Rosa Maria Cusido

Expression of the rolC gene and nicotine production in transgenic roots and their regenerated plants

Abstract Transformation of Nicotiana tabacum cv. Xanthi leaf sections with the pPCV002-ABC (rol genes A, B and C together under the control of their own promoter) or pPCV002-CaMVC (rol gene C alone under the control of the CaMV 35S promoter) construction present in trans-acting Agrobacterium tumefaciens vectors yielded several transgenic root lines. The two types (rolABC and rolC) of transgenic root lines were examined for their nicotine productivity in relation to growth rate and the amount of rolC gene product measured with specific antibodies. In all cases, the changes in the amount of this polypeptide were positively correlated with the capacity of the transgenic roots to grow and produce nicotine. Both capacities were greatly increased when the rolA, rolB and rolC genes were present together, which demonstrates that the activity of the three rol-gene-encoded functions is synergistic. Consistent observations were also made in the corresponding regenerated plants.

Biotechnological production of taxol and related taxoids: current state and prospects.

Taxol is one of the most effective anti-cancer drugs ever developed. The natural source of taxol is the inner bark of several Taxus species, but it accumulates at a very low concentration and with a prohibitively high cost of extraction. Another problem is that the use of inner bark for taxol production implies the destruction of yew trees. For all these reasons, the growing demand for taxol greatly exceeds the supply that can be sustained by isolation from its natural source and alternative sources of the drug are being sought. Although taxol has been prepared by total synthesis, the process is not commercially viable. Taxol can also be semisynthetically produced via the conversion of baccatin III or 10-deacethylbaccatinIII found in Taxus needles but the cost and difficulty of the extraction process of the semisynthetic precursors are also very high. The most promising approach for the sustainable production of taxol and related taxoids is provided by plant cell cultures at an industrial level. Taxol is currently being clinically used against different tumour processes but due to the difficulty of its extraction and formulation, as well as the growing demand for the compound, new taxol analogues with improved properties are being studied. In this revision we discuss current research in the design of new taxol-related compounds, the chemical structure/anti-cancer activity relationship and new formulations of the drug. We also consider the optimizing strategies to improve taxol and related taxoid production in cell cultures, as well as the current knowledge of taxol metabolism, all of which are illustrated with examples, some of them from our own research.

Alkaloid production in Duboisia hybrid hairy roots and plants overexpressing the h6h gene

Abstract In the biosynthetic pathway leading to the tropane alkaloid scopolamine, which is the 6,7β-epoxide of hyoscyamine, the enzyme hyoscyamine 6-hydroxylase (H6H; EC 1.14.11.11) catalyses two consecutive steps, hydroxylation of hyoscyamine followed by epoxide formation, to produce scopolamine. The h6h gene encoding H6H from Hyoscyamus niger , placed under the regulation of the CaMV 35S promoter, was introduced into the genome of a scopolamine-rich Duboisia hybrid by a binary vector system using the Agrobacterium rhizogenes strain LBA9402. The presence and overexpression of the transgene in the resulting kanamycin-resistant hairy roots and the regenerated plants obtained from them were confirmed by polymerase chain reaction and Northern blot analysis, respectively. The scopolamine levels in the resulting engineered hairy root lines increased up to three times compared to wild-type hairy roots, but there was no clear increase in the engineered regenerated plants. The best engineered line (H6H-5) produced 74.50xa0mg/l scopolamine, which to our knowledge is the highest content ever reported for hairy roots of tropane-alkaloid-producing plants containing the 35S- h6h transgene.

Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants

Abstract Inoculation of leaf sections of tobacco, Duboisia hybrid and Datura metel Solanaceae plants with A4 strain of Agrobacterium rhizogenes , induced transformed roots with the capacity to produce putrescine-derived alkaloids. In general, the hairy roots obtained showed two morphologies: typical hairy roots with high capacity to produce alkaloids and callus-like roots with faster growth capacity and lower alkaloid production. The aux 1 gene of A. rhizogenes was detected by PCR analyses in all roots showing callus-like morphology. However, this gene was only detected in 25–60% of the root cultures established showing typical hairy morphology. This fact suggests a significant role of aux genes in the morphology of transformed roots. Inoculation of leaf sections with A. tumefaciens strain C58 C1 carrying the pRiA4TR- (deletion of aux genes) did not produce roots with callus-like morphology.

Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures

Squalene synthase (SS) dimerizes two molecules of farnesyl diphosphate to synthesize squalene, a shared precursor in steroid and triterpenoid biosynthesis in plants. The SS1 gene encoding SS from Arabidopsis thaliana was introduced in Withania coagulans under the control of the CaMV35S promoter together with the T-DNA of Agrobacterium rhizogenes A4. The engineered hairy roots were studied for withanolide production and phytosterol accumulation and the results were compared with those obtained from control roots harbouring only the T-DNA from pRiA4. The increased capacity of the engineered roots for biosynthesizing phytosterols and withanolides was strongly related with the expression level of the transgene, showing the effectiveness of overexpressing 35SS1 to increase triterpenoid biosynthesis.

MANIPULATION BY CULTURE MIXING AND ELICITATION OF PACLITAXEL AND BACCATIN III PRODUCTION IN TAXUS BACCATA SUSPENSION CULTURES

SummaryExperiments were carried out with Taxus baccata cell lines showing different paclitaxel-producing capacities (between 1.74 and 19.91 mgl−1) when growing in a selected product-formation medium that specifically stimulated the production of taxane to the detriment of cell growth. Through mixing low-, medial- and high-producing lines, it could be observed that paclitaxel productivity in the resulting mixed lines was clearly higher than the mean productivity of the individual lines before mixing. This suggests that culture components generated by high-producing individual lines within the population might induce paclitaxel production. Although the accumulation of paclitaxel and baccatin III was higher when 100 μM methyl jasmonate was added to the subcultures of the mixed lines, the results indicate that exogenously applied methyl jasmonate was not the first factor to stimulate taxane production. The possible effects of methyl jasmonate elicitation and paclitaxel accumulation on cell viability are also considered.

Effect of Benzyladenine and Indolebutyric Acid on Ultrastructure, Glands Formation, and Essential Oil Accumulation in Lavandula Dentata Plantlets

Lavandin (Lavandula dentata) axillary buds were grown in Linsmaier-Skoog (LS) medium solidified with 10 % bactoagar (control) and supplemented with 0.1 mg dm−3 benzyladenine (BA), 0.1 mg dm−3 indolebutyric acid (IBA) or both plant growth regulators. In the studied conditions the axillary buds developed into plantlets. The addition of BA inhibited the formation of glands by 44 % as compared with the control plantlets and also inhibited their development: these plantlets had the highest number of unbroken glands (in pre-secretory state) when compared with plantlets grown in the other conditions. The presence of BA stimulated chloroplast formation, and increased the content of essential oils by 150 % with respect to the control plantlets. It also increased their secretion, and the number of lipid droplets in the chloroplasts, cytosol and plasmalemma. On the contrary, the presence of IBA decreased the essential oil concentration in plantlets by 31 % when compared with the control ones and inhibited their secretion capacity.

Growth and tropane alkaloid production inAgrobacterium transformed roots and derived callus ofDatura

Small callus pieces excised from theAgrobacterium transformed root line D2 ofDatura stramonium, were cultured onto solidified MS medium supplemented with a 1.0 μM kinetin and three different concentrations (0.1, 0.5 and 1.0 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D), and were examined for their alkaloid productivity in relation to organization level and growth rate. Growth of transformed roots (in a MS liquid medium without plant growth regulators) was greater than that of transformed calli excised from them and cultured separately. The addition of 1.0 μM 2,4-D to the culture medium had a positive effect on callus biomass production, while it inhibited root formation by this tissue (the lower the 2,4-D concentration in the medium the greater the number of roots which emerged from the calli). Hyoscyamine production was also higher in the transformed roots than in the transformed calli, and in these tissues the production of hyoscyamine was positively correlated with organogenesis index (i.e. its ability for rooting). At the same time, the epoxidation of hyoscyamine to scopolamine only took place in the transformed calli. This occurred to a greater extent at the lower concentrations of 2,4-D in the culture medium. The mode through which the 2,4-D could control the alkaloid production of transformed callus is discussed.

Genetic Transformation of Artemisia carvifolia Buch with rol Genes Enhances Artemisinin Accumulation.

The potent antimalarial drug artemisinin has a high cost, since its only viable source to date is Artemisia annua (0.01–0.8% DW). There is therefore an urgent need to design new strategies to increase its production or to find alternative sources. In the current study, Artemisia carvifolia Buch was selected with the aim of detecting artemisinin and then enhancing the production of the target compound and its derivatives. These metabolites were determined by LC-MS in the shoots of A. carvifolia wild type plants at the following concentrations: artemisinin (8μg/g), artesunate (2.24μg/g), dihydroartemisinin (13.6μg/g) and artemether (12.8μg/g). Genetic transformation of A. carvifolia was carried out with Agrobacterium tumefaciens GV3101 harboring the rol B and rol C genes. Artemisinin content increased 3-7-fold in transgenics bearing the rol B gene, and 2.3-6-fold in those with the rol C gene. A similar pattern was observed for artemisinin analogues. The dynamics of artemisinin content in transgenics and wild type A.carvifolia was also correlated with the expression of genes involved in its biosynthesis. Real time qPCR analysis revealed the differential expression of genes involved in artemisinin biosynthesis, i.e. those encoding amorpha-4, 11 diene synthase (ADS), cytochrome P450 (CYP71AV1), and aldehyde dehydrogenase 1 (ALDH1), with a relatively higher transcript level found in transgenics than in the wild type plant. Also, the gene related to trichome development and sesquiterpenoid biosynthesis (TFAR1) showed an altered expression in the transgenics compared to wild type A.carvifolia, which was in accordance with the trichome density of the respective plants. The trichome index was significantly higher in the rol B and rol C gene-expressing transgenics with an increased production of artemisinin, thereby demonstrating that the rol genes are effective inducers of plant secondary metabolism.

Enhanced artemisinin yield by expression of rol genes in Artemisia annua

BackgroundDespite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.MethodsIn this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4xa0%). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC–MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.ResultsTransgenics of rolB gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4–12-fold increase in artesunate and 1.2–3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.ConclusionThus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.