利用气升式内环流生物反应器培养青蒿毛状根生产青蒿素

利用自制的气升式内环流生物反应器进行青蒿(Artemisia annua L.)毛状根多层培养生产青蒿素。毛状根培养物在培养过程中均匀分布在生物反应器的筛网间,或以不锈钢网为附着点向四周生长,在25 ℃和12 h/d光照周期下,经20 d分批培养获得生物量干重22.57 g/L,青蒿素产量374.4 mg/L,并对培养过程中蔗糖、磷酸盐、硝酸盐和氨盐消耗的动力学进行了分析。     

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。     

利用气升式内环流生物反应器培养青蒿毛状根生产青蒿素

利用自制的气升式内环流生物反应器进行青蒿（ArtemisiaannuaL．）毛状根多层培养生产青蒿素。毛状根培养物在培养过程中均匀分布在生物反应器的筛网间，或以不锈钢网为附着点向四周生长，在25℃和12h／d光照周期下，经20d分批培养获得生物量干重22．57g／L，青蒿素产量374．4mg／L，并对培养过程中蔗糖、磷酸盐、硝酸盐和氨盐消耗的动力学进行了分析。     

不同理化因子对雪莲毛状根生长和总黄酮生物合成的影响

在 1 2MS液体培养基上研究了不同理化因子对水母雪莲毛状根生长和总黄酮生物合成的影响。实验结果表明 :氮源总浓度 (包括NH+4和NO-3)为 30mmol L ;NH⁺₄ NO^-₃比例为 5∶2 5 ;2 %蔗糖和 3%葡萄糖组合 ;0.5mg LGA₃和 0.5mg LIBA ;pH5.8;18h d的光照 (光强为 35.0.0lx) ;2.4℃ ;摇床转速为 100rmin有利于毛状根生长及总黄酮的生物合成。在此培养条件下 ,经过21d的培养毛状根生长量达到 12.8g L(DW) ,总黄酮合成量为 192.2mg L ,即总黄酮含量占毛状根干重的 15 % ,约为干重野生水母雪莲植株总黄酮含量的 2.5倍。     

重金属镉及其与锌组合对黄瓜毛状根生长及其抗氧化酶活性的影响

为了探讨利用黄瓜毛状根来修复重金属镉(Cd)污染的可能性, 研究了重金属Cd单独及其与锌(Zn)组合对黄瓜毛状根生长及其抗氧化酶SOD、POD活性变化的影响。结果表明, Cd≤10 mg/L仅在培养5~15 d间促进黄瓜毛状根生长, 使根增粗; 而Cd≥15 mg/L则抑制黄瓜毛状根生长, 浓度愈高抑制作用愈明显, 侧根变得短而细小。在供试的不同浓度Cd培养的黄瓜毛状根中, 除10 mg/L Cd外, 其余Cd浓度培养的黄瓜毛状根可溶性蛋白含量随着培养时间的延长而逐渐下降; 但其POD和SOD活性则随着培养时间的延长而逐渐升高。与对照(仅添加25 mg/L Zn)相比, 仅1 mg/L Cd+ 25 mg/L Zn组合在培养7~15 d期间促进黄瓜毛状根生长; 其余浓度Cd和25 mg/L Zn组合都抑制黄瓜毛状根的生长, 且Cd浓度愈高抑制作用越强, 侧根数目更少且短小, 侧根根尖变得肿胀; 同时, 除培养5 d外, 25 mg/L Zn和不同浓度Cd组合培养的黄瓜毛状根的生物量、POD和SOD活性均比单独添加对应浓度Cd培养的毛状根降低, 但其可溶性蛋白含量则较之明显提高。结果表明: 黄瓜毛状根具有较强的重金属Cd耐受能力, 高浓度Cd则抑制其生长; 而镉和锌组合会随着培养时间的延长而加重Cd对黄瓜毛状根生长的抑制作用。     

培养条件对新疆紫草毛状根生长及紫草素含量的影响

以发根农杆菌诱导的新疆紫草毛状根为试验材料,采用二阶段液体培养法,首次建立了新疆紫草毛状根培养技术体系。结果显示:采用SH无铵培养基、pH 5.8时有利于毛状根的生长。培养12d时毛状根的增殖倍数达最高,平均10.26倍;毛状根生产的继代周期为25～30d;4种树脂吸附的紫草素及其衍生物含量均较对照(不添加树脂)高,以NKA-9所吸附的紫草素及其衍生物含量最高,为2.38%,较对照提高0.97倍。培养10d时添加NKA-9树脂,紫草素及其衍生物含量平均为3.64%,是对照的3.08倍。研究表明,生长阶段采用液体培养可以使新疆紫草毛状根快速增殖,生产阶段添加大孔吸附树脂能够提高紫草素及其衍生物含量。     

外源激素对何首乌毛状根生长及蒽醌类成分生物合成的影响

研究了外源激素对液体培养何首乌毛状根生长及其蒽醌类化合物生物合成的影响。结果表明外源激素2,4-D、NAA和6-BA对何首乌毛状根的生长及蒽醌生物合成有较大的影响。在MS培养基中添加2,4-D对何首乌毛状根的生长和蒽醌类化合物的生物合成有很强的抑制作用,而添加适量浓度NAA和6-BA则可促进何首乌毛状根的生长以及蒽醌类物质的生物合成。     

褐脉少花龙葵毛状根的诱导、培养及其澳洲茄胺的产生

利用发根农杆茵的遗传转化和液体培养技术,研究了褐脉少花龙葵(solanum nigrum L.Var.Dauciforum)毛状根的诱导和离体培养及其澳洲茄胺的产生以及液体培养过程中培养基中N源和钙的消耗变化.结果表明.发根农杆菌ATCC15834感染褐脉少花龙葵叶片外植体5 d后产生毛状根.感染25d后,约90%的叶片外植体产生毛状根.毛状根能在无外源生长调节剂的MS固体和体培养基上自主生长.PCR扩增结果显示发根农杆菌Ri质粒的rilB和rolC基因已在少花龙葵毛状根基因组中整合并得到表达.所产生的毛状根能产生药用次生物质澳洲茄胺,其含量约为非转化植株根的1.3倍,达到582.05μg/g干重.少花龙葵毛状根液体培养0-5 d内处于生长迟滞期、5-15 d为快速生长期、15d后进入生长平台期.培养基的硝态氮和铵态氮在毛状根液体培养过程中被逐渐吸收和消耗,至培养15 d时铵态氮被消耗殆尽.而硝态氮仍剩余44.7%;培养基中钙的浓度在培养过程中虽逐渐降低,但在培养25d时仍未被完全消耗,其浓度约为起始浓度的43.5%.该结果为今后设计合适的培养基来规模培养褐脉少花龙葵毛状根生产药用次生物质澳洲茄胺提供了可能性.     

青蒿素在露水草毛状根中的生物转化

露水草毛状根培养系中加入青蒿素培养８ｄ后,青蒿素转化去氧青蒿素。根据光谱数据,对去氧青蒿素的结构进行了鉴定。结果表明,通过水草毛状根能将青蒿素进行选择性还原为去氧青蒿素。     

青蒿毛状根合成青蒿素的培养条件研究

对影响青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）毛状根生长及青蒿素合成的培养条件进行了研究,确定最适的培养条件为：初始ｐＨ５．８～６．０,摇瓶转速１３０～１５０ｒ／ｍｉｎ,摇瓶装液量体积分数为２５％,光照周期为１６ｈ／ｄ,温度为３０℃。在此条件下,经过２５ｄ培养获得青蒿素产量为２２３．３ｍｇ／Ｌ。     

促进黄花蒿发根青蒿素合成的内生真菌诱导子的制备

应用酸解法对黄花蒿(ArtemisiaannuaL.)内生胶孢炭疽菌(Colletotrichumgloeosporioides)菌丝体进行提取,在黄花蒿发根培养系统中比较了各制备提取物的青蒿素诱导活性。活性提取物经过SephadexG25层析后,部分纯化的内生菌寡糖提取物(MW<2500)可显著促进发根青蒿素的合成,培养23d的发根经诱导子(0.4mg/mL)处理4d后,青蒿素产量可达13.51mg/L,比同期对照产量提高51.63%,诱导作用与诱导子浓度、作用时间相关。内生菌寡糖诱导子的制备和使用,在青蒿素生物技术生产研究中为首次应用。     

Effects of Fungal Elicitors on Cell Growth and Artemisinin Accumulation in Hairy Root Cultures of Artemisia annua

The artemisinin accumulation in the hairy root cultures of Artemisia annua L. was enhanced via a treatment of three fungal elicitors separately ( Verticillium dahliae Kleb., Rhizopus stolonifer (Ehrenb. ex Fr. ) Vuill and Colletotrichum dematium (Pers.) Grove). Among these three elicitors, V. dahliae had the highest inducing efficiency, but none of them manifests any noticeable effects on the cell growth of the hairy root cultures. The artemisinin content of the hairy root cultures treated with V. dahliae elicitor was 1.12 mg/g DW, which was 45% higher than the control (0.77 mg/g DW). The results showed that elicitation was dependent on the elicitor concentration, the incubation period and the physiological stage at which the hairy root cultures were treated. In addition, the authors found that for V. dahliae, the optimum concentration was 0.4 mg carbohydrate per millilitre medium, the strongest response of A. annua hairy root cultures to the elicitation was at the late exponential growth stage, and the highest artemisinin content of the hairy root cultures was on the 4th day post treatment.     

真菌诱导子对青蒿发根细胞生长和青蒿素积累的影响

３种真菌诱导子（大菌丽花轮枝孢（Ｖｅｒｔｉｃｉｌｌｉｕｍ ｄａｈｉａｅ Ｋｌｅｂ．）、葡枝根霉（Ｒｈｉｚｏｐｕｓ ｓｔｏｌｏｎｉｆｅｒ（Ｅｈｒｅｎｂ．ｅｘＦｒ．）Ｖｕｉｌｌ）和束状刺盘孢（Ｃｏｌｌｅｔｏｒｉｃｈｕｍ ｄｅｍａｔｉｕｍ（Ｐｅｒｓ．）Ｇｒｏｖｅ）处理青蒿（Ａｒｔｅｍｉｓｉａ ａｎｎｕａＬ．）的发根,均能促进发根中青蒿素的积累,其中以大丽花轮枝孢的诱导效果最好;对细胞生长均没有明显影响,     

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.     

Ri质粒转化的青蒿发根培养及青蒿素的生物合成

用发根农杆菌(Agrobacterium rhizogenes)转化药用植物青蒿(Artemisia annua L．)并建立了发根体外培养系统。Southern杂交、NPT Ⅱ酶的检测证明Ri质粒的T—DNA转移并整合到植物的核基因组上。在发根培养系统中,检测了青蒿的重要次生代谢物一青蒿素的含量,检测了不同理化因子对发根生长及青蒿素含量的影响。结果表明：光照(日光灯,12h光周期,20001x)有利于次生产物青蒿素的积累。培养基的pH值为5．4。蔗糖浓度为3％不仅促进发根的生长,而且促进青蒿素的积累。低浓度萘乙酸(NAA)对发根生长具有促进作用,但抑制青蒿素的合成。赤霉素GA,对发根的生长及次生产物的合成都具有促进作用,其最适浓度为4．8mg／L。     

Isolation and production of artemisinin and stigmasterol in hairy root cultures of Artemisia annua

Scaled-up hairy root culture of Artemisia annua L. was established in three-liter Erlenmeyer flask. Both artemisinin and stigmasterol that derive from the common precursors of isopentenyl diphosphate and farnesyl pyrophosphate were isolated from hairy roots. The production rate of artemisinin isolated by column chromatography from hairy root cultures was 0.54% (mg.gDW⁻¹). Stigmasterol was identified by mass spectrometry and nuclear magnetic resonance analysis. The production of stigmasterol isolated by column chromatography from hairy root cultures was 108.3% (mg.gDW⁻¹). In hairy root cultures, the production rate of stigmasterol was estimated to be 201 times greater than that of artemisinin. Our results suggest that investigation of secondary metabolites may provide a new insight to study artemisinin production in hairy root cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Tetraploid Artemisia annua hairy roots produce more artemisinin than diploids

Hairy root cultures of diploid Artemisia annua L. (clone YUT16) grow rapidly and produce the antimalarial sesquiterpene artemisinin. Little is known about how polyploidy affects the growth of transformed hairy roots and the production of secondary metabolites. Using colchicine, we produced four stable tetraploid clones of A. annua L. from the YUT16 hairy root clone. Analysis showed major differences in growth and artemisinin production compared to the diploid clone. Tetraploid clones produced up to six times more artemisinin than the diploid parent. This study provides an initial step in increasing our understanding of the role of polyploidy in secondary metabolite production, especially in hairy roots.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.