection of epigenetic variation in plant cell cultures will be provided and discussed in the light of recent progress in the plant epigenetics field.