长鞭红景天的组织培养和快速繁殖

1植物名称长鞭红景天(Rhodiola fastigiata). 2材料类别幼叶和嫩茎. 3培养条件愈伤组织诱导培养基:(1)WPM 6-BA 1.5 mg·L-1(单位下同) IBA 0.1;分化与增殖培养基:(2)WPM 6-BA 1.5 IBA 0.05;生根培养基:(3)WPM IBA 0.1,(4)WPM IBA 0.1 多效唑0.05.上述培养基均加入5.0g·L-1的琼脂粉、3%蔗糖,pH 5.8.培养温度为(25±2)℃,光照度为1 500～2000 lx,光照时间10 h·d-1.     

藜蒿的组织培养和快速繁殖研究

通过组织培养技术可以诱导藜蒿的幼叶和茎段产生愈伤组织和植株.研究表明,MS NAA 0.5 mg/L 6-BA 0.5 mg/L(蔗糖3%)最适合诱导藜蒿的叶片、茎段形成愈伤组织以及愈伤组织分化成再生植株,而MS NAA 0.5 mg/L最适合外植体诱导生根,1/2MS培养基对幼苗的生根作用十分明显.再生植株水培试验,筛选出了适合于藜蒿工厂化生产的营养液.     

银叶菊的组织培养

1植物名称银叶菊(Senecio cineraria). 2材料类别嫩叶及其叶柄. 3培养条件愈伤组织的诱导和分化培养基:(1)MS 6-BA 1.0 mg·L-1(单位下同) NAA 0.1;(2)MS 6-BA 2.0 NAA 0.1;(3)MS 6-BA 1.0 KT2.0 NAA 0.1.增殖培养基:(4)MS 6-BA 0.5 NAA 0.1.生根培养基:(5)MS NAA 0.2.培养基均加3%蔗糖和0.6%琼脂,pH 5.8.培养温度22～27℃,光照度1500～2000lx,光照12 h·d-1.     

活血丹组织培养与快速繁殖技术研究

以活血丹为材料,应用组织培养和快速繁殖技术,对适于活血丹增殖分化的培养基、培养方式进行了系统的研究。用活血丹叶片作为外植体,在MS添加生长素2,4-D和细胞分裂素BA的培养基上成功诱导出愈伤组织,并对其继代培养条件进行研究,分析了继代培养中褐化的原因。在MS添加NAA和BA的培养基中,活血丹的茎尖和带腋芽茎段能直接诱导出大量丛生芽,随后将不定芽转入MS添加IBA和KT的培养基中,可生成不定根,完成快速繁殖技术体系。结果表明:活血丹愈伤组织诱导的最佳培养基为MS+2,4-D(1.5mg/L)+BA(1.0mg/L),诱导率高达91.38%。丛生芽诱导的适宜培养基为MS+NAA(0.1mg/L)+BA(1·0mg/L),在此培养基上,出芽率达100%,芽增殖系数接近于10,有利于生物量的积累。而根的诱导则在MS+IBA(1.0mg/L)+KT(1.0mg/L)培养基上进行最好,此基础上能诱导出健康、粗壮的根。试管苗炼苗后移栽,成活率达100%。     

虎耳兰的组织培养和快速繁殖

1植物名称虎耳兰(Haemanthus albiflos). 2材料类别幼嫩叶片. 3培养条件诱导愈伤组织和芽分化培养基:(1)MS 6-BA 3.0 mg.L-1(单位下同) NAA 0.1;(2)MS 6-BA 2.0 NAA 0.1;(3)MS 6-BA 2.0 NAA 1.0.增殖培养基:(4)MS 6-BA2.0 NAA0.1 0.2?(活性炭).诱导生根培养基:(5)1/2MS NAA 0.2;(6)1/2MS NAA 0.2 0.2?.以上培养基均加入琼脂6.2 g·L-1,pH 5.8.培养基(1)～(3)中蔗糖为3.0%,(5)和(6)中蔗糖为1.5%.培养温度(25±2)℃,光照度1 500～2 000lx,光照12 h.d-1.     

华南可爱花的组织培养和快速繁殖

1植物名称华南可爱花(Eranthemum austrosinensis). 2材料类别带侧芽的茎段. 3培养条件以Ms为基本培养基.愈伤组织诱导培养基:(1)MS 6-BA 2.0 mg.L-1(单位下同) NAA0.1 TDZ 0.01.愈伤组织分化培养基:(2)MS 6-BA 2.0 NAA 0.1.不定芽诱导培养基:(3)MS 6-BA 0.2 NAA 0.1;(4)MS 6-BA 0.4 NAA 0.1;(5)MS 6-BA 0.6 NAA 0.1;(6)MS 6-BA 0.8 NAA 0.1;(7)MS 6-BA 1.0 NAA 0.1;(8)MS 6-BA 1.2 NAA0.1;(9)MS 6-BA 1.5 NAA 0.1.生根培养基:(10)Ms NAA 0.2;(11)MS NAA 0.4.以上培养基均添加3%蔗糖、0.65%卡拉胶,pH 5.8～6.2.培养温度为(25±1)℃,光照度1 500～2 000 lx,光照时间为12 h.d-1.     

蓝花楹组织培养与快速繁殖研究

以蓝花楹(Jacaranda mimosifolia Humb.et Bonpl.)胚轴为外植体进行组织培养和快繁体系建立的研究。结果表明,蓝花楹种子经40℃-45℃温水浸泡后发芽率较高,达到55.7%。蓝花楹不定芽和愈伤组织诱导的最适培养基分别为MS+6-BA2.0 mg L-1+NAA 0.1 mg L-1+2,4-D 0.1 mg L-1和M S+6-BA 0.5 mg L-1+NAA 1.0 mg L-1+2,4-D 1.0 mg L-1。不定芽和愈伤组织增殖的最适培养基分别为改良MS培养基+6-BA 0.5 mg L-1+NAA 0.5 mg L-1+IBA 0.5 mg L-1和MS+6-BA 1.0 mg L-1+NAA 0.5 mg L-1+ZT 3.0 mg L-1。愈伤组织分化最适培养基为M S+BA 1.0 mg L-1+NAA 0.5 mg L-1+2,4-D 0.5 mg L-1。最适生根培养基为1/2MS+蔗糖20 g L-1+NAA 0.1 mg L-1+活性炭2.0 g L-1,生根率达78.3%。     

珠芽魔芋的组织培养与快速繁殖

1植物名称珠芽魔芋[Amorphophallus bulbifer (Roxb.)Blume]。2材料类别芽鞘。3培养条件(1)愈伤组织诱导培养基:MS 6-BA 0.6mg·L~(-1)(单位下同) NAA 0.1;(2)不定芽诱导和增殖培养基:MS 6-BA 1.0 NAA 0.4:(3)生根培养基:1/2MS NAA 0.1。以上培养基均加入3%蔗糖和0.6%琼脂,pH 6.0。培养温度(25±2)℃。     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L., is an effective antimalarial agent, especially for multi-drug resistant and cerebral malaria. To date, A. annua is still the only commercial source of artemisinin. The low concentration of artemisinin in A. annua, ranging from 0.01 to 0.8% of the plant dry weight, makes artemisinin relatively expensive and difficult to meet the demand of over 100 million courses of artemisinin-based combinational therapies per year. Since the chemical synthesis of artemisinin is not commercially feasible at present, another promising approach to reduce the price of artemisinin-based antimalarial drugs is metabolic engineering of the plant to obtain a higher content of artemisinin in transgenic plants. In the past decade, we have established an Agrobacterium-mediated transformation system of A. annua, and have successfully transferred a number of genes related to artemisinin biosynthesis into the plant. The various aspects of these efforts are discussed in this review.     

栽培青蒿中总黄酮提取工艺

利用超声波辅助技术,获得最大限度提取青蒿中总黄酮的新工艺。用正交设计理论,结合分光光度法,优化超声波辅助醇提法提取青蒿总黄酮工艺中的关键技术参数。最佳提取．工艺为：超声波频率59kHz,乙醇体积分数60％,提取时间40min,料液比1：40。超声波辅助提取法能够实现青篙中总黄酮的高效提取,产率达1．497％。     

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.     

黄花蒿组培快繁与种质离体保存的研究

以带侧芽的黄花蒿(Artemisia annua L.)茎段为外植体,以MS为基本培养基,进行组织培养和种质保存研究.结果表明,培养基MS 6-BA 1.0 mg L-1 IBA 0.1 mg L-1、MS 6-BA 0.5 mg L-1 IBA 0.1 mg L-1和MS NAA0.1 nag L-1 IBA 0.5 rng L-1可分别用于黄花蒿的芽诱导、增殖和生根培养,培养20 d的增殖倍数为5.5倍,生根率98.3%.培养基MS CCC 1.0 mg L-1、MS CCC 2.0 nag L-1、MS PP3334.0 mg L-1可用作离体保存,连续保存200 d的存活率分别达72.3%、77.0%、69.2%.活力检测表明,黄花蒿种质经保存后的增殖、生根能力没有下降.因此,可通过诱导腋芽增殖建立黄花蒿快繁体系,及在培养基中添加CCC或PP333拼能使材料长期保存.     

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.     

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。     

Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L.

Following our previously described Agrobacterium tumefaciens-mediated transformation procedure for Artemisia annua L., we have undertaken several additional experiments to establish the importance of some parameters such as explant type, age of explant source, A. tumefaciens strain and type of binary vector. Several binary vectors were useful for the production of transgenic callus on explants of different ages. In transformed calli, a good correlation between integration and expression of foreign DNA was observed: different assays showed expression of β-Glucuronidase, neomycin phosphotransferase II, superoxide dismutase and bleomycin acetyl transferase. The regeneration of transgenic plants required more restricted conditions. Only with the pTJK136 vector could transgenic plants be obtained from leaf and stem explants from 12- to 18-week-old plants. Co-cultivation for 48 h seemed favorable for the regeneration of transgenic plants. Stable integration and expression of the transgenes was also shown in the progeny. Received: 18 August 1997 / Revision received: 3 December 1997 / Accepted: 3 July 1998     

TLC-densitometric analysis of artemisinin for the rapid screening of high-producing plantlets of Artemisia annua L

A simple TLC-densitometric technique has been developed for the rapid and accurate analysis of artemisinin in a large number of Artemisia annua plantlets cultured in vitro. This new analytical method is based on the structural conversion of artemisinin on a silica gel layer by ammonia vapour to form 10-azadesoxyartemisinin, a chromophore-containing compound (lambdamax 320 nm) that can be detected by UV-based TLC densitometry. The TLC system was evaluated quantitatively in terms of product stability, precision, accuracy and calibration. Good linearity was obtained in the range of 0.01-0.12 microg artemisinin. The technique appeared to be accurate and sensitive as compared with the complicated pre-column reaction-HPLC technique. Among 90 samples of A. annua plantlets, the artemisinin content in the leaves appeared to be highly variable, ranging from 0.02 to 0.67% w/w dry weight. These results demonstrate that densitometric TLC can be a cheap and simple technique for the accurate screening of high-artemisinin-producing plants.