Non-transformed Cultures Research Articles

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

Over-Expression of the Cell-Cycle Gene LaCDKB1;2 Promotes Cell Proliferation and the Formation of Normal Cotyledonary Embryos during Larix kaempferi Somatic Embryogenesis.

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.

Organoids to Dissect Gastrointestinal Virus-Host Interactions: What Have We Learned?

Historically, knowledge of human host–enteric pathogen interactions has been elucidated from studies using cancer cells, animal models, clinical data, and occasionally, controlled human infection models. Although much has been learned from these studies, an understanding of the complex interactions between human viruses and the human intestinal epithelium was initially limited by the lack of nontransformed culture systems, which recapitulate the relevant heterogenous cell types that comprise the intestinal villus epithelium. New investigations using multicellular, physiologically active, organotypic cultures produced from intestinal stem cells isolated from biopsies or surgical specimens provide an exciting new avenue for understanding human specific pathogens and revealing previously unknown host–microbe interactions that affect replication and outcomes of human infections. Here, we summarize recent biologic discoveries using human intestinal organoids and human enteric viral pathogens.

Nanoceria-curcumin conjugate: Synthesis and selective cytotoxicity against cancer cells under oxidative stress conditions

A water- and alcohol-soluble cerium oxide-curcumin conjugate was obtained by co-evaporation with poly(N-vinylpyrrolidone) (PVP). A nanocomposite consisting of hybrid organic-inorganic particles was stable in a wide range of pH values. Its properties were evaluated using nine cell lines: normal (MDBK, ST, Vero) and malignant (L929, T98G, HEp-2, A549, RIN-m5F, Hep G2). PVP-stabilised nanoceria was shown to inhibit autoxidation of curcumin, to enhance curcumin photostability, to promote bioaccumulation and to affect curcumin cytotoxicity and photocytotoxicity, depending on cell type, being more toxic to cancer cells in a selective manner. Under the conditions of UVA/UVC or H2O2-induced oxidative stress, the nanoceria-PVP-curcumin (NPC) conjugate was found to possess a selective cytotoxicity: it caused drastic inhibition of metabolic activity or a decrease in the total number of tumour cells, while in non-transformed cultures under the same conditions, the nanoceria-PVP-curcumin conjugate protected cells from these damaging factors. The NPC-conjugate, unlike curcumin itself, demonstrated a photosensitising effect in tumour cell cultures, while protecting non-transformed cultures from the damaging effects of UV radiation or oxidative stress. Based on the results obtained, we strongly believe that this novel hybrid material has enhanced characteristics compared to other curcumin formulations, and can be considered as a potent drug for biomedical applications, including cancer therapy.

Determination of lignans, phenolic acids and antioxidant capacity in transformed hairy root culture of Linum usitatissimum

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙+ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.

Enhancement of silymarin and phenolic compound accumulation in tissue culture of Milk thistle using elicitor feeding and hairy root cultures.

In the present study, the effects of the metabolite elicitors chitosan, methyl jasmonate (MeJA) and salicylic acid (SA) as well as the hairy root transformation were tested for silymarin and phenolic compound accumulation in in vitro cultures of Milk thistle. For callus induction, leaf explants were cultured on MS medium supplemented with 5 mg/l NAA + 2 mg/l Kin + 0.1 mg/l GA3. Chitosan, SA and MeJA were added separately in three concentrations 200, 400 and 800 mg/l; 10, 20 and 40 mg/l; 20, 40 and 80 mg/l, respectively, to hormone free B5 medium. Alternatively, cotyledons of 12 day old seedlings were transformed with Agrobacterium rhizogenes A4 strain. Overall, increasing the concentrations of the three elicitors dramatically increased the total silymarin content. Remarkably, the elicitors mainly enhanced the accumulation of silybine A&B that were not detected in un-treated callus culture (control). In addition, the hairy root culture triggered the accumulation of silybine A&B, and silydianin, which was not detected in the non-transgenic roots. The hairy root culture was superior in production of the phenolic compounds in comparison to the control and elicitor treatments. The hairy root cultures showed also higher antioxidant capacities than non-transformed cultures and/or chemically elicited-callus cultures. Thus hairy root provide instrumental in enhancing the production of economically valuable metabolite.

Lignan accumulation in callus and Agrobacterium rhizogenes-mediated hairy root cultures of flax (Linum usitatissimum)

Agrobacterium rhizogenes is gaining intensity in research to develop and produce a large number of commercially important secondary metabolites, such as lignans. Worldwide, lignans are receiving great attention because of their putative beneficial effects on human health. In this study, we investigated the potential of callus and A. rhizogenes, hairy roots, of flax Linum usitatissimum in accumulating lignans, namely, secoisolariciresinol diglucoside (SDG), secoisolariciresinol (SECO) and matairesinol (MAT). Callus cultures were established on Gamborg B5 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with 6-benzyladenine or gibberellic acid (GA3). These callus cultures were transformed with A. rhizogenes, which were initiated on 1.0 mg l−1 2,4-D + 0.5 mg l−1 GA3 or 1.0 mg l−1 GA3 media. Both hairy root culture 1 and hairy root culture 2 accumulated SDG, SECO and MAT with a total lignan concentration of 1.227 and 1.057 µmol g−1, respectively. However, non-transformed cultures did not accumulate MAT but accumulated SECO. Determination of total phenolic content by high-performance liquid chromatography revealed higher phenolic acid content in hairy root cultures compared with that in non-transformed cultures. Antioxidant activity, as measured by 2,2-diphenyl-1-picryl-hydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid-free radical scavenging assays, was significantly higher in hairy root cultures than in non-transformed cultures. Furthermore, the extract of hairy root culture showed inhibition of proliferation of human breast cancer cell line (MCF-7).

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l−1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.

Teratomas of Drosera capensis var. alba as a source of naphthoquinone: ramentaceone

Plants belonging to genus Drosera (family Droseraceae) contain pharmacologically active naphthoquinones such as ramentaceone and plumbagin. Hairy root cultures obtained following Agrobacterium rhizogenes-mediated transformation have been reported to produce elevated levels of secondary compounds as well as exhibit desirable rapid biomass accumulation in comparison to untransformed plants. The aim of this study was to establish hairy root or teratoma cultures of Drosera capensis var. alba and to increase the level of ramentaceone in transformed tissue by application of abiotic and biotic elicitors. The appearance of transformed tissues—teratomas but not hairy roots was observed 18 weeks after transformation. The transformation efficiency was 10% and all teratoma cultures displayed about 3 times higher growth rate than non-transformed cultures of D. capesis. The transformation was confirmed by PCR and Southern hybridization using primers based on the A. rhizogenesrolB and rolC gene sequences. HPLC analysis of ramentaceone content indicated 60% higher level of this metabolite in teratoma tissue in comparison to non-transformed cultures. Among the elicitors tested jasmonic acid (2.5 mg l−1) turned out to be the most effective. The productivity of ramentaceone in elicited teratoma cultures was about sevenfold higher than in liquid cultures of D. capensis var. alba and amounted to 2.264 and 0.321 mg respectively during 4 weeks of cultivation. This is the first report on the transformation of Drosera plant with A. rhizogenes.

Butyrate in vitro immune-modulatory effects might be mediated through a proliferation-related induction of apoptosis

Survival and proliferation signals are two processes closely interrelated and finely controlled in most cell types, whose deregulation may lead to carcinogenesis. In the last decade, different studies have suggested that both cellular functions are also intimately associated with other cellular activities such as differentiation and cellular activation, especially in immune cells. The aim of this study was to evaluate the effects of the short-chain fatty acid (SCFA) butyrate on the proliferation and activation state of different cell types involved in inflammatory bowel disease. We focused on intestinal epithelial cells, macrophages and T-lymphocytes, using both primary non-transformed cultures and established cell lines. The results showed that low concentrations of butyrate inhibited the proliferation of all the immune cell types tested in this work, whereas it only induced apoptosis in activated T-lymphocytes, non-differentiated epithelial cells and macrophage cell lines, but not in differentiated epithelial cells or primary macrophages. Butyrate apoptosis induction was mediated by caspase-3/7 activation. This SCFA was only able to modify cell activation, measured as expression of inflammatory cytokines, in those cell types in which apoptosis was induced. In conclusion, our results suggest a cell type-specificity of the immune-modulatory effects of butyrate based on the proliferation/activation characteristic physiology of these processes in different cells types.

Utility of Nicotiana tabacum Cell Suspension Cultures Expressing Human CYP1A1, CYP1A2 and CYP3A4 to Study the Oxidative Metabolism of the Herbicide 14C-Fluometuron

The metabolism and biotransformation of the (14)C-labeled phenylurea herbicide fluometuron was examined using tobacco cell suspension cultures transformed separately with human cyp1a1, cyp1a2 and cyp3a4, and corresponding non-transformed cultures in order to screen and predict metabolic patterns. Experimental parameters modified were concentration of (14)C-fluometuron, incubation period, and additional application of inhibitor carbaryl. Media and cell extracts were analyzed by radio-TLC and radio-HPLC, isolated metabolites by LC-MS, and non-extractable residues by combustion. During 48 hours, the CYP1A1 expressing cultures metabolized 90.0 % of applied fluometuron, while the non-transgenic controls transformed 67.0 %. The CYP1A2 expressing cultures exhibited highest rates (95.1 %), CYP3A4 expressing cultures lowest rates (43.0 %). The primary metabolites identified were mono-demethyl (main metabolite in controls) and di-demethyl fluometuron (mainly in CYP1A2 cultures), besides a non-identified primary product (mainly in CYP1A1 cultures); metabolic profiles differed distinctly among cultures. After addition of carbaryl, rates of fluometuron decreased noticeably in controls and not in CYP3A4 expressing cultures. This may indicate inhibition of endogenous tobacco P450s involved in fluometuron metabolism but not of CYP3A4. Additionally, the P450-transgenic cultures proved to be valuable tools to produce large amounts of metabolites for thorough identification.

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice α-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.

Comparison of the Biotransformation of the14C-Labelled Insecticide Carbaryl by Non-Transformed and Human CYP1A1-, CYP1A2-, and CYP3A4-Transgenic Cell Cultures ofNicotiana tabacum

Transgenic tobacco-cell-suspension cultures expressing separately the human cytochrome P450 monooxygenases CYP1A1, CYP1A2, and CYP3A4 were utilized to study the biotransformation of the 14C-labelled insecticide carbaryl (=naphthalen-1-yl methylcarbamate). The resulting data were compared to similar data from the corresponding non-transformed (NT) tobacco-cell culture and commercially available membrane preparations (Bactosomes) of genetically modified bacteria separately containing the same human P450s. A rapid conversion rate of carbaryl was observed with the CYP1A1 and CYP1A2 cells, where only 49.7 and 0.2% of applied carbaryl (1 mg/l), respectively, remained after 24 h, as compared to 77.7% in the non-transformed culture. Unexpectedly, the corresponding results obtained from the CYP3A4 cultures were not definite. With 25 mg/l of carbaryl and 96 h of incubation, it was proven that the insecticide is also substrate of CYP3A4. This finding was supported by GC/EI-MS analysis of the primary metabolite pattern produced by the isozyme. This consisted of naphthalene-1-ol, N-(hydroxymethyl)carbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl, whereas the main product in non-transformed cells was N-(hydroxymethyl)carbaryl. Data obtained from the CYP1A1, CYP1A2, or CYP3A4 Bactosomes agreed with those of the P450-transgenic tobacco cells. Problems with GC/EI-MS analysis of carbaryl and its metabolites are discussed.

Biotransformation of Metamitron by Human P450 Expressed in Transgenic Tobacco Cell Cultures

In the present investigation, the oxidative metabolism of 14C-labeled metamitron was examined in plant cell cultures of tobacco overexpressing human P450 enzymes CYP1A1 or CYP1A2; special interest was in the aromatic hydroxylation of the herbicide. The oxidative metabolites deaminometamitron (DAM) and 4-hydroxydeaminometamitron (4-HDAM) were found in the untransformed control culture as well as in the transgenic culture. The transgenic cultures, however, exhibited higher turnover rates after 48 h of incubation with 20 μg 14C-metamitron per assay (untransformed: 40%, CYP1A1: 80%, CYP1A2: 100%). Primary metabolite 4-HDAM was partially found in glucosylated form in the transgenic cultures. As minor oxidative metabolites, 6-hydroxyphenyl-3-methoxymethyl-1,2,4-triazine-5(4H)-one and 3-hydroxymethyl-6-phenyl-1,2,4-triazine-5(4H)-one were identified in the transgenic cultures by GC-MS, LC-MS. Additionally, it could be demonstrated that both foreign enzymes (CYP1A1, CYP1A2) also catalyzed the deamination of metamitron. In a large-scale study (up to 400 μ g per assay) with the transgenic culture expressing CYP1A2, the high efficiency of this P450 system toward metamitron was demonstrated: turnover of the xenobiotic was almost complete with 400 μ g. Since large portions of unglucosylated 4-H-DAM were found, the activity of foreign CYP1A2 apparently exceeded that of endogenous O-glucosyltransferases of the tobacco cell culture. We concluded that in comparison to the nontransformed cell culture, the extent of metabolism was considerably higher in the transgenic cultures. The transgenic cell cultures expressing human CYP1A1 or CYP1A2 are thus suitable tools for the production of large quantities of primary oxidized metabolites of metamitron.

Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specific transactivators.

In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.

Greenhouse evaluation of fitness-related reproductive traits in roundup®-tolerant transgenic creeping bentgrass (Agrostis Stolonifera L.)

Creeping bentgrass is a very important turfgrass species used extensively on golf course greens, fairways, and tees. One of the challenges of creeping bentgrass management is the control of grassy weeds, most of which respond to herbicides in a similar manner to that of creeping bentgrass. As part of a weed management program for golf courses, Roundup®-tolerant creeping bentgrass will be simple to employ and more effective in controlling problem weeds than currently available methods. The goal of this research was to evaluate fitness-related reproductive traits in four transgenic creeping bentgrass events modified to express a Roundup®-tolerant gene, cp4 epsps, to determine if these creeping bentgrass events had gained an unexpected reproductive fitness advantage. We compared transgenic events ASR 333, ASR801 with their nontransformed tissue culture line, C99056L and transgenic events ASR365, ASR368 with their non-transformed tissue culture line, B99061R. Populations of plants from three conventional cultivars were also included for comparison to determine whether significant variations, if present in transgenic events, were novel to the non-transformed organism, Agrostis stolonifera L. Our results showed that none of the four transgenic events surveyed were significantly different from the respective non-transformed tissue culture line plants for the following characteristics: first heading date, anthesis duration, inflorescence length, number of florets per inflorescence, pollen size, and seed-set capacity through open-pollination. One of the transgenic events, ASR333, needed significantly more days for anthesis initiation than the nontransformed tissue culture line, C99056L; while another transgenic event, ASR801, exhibited significantly shorter pollen longevity than plants of the tissue culture line, C99056L. However, ASR801 was not significantly different from the conventional cultivars ‘Penn A-4’ and ‘Penncross’ for pollen longevity. Plants of both transgenic events ASR365 and ASR368 did not differ significantly from plants of the tissue culture line, B99061R, for all characters measured.

Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes.

The transformation of Rubia cordifolia L. cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.

Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway.

It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells. The activator mechanism is still unknown. In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes. It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins. Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca2+ channel blockers and calcium deficiency. The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment. The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells. Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca2+ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine. These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca2+-dependent NADPH oxidase pathway.

Studies on the alkaloid production of genetically transformed and non-transformed cultures of Lobelia inflata L.

The investigations of the growth and alkaloid production of cell suspension-, callus-, organized- and hairy root cultures from Lobelia inflata L. proved that these cultures are able to synthesize the characteristic piperidine alkaloids of the intact plant. Alkaloid precursor amino acids (Phe, Lys) and plant growth regulators affect not only the growth and differentiation of tissue cultures but also their secondary metabolism. The synthetic regulator Sz/I I combined with Phe increased the total alkaloid content considerably in callus- and organized cultures; regulator Sz/28 especially increased the lobeline content (in organized cultures in response to Lys, in callus tissues as a result of Phe application). With the aim of optimizing growth and alkaloid production of the genetically transformed hairy root cultures of Lobelia inflata L. we studied the effect of some growth regulators (NAA, IAA, kinetin) and precursor amino acids (Lys, Phe). The kinetin had inhibiting effect on the growth and lobeline production of the hairy roots. The IAA and NAA increased the biomass formation and lobeline production. The highest lobeline level was detected in tissues cultivated on hormone-free medium containing Phe.