Production and metabolic engineering of bioactive substances in plant hairy root culture

In the past three decades, hairy roots research for the production of valuable biological active substances has received a lot of attention. The addition of knowledge to enhance the yields of desired substances and the development of novel tools for biomass engineering offer new possibilities for large-scale cultivation of the plant hairy root. Hairy roots can also produce recombinant proteins through the transfer of Agrobacterium T-DNA into the plant genome, and thereby hold immense potential for the pharmaceutical industry. This review highlights some of the significant progress made in the past few years and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.     

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.     

Neuroprotection by bioactive components in medicinal and food plant extracts

Neurodegenerative diseases of the human brain comprise a variety of disorders that affect an increasing percentage of the population. Some of these are age dependent (e.g. Alzheimer's and Parkinson's diseases) and some are infection dependent, e.g. human immunodeficiency virus (HIV/AIDS). The vulnerable brain regions in HIV/AIDS individuals include the dentate nucleus in the cerebellum, the red nucleus, substantia nigra (SN) in the mid-brain, the subthalamic nucleus, thalamic fasciculus in the diencephalons, the globus pallidus and striatum (or neostriatum, which consists of caudate and putamen) in the forebrain. Lesion in these regions may lead to progressive dementia, which is similar to what is observed in Alzheimer's disease and Parkinson's disease. The entry of calcium into the cytoplasm of cells at concentrations that can activate oxidative enzymes such as phospholipase A(2) and xanthine oxidase, deplete cells of cysteine and glutathione, cause mitochondrial release of free radicals and cell death. Glutamate and its receptors are key molecular elements at the interface between neurons and glia. Dietary factors can modulate physiological functions (including brain function) thereby increasing the economic productivity of a population as a function of health. A greater understanding of the molecular mechanisms of neuroprotection, oxidative stress and immune function will facilitate definition of the prophylactic potentials of diet, nutritional/food supplements, medicinal plants and herbal extracts.     

Production of ecdysteroids by plant cell culture of Pteridium aquilinum

Summary Cells of the fernP. aquilinum, both callus and suspension cultures, are able to produce ecdysterone, 5--OH-ecdysterone, ecdysone, ponasterone and further five unidentified ecdysteroids. Under the cultivation conditions there appears to be an overproduction of ponasterone and total ecdysteroids, in comparison to the original plant.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Production of recombinant plant gum with tobacco cell culture in bioreactor and gum characterization

Many plant gums, such as gum arabic, contain hydroxyproline-rich glycoproteins (HRGPs), which are also abundant components of the plant cell extracellular matrix. Here we expressed in transgenic BY2 Nicotiana tabacum (tobacco) cells, a synthetic gene encoding a novel HRGP-based gum, designated gum arabic-8 or (GA)(8). (GA)(8) encoded eight repeats of the consensus polypeptide sequence of gum arabic glycoprotein (GAGP): Gly-Pro-His-Ser-Pro-Pro-Pro-Pro-Leu-Ser-Pro-Ser-Pro-Thr-Pro-Thr-Pro-Pro-Leu, in which most of the Pro residues were posttranslationally modified to hydroxyproline (Hyp). (GA)(8) was expressed as a green fluorescent protein (GFP) fusion protein targeted to the culture medium, (GA)(8)GFP. The culture of the transgenic cells in a 5-L bioreactor showed that the production of (GA)(8)GFP was cell growth-associated. The extracellular yield of (GA)(8)GFP was 116.8 mg/L after 14 days of culture and accounted for 87% of the total fusion protein expressed. (GA)(8)GFP was purified from the culture medium by a combination of hydrophobic interaction, gel permeation, and reversed phase chromatography. Biochemical characterization indicated that the amino acid composition of the (GA)(8) module, after removal of GFP by proteolysis, was virtually identical to that of predicted by the GAGP consensus sequence and that carbohydrate, which occurred as arabinogalactan polysaccharides and small oligoarabinosides O-linked through the Hyp residues, accounted for 84% of the molecules' dry weight. Functional assays showed that (GA)(8) exhibited low viscosity in aqueous solution similar to native GAGP. However, neither GFP alone nor the (GA)(8) module could emulsify orange oil. However, the fusion protein (GA)(8)GFP possessed 1.28-fold better emulsification properties than native GAGP. This work demonstrates the feasibility and potential of a synthetic gene approach to the de novo design of novel glycoprotein-based gums and emulsifiers.     

Production of plant bioactive triterpenoid saponins: from metabolites to genes and back

Phytochemistry Reviews - Saponins are specialized plant terpenoids derived from the mevalonic acid pathway. Triterpenoid saponin cores are decorated with sugar residues, conferring a highly...     

Plastids: dynamic components of plant cell development

The gravitropic bending of maize roots, as a response to reorientation of the root within a gravitational field, was examined for sensitivity to exogenous applications of the cytoskeletal inhibitor, cytochalasin D. Agar blocks were impregnated with this inhibitor, and were applied either to the root cap or to the zone of root cell elongation. Root growth was normal with either treatment, if the roots were not repositioned with respect to the gravitational vector. When untreated roots were placed in a horizontal position with respect to gravity, a 40 degree bending response was observed within one hour. This bending also occurred when cytochalasin D was applied at high concentrations to the zone of root cell elongation. However, when cytochalasin D above 40 micrograms/ml was applied to the root cap, roots lost the ability of directional reorientation within the gravitational field, causing a random bending.     

培养基和栽培方式对蛹虫草子实体活性成分的影响

采用麦粒（W）、麦粒加蚕蛹粉（WW）、大米（R）、大米加蚕蛹粉（RW）的配料栽培方式,以及蚕蛹（CY）活体接种栽培方式培养蛹虫草子实体,比较蛹虫草子实体中多糖及核苷、游离糖醇和小分子糖类含量。结果表明：培养基和栽培方式影响蛹虫草多糖含量及其单糖组成和分子量分布,多糖含量最高的为蚕蛹活体接种培养的处理（CY）,多糖含量为6.19%,其他处理的多糖含量仅为CY的44.4%-62.8%;蚕蛹（CY）上培养的蛹虫草子实体中核苷类成分含量最高,在麦粒上培养获得的子实体中核苷含量显著高于在大米上的处理,并且麦粒添加蚕蛹粉后培养的子实体中尿苷、鸟苷、N⁶-(2-羟乙基)腺苷含量显著上升,大米添加蚕蛹粉后培养的子实体中尿苷、虫草素、N⁶-(2-羟乙基)腺苷含量显著上升;配料栽培的4个处理,其子实体中海藻糖含量为20.07%-23.40%,蚕蛹活体接种的处理（CY）海藻糖含量显著降低,为8.41%;添加蚕蛹粉后子实体中甘露醇含量显著降低。对各成分含量的数据进行归一化处理后进行蛹虫草品质的综合评价,在蚕蛹上培养的蛹虫草综合评分最高,麦粒为主要培养基培养的蛹虫草品质好于大米为主要培养基的,且添加蚕蛹粉的处理优于未添加的处理。     

Safety aspects of insect cell culture

Conclusions The hazards posed to the environment by the accidental release of baculovirus expression vectors can be put into perspective by the results obtained from experiments in which AcNPV was released deliberately into the field (Bishop et al., 1992). Polyhedrin positive viruses will persist in soil and on leaf surfaces for periods comprising weeks and months. However, polyhedrin negative viruses (similar to those used as expression vectors) do not survive in similar situations. In consequence, accidental release of baculovirus expression vectors poses negligible hazard. The risk of such a release will largely depend on the skill of the operators. This does not take into account the hazard posed by the recombinant product which is being made by the virus-infected insect cell. Synthesis of a mammalian-specific toxin, of course, would require particularly careful manipulation of the virus-infected cell culture.The fact that insect cell lines represent an undefined risk, in terms of carriage of adventitious agents means that their containment should be maintained at a minimum of the European containment level 2. Where the tissue of origin has a high risk of infection with human pathogens or where cells may have been used in a virus culture laboratory then appropriate testing is advisable. Careful risk assessment respecting the scale of work and whole procedures (in addition to individual assessment of agents and reagents) will ensure safe working conditions for laboratory staff. If applied properly safety procedures will also succeed in encouraging clean, efficient and well documented work procedures which are synonymous with the economical use of time and resources and good science.     

植物干细胞培养研究进展

植物干细胞位于分生组织,是处于未分化状态的细胞,液泡化程度低,具有较高的线粒体活性,遗传稳定,具有很强的自我更新和再生能力。植物干细胞培养在下游制药和功能性食品以及化妆品行业具有广泛的应用潜质。文中综述了植物干细胞的基本培养技术、鉴别技术,为该领域的深入研究提供参考。     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

Analytical aspects of plant metabolite profiling platforms: current standings and future aims

Over the past years, metabolic profiling has been established as a comprehensive systems biology tool. Mass spectrometry or NMR spectroscopy-based technology platforms combined with unsupervised or supervised multivariate statistical methodologies allow a deep insight into the complex metabolite patterns of plant-derived samples. Within this review, we provide a thorough introduction to the analytical hard- and software requirements of metabolic profiling platforms. Methodological limitations are addressed, and the metabolic profiling workflow is exemplified by summarizing recent applications ranging from model systems to more applied topics.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

Interferon-inducing action of polysaccharide-containing biopolymers from ginseng root and cell culture]

Induction of interferon (IF) and tumor necrosis factor (TNF) under the action of two polysaccharide preparations of ginseng i.e. panaxan-1 (from ginseng root) and panaxan-2 (from ginseng cell culture) was studied. Both the preparations induced production of TNF and IF in human leukocytes. By its properties and the typing results the induced IF proved to be gamma-IF. The preparation from the ginseng cell culture in the doses used had a higher IF inducing activity which could be explained by the difference in the polysaccharide composition of the preparations.     

Effect of plant growth regulators and medium composition on cell growth and saponin production during cell-suspension culture of mountain ginseng (Panax ginseng C. A. mayer)

We have established cell-suspension cultures of mountain ginseng (Panax ginseng G A. Mayer), and have attempted to increase the yield of saponin by manipulating our processing method and culturing factors (e.g., media strengths; the presence of plant growth regulators or sucrose; ratios of NO⁺ ₃/ NH^- ₄). Maximum biomass yield was obtained in media containing 2,4-D. However, saponin productivity was much higher in a medium comprising either IBA or NAA; 7.0 mg/L IBA was optimal for promoting both cell growth (10.0 g/L dry weight) and saponin production (7.29 mg/g DW total ginsenoside). Although the addition of cytokinins (BA and kinetin) did not affect cell growth, the level of saponin (particularly in the Rb group) was enhanced when the media were supplemented with either 0.5 mg/L BA or 0.5 mg/L kinetin. Half- and full-strength MS media were equally suitable for inducing both biomass as well as saponin production. We also investigated the effect of various concentrations of sucrose and nitrogen, and found that 30 g/L sucrose enhanced biomass yield as well as saponin content However, further increases (i.e., up to 70 g/L) led to a decrease in saponin accumulation and biomass production. Maximum growth and saponin productivity were reported from treatments with an initial nitrogen concentration of 30 mM. In general, the amount of saponin increased when the test media had high NO⁺ ₃/ NH^- ₄ ratios; in fact, saponin production was greatest when nitrate was the sole nitrogen source.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

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

Summary 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 μm 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 highly-pigmented small aggregates, 18 to 238 μm 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.