Influence of nano‐zinc oxide on tropane alkaloid production,h6h gene transcription and antioxidant enzyme activity in Hyoscyamus reticulatus L. hairy roots

The use of nanotechnology and biotechnology to improve the production of plant bioactive compounds is growing. Hyoscyamus reticulatus L. is a major source of tropane alkaloids with a wide therapeutic use, including treatment of Parkinson's disease and to calm schizoid patients. In the present study, hairy roots were obtained from two‐week‐old cotyledon explants of H. reticulatus L. using the A7 strain of Agrobacterium rhizogenes. The effects of different concentrations of the signaling molecule nano‐zinc oxide (ZnO) (0, 50, 100 and 200 mg/L), with three exposure times (24, 48 and 72 h), on the growth rate, antioxidant enzyme activity, total phenol contents (TPC), tropane alkaloid contents and hyoscyamine‐6‐beta‐hydroxylase (h6h) gene expression levels were investigated. Growth curve analysis revealed a decrease in fresh and dry weight of ZnO‐treated hairy roots compared to the control. ANOVA results showed that the antioxidant activity of the enzymes catalase, guaiacol peroxidase and ascorbate peroxidase was significantly higher in the ZnO‐treated hairy roots than in the control, as was the TPC. The highest levels of hyoscyamine (37%) and scopolamine (37.63%) were obtained in hairy roots treated with 100 mg/L of ZnO after 48 and 72 h, respectively. Semi‐quantitative RT‐PCR analysis revealed the highest h6h gene expression was in hairy roots treated with 100 mg/L of ZnO after 24 h. It can be concluded that ZnO is as an effective elicitor of tropane alkaloids such as hyoscyamine and scopolamine due to its enhancing effect on expression levels of the biosynthetic h6h gene.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Influence of chitosan,acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida Effect of medium pH and growth phase

The effects of chitosan, acetic acid and citric acid on production and release of hyoscyamine and scopolamine in hairy root cultures of Brugmansia candida were studied. Chitosan and acetic acid were tested at different concentrations and also at different media pH values. At pH 5.5, and at certain concentrations, acetic acid and chitosan increased the content of root scopolamine and hyoscyamine, and promoted the release of both alkaloids. Lowering the pH to 3.5 and 4.5 reduced the accumulation of both alkaloids in the roots, but at a pH of 4.5, their release increased significantly. Acetic and citric acid stimulated the release of scopolamine and hyoscyamine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering

 In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems. Received: 24 December 1998 / Accepted: 13 January 1999     

Increase in the indole alkaloid production and its excretion into the culture medium by calcium antagonists inCatharanthus roseus hairy roots

Treatment of Catharanthus roseus hairy roots with antagonists, like verapamil and CdCl₂, that block the Ca²⁺ flux across the plasma membrane enhanced the total alkaloid content by 25% and their secretion 10 times. The specific Ca²⁺ chelator, EGTA, stimulated 90% of the total alkaloid secretion. Treatment with inhibitors of intracellular Ca²⁺ movement, like TMB-8 and trapsigargin, enhanced the total alkaloid content by 74% and their secretion into the culture media by 4- to 6-fold. The results suggest that an inhibition of external and internal Ca²⁺ fluxes induces an increase in the indole alkaloid accumulation and secretion in C. roseus hairy roots.     

青蒿毛状根生长、青蒿素合成以及 营养物消耗的动力学

诱导产生的青蒿毛状根培养物置于MS培养基(含30 g/L蔗糖)进行悬浮培养,并对悬浮培养过程中毛状根生长、青蒿素合成、蔗糖、磷酸盐和不同氮源的消耗、pH和电导率的动力学过程进行分析。经30 d培养,生物量干重和青蒿素产量分别达到13.7 g/L和0.23 g/L,碳源和氮源在培养过程中被逐渐利用,而磷酸盐的利用速率最快,培养至15 d所有的磷酸盐均被吸收,pH在培养初期降低,后又逐渐上升,电导率由于毛状根生长对无机离子的吸收而逐渐减低。     

镉及其与钙组合对褐脉少花龙葵毛状根生长、抗氧化酶活性和吸收镉的影响

为了探讨利用褐脉少花龙葵毛状根来修复重金属镉(Cd)污染的可能性,采用溶液培养法研究了Cd单独及其与钙(Ca)组合对褐脉少花龙葵毛状根生长、抗氧化酶超氧化物歧化酶(SOD)和过氧化物酶(POD)活性及对Cd吸收的影响。结果表明,Cd≤50μmol/L时能促进毛状根生长,而高于100μmol/LCd则抑制毛状根生长,使其侧根根尖变褐和变短,数目减少。与对照相比,不同浓度Cd培养的毛状根可溶性蛋白含量和SOD活性先升高后逐渐下降;其丙二醛(MDA)含量显著提高;100μmol/LCd使毛状根POD活性逐渐升高,但300μmol/LCd则使毛状根POD活性逐渐降低。与对照(仅添加100μmol/L或300μmol/LCd的毛状根)相比,Cd和10～30mmol/LCaCl2组合培养使毛状根可溶性蛋白含量和MDA含量降低;但提高其SOD活性;而100μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性均比对照低;而300μmol/LCd和10～30mmol/LCaCl2结合培养的毛状根POD活性则均比对照提高。原子吸收分光光度法测定结果表明,毛状根吸收和吸附的重金属Cd含量随着培养基中Cd浓度的升高而增加。但外源加入10～30mmol/LCaCl2能减少毛状根对Cd的吸收,并调节其抗氧化酶SOD和POD活性,降低其膜脂过氧化水平而解除重金属Cd对毛状根生长的抑制或毒害。     

Modulation of artemisinin biosynthesis by elicitors,inhibitor, and precursor in hairy root cultures of Artemisia annua L.

Artemisinin is frequently used in the artemisinin-based combination therapy to cure drug-resistant malaria in Asian subcontinent and large swath of Africa. The hairy root system, using the Agrobacterium rhizogenes LBA 9402 strain to enhance the production of artemisinin in Artemisia annua L., is developed in our laboratory. The transgenic nature of hairy root lines and the copy number of transgene (rol B) were confirmed using polymerase chain reaction and Southern Blot analyses, respectively. The effect of different concentrations of methyl jasmonate (MeJA), fungal elicitors (Alternaria alternate, Curvularia limata, Fusarium solani, and Piriformospora indica), farnesyl pyrophosphate, and miconazole on artemisinin production in hairy root cultures were evaluated. Among all the factors used individually for their effect on artemisinin production in hairy root culture system, the maximum enhancement was achieved with P. indica (1.97 times). Increment of 2.44 times in artemisinin concentration by this system was, however, obtained by combined addition of MeJA and cell homogenate of P. indica in the culture medium. The effects of these factors on artemisinin production were positively correlated with regulatory genes of MVA, MEP, and artemisinin biosynthetic pathways, viz. hmgr, ads, cyp71av1, aldh1, dxs, dxr, and dbr2 in hairy root cultures of A. annua L.     

Enhanced ethylene production by primary roots of Zea mays L. in response to sub-ambient partial pressures of oxygen

Ethylene production by primary roots of 72–h-old intact seedlings of Zea mays L. cv. LG11 was studied under ambient and sub-ambient oxygen partial pressures (pO₂) using a gas flow-through system linked to a photoacoustic laser detector. Despite precautions to minimize physical perturbation to seedlings while setting-up, ethylene production in air was faster during the first 6h than later, in association with a small temporary swelling of the roots. When roots were switched from air (20–8kPa O₂) to 3 or 5kPa O₂ after 6h, ethylene production increased within 2—3 h. When, the roots were returned to air 16 h later, ethylene production decreased within 2—3 h. The presence of 10kPa CO₂ did not interfere with the effect of 3kPa O₂. Transferring roots from air to 12–5kPa did not change ethylene production, while a reduction to 1 kPa O₂ induced a small increase. The extra ethylene formed in 3 and 5 kPa O₂ was associated with plagiotropism, swelling, root hair production, and after 72 h, increased amounts of intercellular space (aerenchyma) in the root cortex. Root extension was also slowed down, but the pattern of response to oxygen shortage did not always match that of ethylene production. On return to air, subsequent growth patterns became normal within a few hours. In the complete absence of oxygen, no ethylene production was detected, even when anaerobic roots were returned to air after 16 h.     

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. CV. Lucknow local

Summary The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l⁻¹ level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.     

Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding

We have studied the effects of precursor amino acids (phenylalanine and cystein), elicitors (salicylic and acetylsalicylic acids, methyl jasmonate, β-aminobutyric acid, yeast extract), PAL-inhibitor (1-amino-2-phenylethylphosphonic acid) applied alone or in combination on glucotropaeolin production and myrosinase activity in hairy root cultures of Tropaeolum majus. Short, 24-h treatment, and subsequent transfer of hairy roots to the fresh medium enabled avoiding detrimental effect of studied stimulators on biomass growth. In control cultures the highest glucotropaeolin content, 58.1 ± 6.7 mg g⁻¹ DW, was detected on the 3rd day after transfer of the roots to the fresh medium but glucotropaeolin yield (mg 100 ml^–1 culture volume) had been increasing until the 9th day after transfer as a result of continuous biomass growth. Glucotropaeolin content and yield were 2-fold enhanced after treatment with precursor amino acids or PAL-inhibitor alone, but their combination additively led to 4-fold increase in glucotropaeolin production. Among the studied elicitors acetylsalicylic acid induced the highest, 3-fold increase in glucotropaeolin production, it also enhanced myrosinase activity, but to a smaller extend (by about 50%). Acetylsalicylic acid also potentiated induced by precursors, PAL-inhibitor, methyl jasmonate and yeast extract production of glucotropaeolin. The highest, 4.8-fold increase in glucotropaeolin production was found after combined acetylsalicylic acid and precursors treatment. Additive effect of acetylsalicylic acid-combined treatment on myrosinase activity was not detected. The obtained results indicate that amino acid precursors, phenylalanine and cystein, availability may be a limiting factor in the process of stimulation of glucotropaeolin production in T. majus hairy root cultures.     

Ferritin enhances production of DNA strand breaks by 6-hydroxydopamine,ascorbic acid and H2O2 in CCC PM2 bacteriophage DNA

Damage of CCC PM2 DNA by 6-hydroxydopamine (6-OHDA) and ascorbic acid (AA), compounds that are both able to release iron from ferritin, was significantly enhanced in the presence of ferritin. H₂O₂, a product of 6-OHDA autoxidation, did not induce DNA strand breaks in the absence of ferritin and only to a minor extent in the presence of ferritin. DNA damage by 6-OHDA and AA could be reduced by the hydroxyl radical scavenger mannitol, the iron chelator desferrioxamine, and, partly, by a combination of superoxide dismutase and catalase. These inhibitory effects were clearly less pronounced in the presence of ferritin. Ferritin obviously played an important role as a source of iron in the pro-oxidative processes of 6-OHDA and AA. These features might be of importance in cancer therapy since many tumor cells contain elevated ferritin levels.     

Synergistic effect of auxins and polyamines in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. during growth,coumarin production and morphogenesis

Hairy roots of Cichorium intybus obtained by infecting with different Agrobacterium rhizogenes strains (LMG-150 and A20/83) were studied for total endogenous indole-3-acetic acid (IAA) levels and indole-3-acetic acid oxidase (IAAO) activity. The roots initiated by LMG- 150 showed higher endogenous IAA levels as well as IAAO activity as compared to the roots from A20/83. Coumarin production in roots obtained by both of these strains strictly correlated with growth, with higher content in the roots obtained by LMG- 150. Moreover roots from LMG-150 showed increased growth index, length of primary roots and number of secondary and tertiary roots. The roots derived from LMG-150 were studied for total endogenous IAA and IAAO activity under the exogenous administration of polyamines and fungal elicitors. The treatment with putrescine (Put) at 1.5 mM level showed maximum endogenous IAA levels and IAAO activity as compared to the control and other polyamine administration, it also supported faster growth in terms of biomass accumulation, and total coumarin production. Of the various treatments, mycelial extract (ME) and culture media filtrate (CMF) of Pythium aphanidermatum and Phytopthora parasitica var. nicotiana, the treatment with 1 % CMF of P. parasitica var. nicotiana, resulted in maximum IAA levels and IAAO activity, which was supported by maximum biomass, coumarin production as compared to the control and other elicitor treatments. Two different regenerants of chicory obtained through A. rhizogenes LMG-150 designated as T-I and T-II, were studied for total endogenous IAA levels and IAAO activity. T-II showed higher titers of IAA with higher activity of IAAO as compared to T-I. Endogenous titer of IAA and IAAO activity was found to be maximum in transformed roots as compared to T-I, T-II, normal roots and normal plants. Our work showed a variation in endogenous auxin levels in these transformed plants. There exists a synergistic effect of endogenous IAA titers and polyamines in regulating root morphogenesis. Fungal elicitors influenced growth and coumarin production and an elicitor preparation of 1 % CMF of P. parasitica var. nicotiana gave spontaneous regeneration of shoots. The implications of these results are discussed.     

Plant water status, ethylene evolution, N(2)-fixing efficiency, antioxidant activity and lipid peroxidation in Cicer arietinum L. nodules as affected by short-term salinization and desalinization

Salinity induced changes in ethylene evolution, antioxidant defense system, N(2)-fixing efficiency and membrane integrity in relation to water and mineral status in chickpea (Cicer arietinum L.) nodules were studied under screen house conditions. At vegetative stage (55-65 DAS) plants were exposed to single saline irrigation (Cl(-) dominated) of levels 0, 2.5, 5.0 and 10.0dSm(-1) and sampled after 3d. The other set of treated plants was desalinized by flooding and the plants were sampled after further 3d. Water potential (Psiw) of leaf and osmotic potential (Psis) of leaf and nodules significantly decreased from -0.44 to -0.56MPa and from -0.65 to -1.15MPa and from -0.75 to -1.77MPa, respectively upon salinization. RWC of leaf and nodules also reduced from 86.05% to 73.30% and 94.70% to 89.98%, respectively. The decline in Psis of nodules was due to accumulation of proline and total soluble sugar. In comparison to control, the increase in ethylene (C(2)H(4)) production was 35-108% higher and correspondingly increase in 1-aminocycloprane-1-carboxylic acid (ACC) content (37-126%) and ACC oxidase activity (31-118%) was also noticed. Similarly, marked increase in H(2)O(2) (25-139%) and thiobarbituric acid substances (TBRAS, 11-133%) contents was seen. N(2)-fixing efficiency i.e. N(2)-ase activity, leghemoglobin and N contents of nodules declined significantly after saline irrigation. The induction in specific activity of antioxidant enzymes was confirmed by the increase in activity of superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione reductase and glutathione transferase, whereas reverse was true for catalase. These activated enzymes could not overcome the accumulation of H(2)O(2) in nodules. Ascorbic acid content also declined from 20 to 38%, whereas Na(+)/K(+) ratio and Cl(-) content were significantly enhanced. Upon desalinization, a partial recovery in all above metabolic processes and water relations parameters was noticed. It is suggested that ethylene in relation to water status and lipid peroxidation and along with other metabolic processes has an important role in induced nodules senescence under salinity.