Yew (Taxus x media Rehd.) cell suspension cultures as a source of taxanes

Natural resources of paclitaxel, an effective anticancer compound, were threatened with extinction soon after the discovery of this valuable substance. Cell suspension cultures derived from different Taxus species have rapidly become an alternative source of paclitaxel and other taxanes. In this paper we provide some insight into cell growth characteristics in cell suspension culture of Taxus x media cv. Hicksii, with emphasis on the effects of jasmonic acid (JA) on taxane production in cell lines with different initial taxane content. Additionally cell growth characteristics of two cell lines was followed during cultivation of cell suspension culture of Taxus x media cv. Hicksii. Packed cell volume (PCV) was shown to be a reliable and efficient alternative for measuring cell growth instead of fresh and dry weight. The initial total taxane content was screened in a number of cell lines, followed by observing the effect of JA on cell mass and total taxane production of selected lines. We showed a great variability in initial taxane content in different cell lines, which decreased during cell suspension maintenance. JA was shown to inhibit cell growth and increase total taxane production (14 to 106 fold).     

A rational approach to improving the biotechnological production of taxanes in plant cell cultures of Taxus spp.

Taxol is a complex diterpene alkaloid scarcely produced in nature and with a high anticancer activity. Biotechnological systems for taxol production based on cell cultures of Taxus spp. have been developed, but the growing commercial demand for taxol and its precursors requires the optimization of these procedures. In order to increase the biotechnological production of taxol and related taxanes in Taxus spp. cell cultures, it is necessary not only to take an empirical approach that strives to optimize in-put factors (cell line selection, culture conditions, elicitation, up-scaling, etc.) and out-put factors (growth, production, yields, etc.), but also to carry out molecular biological studies. The latter can provide valuable insight into how the enhancement of taxane biosynthesis and accumulation affects metabolic profiles and gene expression in Taxus spp. cell cultures.     

红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: application to taxane production byTaxus sp.

TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol®) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.Abbreviations TLC Thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - HPLC high pressure liquid chromotography - UV ultraviolet - Rf retention factor     

Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey

The literature concerning the tissue culture of Taxus sp. as an alternative source for taxoid production is reviewed. The aim of this review is to summarize and discuss the progress achieved with the approaches and methods used for the establishment of various Taxus culture systems, the methods used for the evaluation of taxoid production, the multiple factors which control taxoid production and the feasibility of the in vitro production of taxoids on a commercial scale.     

Effects of paclitaxel-producing fungal endophytes on growth and paclitaxel formation of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells

Effects of a fungal endophyte, Fusarium mairei, on growth and paclitaxel formation of Taxus cuspidata cells were investigated by adding fungal endophyte culture supernatant (FECS) to suspension cultures of T. cuspidata cells. The main effective chemical responsible for paclitaxel formation in FECS was an exopolysaccharide (EPS) of molecular weight ~2 kDa. FECS fractions except EPS stimulated growth of Taxus cells but had no effects on paclitaxel accumulation. Additionally, elicitation efficiency of FECS based on different culture conditions was studied. EPS content in FECS was related to FECS culture conditions. FECS with long cultivation and high-aeration cultivation contained higher EPS content and resulted in higher paclitaxel yield than that with short cultivation and low-aeration cultivation. The maximum yield of paclitaxel from Taxus cultures, elicited by FECS with 9-day cultivation, was 4.7-fold that of the control cultures.     

Establishment of cell suspension cultures of yew (<Emphasis Type="Italic">Taxus × media</Emphasis> Rehd. and assessment of their genomic stability

Summary Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inibited growth while no effect on nuclear DNA content was noted.     

Culture of Isolated Single Cells from <Emphasis Type="Italic">Taxus</Emphasis> Suspensions for the Propagation of Superior Cell Populations

Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4×10⁵ cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mm), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days⁻¹ was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

<Emphasis Type="Italic">Cupressus</Emphasis> callus and cell suspension cultures: Effect of seiridins on their growth and sensitivity

Summary Cupressus macrocarpa and C. arizonica were examined for callus and cell culture production in vitro. Both species produced callus on agar-solidified MSCY medium supplemented with vitamins, antioxidants, 0.14 μM kinetin (KIN), and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures of both species were established in liquid MSCY medium. Seiridin (SE) and iso-seiridin (ISE), two phytotoxic butenolides produced by Seiridium cardinale, S. cupressi, and S. unicorne, the causal agents of many canker diseases of cypress, were tested on callus or cell suspension cultures. In the medium without other plant growth regulators (PGR), SE promoted cell proliferation of cypress better than ISE, for callus initiation, callus maintenance, and cell suspension cultures. The growth rates of cypress callus tissues and suspension cultures of both cypress species on media containing 50–150 μM SE or ISE were measured. At concentrations of 50 μM and higher, growth rates increased exponentially with the SE concentration. A comparison with KIN and 2,4-D indicated that 50 μM SE promoted growth of callus tissues and cell suspension cultures more than 100 μM ISE. SE can also interact with, or counteract, KIN and 2,4-D. It was demonstrated that SE could replace KIN in the medium for C. arizonica. SE could be involved in cell enlargement and proliferation processes. The less susceptible cypress species (C. arizonica) had a high content of terpenoids than that of the more susceptible species (C. macrocarpa). SE could be a useful tool as a phytohormonal-like regulator to manipulate physiological changes at the cellular level and as an elicitor of sensitivity or tolerance of cypress germplasm to the phytotoxin.     

Establishment of <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension cultures for the production of Juvenile hormone III

Cell suspension cultures of Cyperus aromaticus were established from the yellow friable callus derived from the root explants of in vitro plantlets. Four callus cell lines were selected based on their growth index from two populations of callus cultures originated from the mother plants grown in two different locations. The selected four cell lines (Z1, Z6, P4, P9) showed uniform cell growth but produced different amounts of juvenile hormone III (JHIII). The Z1 cell line possessed fast-growing characteristics, produced a high JHIII content, and was chosen as the elite cell line for an optimization study of C. aromaticus cell suspension cultures. An inoculum cell mass of 0.3 g from 12-d cultures in 30 ml culture medium was found to be the optimum inoculum size and culture age for establishing the cell suspension culture of C. aromaticus. MS basal medium supplemented with 4.5 mg/l 2,4-D and 5.5 mg/l NAA was found to be the best medium for production of maximum cell biomass and JHIII. These results indicated that JHIII can be produced from suspension culture of C. aromaticus using a single-stage cell-culture system.     

Stable transformation and long-term maintenance of transgenic <Emphasis Type="Italic">Taxus</Emphasis> cell suspension cultures

A cell line of Taxus cuspidata has been transformed with wild-type Agrobacterium rhizogenes ATCC strain 15834 containing binary vector pCAMBIA1301 and, separately, with A. tumefaciens strain EHA105 containing binary vector pCAMBIA1305.2. Additionally, a cell line of T. chinensis has been transformed with wild-type A. rhizogenes ATCC strain 25818 containing binary vector pCAMBIA1301. The two transgenic T. cuspidata cell lines have been maintained in culture for more than 20 months, and the transgenic T. chinensis cell line for more than 9 months, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernable effect on growth or Taxol production in the transgenic cell lines when compared to the parent control. The methods for transforming non-embryogenic Taxus suspension cultures are described.     

Molecular cloning and characterization of a cytochrome P450 taxoid 9á-hydroxylase in Ginkgo biloba cells

Taxol is a well-known effective anticancer compound. Due to the inability to synthesize sufficient quantities of taxol to satisfy commercial demand, a biotechnological approach for a large-scale cell or cell-free system for its production is highly desirable. Several important genes in taxol biosynthesis are currently still unknown and have been shown to be difficult to isolate directly from Taxus, including the gene encoding taxoid 9α-hydroxylase. Ginkgo biloba suspension cells exhibit taxoid hydroxylation activity and provides an alternate means of identifying genes encoding enzymes with taxoid 9α-hydroxylation activity. Through analysis of high throughput RNA sequencing data from G. biloba, we identified two candidate genes with high similarity to Taxus CYP450s. Using in vitro cell-free protein synthesis assays and LC–MS analysis, we show that one candidate that belongs to the CYP716B, a subfamily whose biochemical functions have not been previously studied, possessed 9α-hydroxylation activity. This work will aid future identification of the taxoid 9α-hydroxylase gene from Taxus sp.     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Paclitaxel production using co-culture of <Emphasis Type="Italic">Taxus</Emphasis> suspension cells and paclitaxel-producing endophytic fungi in a co-bioreactor

The co-culture of the suspension cells of Taxus chinensis var. mairei and its endophytic fungi, Fusarium mairei, in a 20-L co-bioreactor was successfully established for paclitaxel production. The co-bioreactor consists of two-unit tanks (10 L each) with a repairable separate membrane in the center, culturing Taxus suspension cells in one tank and growing fungi in another. By optimizing the co-culture conditions, there was a desirable yield of paclitaxel in Taxus cell cultures. The Taxus cell cultures by co-culture produced 25.63 mg/L of paclitaxel within 15 days; it was equivalent to a productivity of 1.71 mg/L per day and 38-fold higher than that by uncoupled culture (0.68 mg/L within 15 days). The optimum conditions for co-culture in the co-bioreactor were: B5 medium, inoculating fungi when Taxus cells had grown for 5 days in the co-bioreactor, hydrophilic separate membrane in the center of the co-bioreactor, and air flow rate of 1:0.85 v/v/m in fungus cultures.     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.