High-frequency shoot multiplication in Artemisia vulgaris L. using thidiazuron

An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory.     

Artemisinin production by transformed roots of Artemisia annua

Summary Transformed root cultures of several strains of Artemisia annua were obtained by infection with Agrobacterium rhizogenes ATCC 15834. Production of artemisinin, measured by HPLC, ranged from 0–0.42 % of dry weight (DW) in 10 different clones. Artemistene, artemisinic acid, and arteannuin B were also measured. Comparisons to literature reports suggest that the commercial production of artemisinic compounds using transformed roots is feasible.     

Factors influencing artemisinin production from shoot cultures of Artemisia annua L.

Artemisinin, an anti-malarial drug isolated from the annual wormwood Artemisia annua L., has a marked activity against chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum, and is useful in treatment of cerebral malaria. Shoot cultures of Artemisia annua L. were established on Murashige and Skoog basal medium which contained (per litre) 30 g sucrose, 0.5 mg 6-benzyladenine and 0.05 mg naphthaleneacetic acid. Using an optimized combination of sucrose (30 g/l), nitrate (45 mM), inorganic phosphate (200 mg/l), gibberellic acid (7 mg/l) and the ratio of NH₄⁺-N to NO₃^–-N of 1:3, artemisinin production reached 26.7 mg/l after 30 days. This procedure provides a potential alternative for production of artemisinin from in vitro tissue cultures.     

Artemisinin content in Artemisia annua L. extracts obtained by different methods

Isolation of artemisinin from Artemisia annua L. and its quantification by the HPLC-MS method are considered. Different extraction methods were used for the isolation of artemisinin: maceration, ultrasonic and subcritical CO₂-extraction. The component content of the CO₂- and hexane extracts was studied by the GC-MS method.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants

Summary A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N⁶-benzyladenine after transformation with the C58C1 Rif^R (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a -glucuronidase-positive response.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GM germination medium - GMVIT germination medium with vitamins - GUS -glucuronidase - Kin kinetin (N⁶-furfurylaminopurine) - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - PCR Polymerase Chain Reaction - T-DNA transfer-DNA - X-glucuronide 5-bromo-4-chloro-3-indolyl -D-glucuronide     

不同土壤环境对黄花蒿生长和青蒿素含量的影响研究

通过田间小区试验,比较研究了施肥与不施肥条件下,4种土壤环境（沙土、旱地土、水稻土和棕色石灰土）对黄花蒿的生长、生物量分配和青蒿素含量的影响。结果表明：黄花蒿对土壤养分的适应性较强,在沙土、旱地土、水稻土和石灰土上均能生长发育,养分水平低时,分配更多的生物量到根,根生物量分数和根/冠比增大;养分水平高时,分配更多的生物量到叶,叶生物量分数增加。黄花蒿的生长和青蒿素含量显著受土壤养分的影响,不施肥时,石灰土和水稻土栽培黄花蒿的株高、地径、总生物量、叶生物量和青蒿素含量显著大于旱地土,而旱地土又显著大于沙土。但在施肥条件下,以上参数不同土壤间无显著差异,且显著高于不施肥。因此,只要根据土壤养分状况合理施肥,黄花蒿在不同养分土壤栽培均能获得较高的青蒿素产量。     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

Effects of Overexpression of the Endogenous Farnesyl Diphosphate Synthase on the Artemisinin Content in Artemisia annua L.

Artemisinin is a novel effective antimalarial drug extracted from the medicinal plant Artemisia annua L. Owing to the tight market and low yield of artemisinin, there is great interest in enhancing the production of artemisinin. In the present study, farnesyi diphosphate synthase （FPS） was overexpressed in high-yield A. annua to Increase the artemisinin content. The FPS activity in transgenic A. ennue was twoto threefold greater than that In non-transgenic A. annua. The highest artemisinin content in transgenic A. annua was approximately 0.9% （dry weight）, which was 34.4% higher than that in non-transgenic A. annua. The results demonstrate the regulatory role of FPS in artemisinin biosynthesis.     

Artemisinin Production by Adventitious Shoots of Artemisia annua in a Novel Mist Bioreactor

A novel ultrasonic inner-loop bioreactor was used for artemisinin production by adventitious shoots in a multiplate culture of Artemisia annua L. The bioreactor was designed to allow the nutrient mist to uprise along a concentric draught-cylinder until it overflows from the top opening and the side-holes of the central tube downward and out of the annulus, so that the nutrient mist can be fulfilled in the bioreactor within 2 ~ 3 minutes. Under the misting cycles of every 3-minute misting in every 90 minute interval, artemisinin production reached totally 46.9 mg DW/L of culture medium at an airflow rate of 0.5 L/min for 25 d of culture in batches. The product amounted 2.9 and 3.2 folds of those obtained from culturing in solid medium and in shaking flasks respectively.     

Role of Salicylic Acid in Promoting Salt Stress Tolerance and Enhanced Artemisinin Production in Artemisia annua L.

In the present investigation, the role of salicylic acid (SA) in inducing salinity tolerance was studied in Artemisia annua L., which is a major source of the antimalarial drug artemisinin. SA, when applied at 1.00 mM, provided considerable protection against salt stress imposed by adding 50, 100, or 200 mM NaCl to soil. Salt stress negatively affected plant growth as assessed by length and dry weight of shoots and roots. Salinity also reduced the values of photosynthetic attributes and total chlorophyll content and inhibited the activities of nitrate reductase and carbonic anhydrase. Furthermore, salt stress significantly increased electrolyte leakage and proline content. Salt stress also induced oxidative stress as indicated by the elevated levels of lipid peroxidation compared to the control. A foliar spray of SA at 1.00 mM promoted the growth of plants, independent of salinity level. The activity of antioxidant enzymes, namely, catalase, peroxidase, and superoxide dismutase, was upregulated by salt stress and was further enhanced by SA treatment. Artemisinin content increased at 50 and 100 mM NaCl but decreased at 200 mM NaCl. The application of SA further enhanced artemisinin content when applied with 50 and 100 mM NaCl by 18.3 and 52.4%, respectively. These results indicate that moderate saline conditions can be exploited to obtain higher artemisinin content in A. annua plants, whereas the application of SA can be used to protect plant growth and induce its antioxidant defense system under salt stress.     

适于青蒿芽生长和青蒿素积累的光、温和培养方式探讨

探讨了光照、温度和培养方式对青蒿芽生长和青蒿素合成的影响。适宜芽生长和青蒿素积累的光照强度约为３ ０００ ｌｘ,照光时间为２０ ｈ／ｄ ;芽生长和青蒿素积累的最适温度分别为２５ ℃和３０ ℃,通过先２５ ℃（２５ ｄ）后３０ ℃（５ ｄ） 的温度转变二步培养法可以提高青蒿素的产量;青蒿芽生长和青蒿素积累的最佳培养方式为非浸没低转速摇瓶培养。     

反相高效液相色谱法测定青蒿中青蒿酸的含量

本文建立了反相高效液相色谱快速测定青蒿中青蒿酸含量的方法。色谱条件为:Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),柱温为(30±1)℃,流动相采用乙腈与0.2%磷酸水溶液混合液(体积比65:35),流速1 mL/min,检测波长220 nm。标准曲线回归方程:Y=8.784573×10-8X-6.443559×10-5,r=0.9997,青蒿酸回收率为102.4%。实验证明该方法稳定可靠、精密度高、重现性好、简单可行,适用于青蒿酸的分析检测。     

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO ₃ ^– /NH ₄ ⁺ and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO ₃ ^– /NH ₄ ⁺ at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l^–1, a 57% increase over that in the standard MS medium.     

Artemisinin biosynthesis in growing plants of Artemisia annua. A 13CO2 study

Artemisinin from Artemisia annua has become one of the most important drugs for malaria therapy. Its biosynthesis proceeds via amorpha-4,11-diene, but it is still unknown whether the isoprenoid precursors units are obtained by the mevalonate pathway or the more recently discovered non-mevalonate pathway. In order to address that question, a plant of A. annua was grown in an atmosphere containing 700 ppm of ¹³CO₂ for 100 min. Following a chase period of 10 days, artemisinin was isolated and analyzed by ¹³C NMR spectroscopy. The isotopologue pattern shows that artemisinin was predominantly biosynthesized from (E,E)-farnesyl diphosphate (FPP) whose central isoprenoid unit had been obtained via the non-mevalonate pathway. The isotopologue data confirm the previously proposed mechanisms for the cyclization of (E,E)-FPP to amorphadiene and its oxidative conversion to artemisinin. They also support deprotonation of a terminal allyl cation intermediate as the final step in the enzymatic conversion of FPP to amorphadiene and show that either of the two methyl groups can undergo deprotonation.     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.