Short Link|短链接：gettr.ink/WNbRtQ

 

Chapter 4 Efficient Cultivation Techniques for Artemisia annua

The research and application of standardized cultivation techniques for traditional Chinese medicinal materials are the foundation and prerequisite for ensuring reliable quality, stable efficacy, and safe use of these materials. The study of cultivation techniques for medicinal materials involves systematically understanding their growth and development patterns and then artificially and reasonably controlling and managing each stage of the planting process to improve both the quality and yield of the medicinal materials.

Over the past 30-plus years since the discovery of artemisinin, domestic experts, especially our researchers, have conducted extensive scientific research on the distribution of Artemisia annua resources, growth and development patterns, cultivation management techniques, and the selection of superior varieties, achieving significant results. These foundational systematic studies have provided strong support for the healthy development of the Artemisia annua cultivation industry in China.

4.1 Basic Research on Artemisia annua Cultivation

4.1.1 Growth and Development Studies

Observations from artificial cultivation of Artemisia annua in northern Guangxi show that Artemisia annua can be sown from late October to mid-May of the following year, and its seeds do not have a dormancy period. Artemisia annua is a shallow-rooted plant. From germination to the appearance of lateral branches, its growth is relatively slow; the rapid growth period is from May to July, and growth stops at the bud stage in early September, with a total growth period of approximately 240 days. Artemisia annua is highly adaptable and has strong drought and flood resistance, though seedlings before the six-true-leaf stage have weak drought resistance. Ferreira's experiments found that Artemisia annua exhibits self-incompatibility, with self-pollination rates lower than 5% when pollen from the same plant lands on the stigma. It is also a strict short-day plant; continuous exposure to a critical day length of less than 13.5 hours for two weeks can induce bud differentiation and flowering. McVaugh's experiments concluded that Artemisia annua is a typical cross-pollinated plant, primarily pollinated by wind and insects, unlike other highland plants. Wallaart discovered that when Artemisia annua content is low, the artemisinin content increases after a single stem immersion. Genetic studies by Chen Xiaotian et al. suggest that artemisinin may be a metabolic product in the cytoplasm or stored in a specific organelle.

4.1.2 Breeding Research on Artemisia annua Varieties

Although the artificial and biosynthetic pathways of artemisinin were successfully developed in the 1980s, these methods are complex, yield low production rates, and are costly, making industrial-scale production challenging. Currently, extracting artemisinin from Artemisia annua remains the primary source of artemisinin-based drugs. Therefore, cultivating high-artemisinin content varieties of Artemisia annua is crucial. As of now, there are no genuinely high-artemisinin varieties. In recent years, foundational work has been done in breeding high-content Artemisia annua varieties.

Ferreira and others analyzed the extensive genetic traits of artemisinin production under greenhouse and field conditions and found that the artemisinin content is determined by genetic traits. Using colchicine induction, Wallaart found that plant height and artemisinin content in colchicine-induced plants were 38% higher than in diploid plants, and the plants retained barley-like leaf characteristics and had lower disease susceptibility. Magalhaes reported that hybridization of Artemisia annua could increase artemisinin yield by 5 kg/ha. Indian researchers used molecular markers to discover high polymorphism among different genotypes of Artemisia annua, revealing that the chemical differences in plants are due to genetic polymorphism. Zhu Weiping from Huzhou Agricultural University's Traditional Chinese Medicine Research Laboratory conducted in-depth research on breeding and selecting high-artemisinin content varieties, finding that selective breeding could increase artemisinin content annually. The study also found a correlation between leaf morphology and artemisinin content, with narrow-lobed leaves having higher content than broad-lobed leaves. Microscopic observation and histochemical localization indicated that glandular trichomes in leaves are the storage sites of artemisinin, with a positive correlation between trichome density and artemisinin content. However, the progress in breeding and selecting Artemisia annua varieties is slow due to its complex metabolism and unclear genetic mechanisms.

From 1991 to 1997, the Chongqing Institute of Medicinal Plant Cultivation conducted the "Artemisia annua Selection and Cultivation Technology Research," selecting Artemisia annua samples with 1.0% artemisinin content from Sichuan Province, though the yield was only 200 kg/ha. The lack of stable artemisinin extraction technology hindered the promotion of these results. In 1992, Chen Herong used colchicine to treat Artemisia annua seeds, developing a new strain called "Jingxia No. 1" with high artemisinin content, significantly increased biomass, tall and robust plants, strong growth adaptability, and good stability. The artemisinin content was 1.0%, but the content decreased annually after planting in Xiyang, and the strain did not become an industry. In 1989, India reported the cultivation of high-artemisinin content varieties using mass selection methods, with dry leaf yield of 2.9 t/ha and artemisinin yield of 31.9 kg/ha.

In 1993, Delabays N conducted interspecific hybridization between Chinese Artemisia annua and northeastern varieties in Switzerland, obtaining hybrids with dry matter yield of 2 t/ha and artemisinin content ranging from 0.64% to 0.95%. In 1994, Delabays N studied the optimal planting density for Artemisia annua in Switzerland, finding that 3 plants/m² planted at the end of April resulted in high artemisinin yields. The hybrid variety "Chine X Yougoslavie 49" achieved an artemisinin yield of 26.64 kg/ha. In 1996, Debrunner N selected five high-artemisinin types from two groups of Vietnamese Artemisia annua and hybridized them with high-artemisinin Chinese varieties, obtaining hybrids with artemisinin yield of 38 kg/ha. In 1999, Magalhaes, P.Mde, developed a new Artemisia annua variety suitable for Brazilian cultivation, with a maximum artemisinin yield of 21.38 kg/ha.

In 1997, Huang Zhengfang and others studied the relationship between plant morphology, stem color, leaf color, leaf shape, and artemisinin content. They cultivated Artemisia annua under different ecological conditions to observe changes in artemisinin content, but the study lacked systematic and comparative data.

In conclusion, although there have been reports of successful breeding of new Artemisia annua varieties over the past twenty years, reports on the promotion and industrialization of these varieties are rare. This is because Artemisia annua is a cross-pollinated plant with low self-pollination rates and high genetic polymorphism between different genotypes, making it difficult to obtain genetically stable high-artemisinin varieties. Moreover, the artemisinin content in high-content varieties fluctuates significantly due to planting years, ecological environments, and cultivation techniques. Therefore, solving the issue of stable production of high-artemisinin Artemisia annua is the primary problem for the healthy and rapid development of the Artemisia annua industry. Only by combining variety selection with stable artemisinin content technology can we establish a unique artemisinin breeding system and cultivate high-quality varieties with promising industrial prospects.

4.1.5 Harvesting and Processing

Zhong Fenglin and others concluded through experiments that the optimal harvesting period for Artemisia annua is between the growth phase and the budding stage when artemisinin content is highest and vegetative volume is maximized. Woerdenbag and El Sohly both believe that Artemisia annua has the highest content during peak flowering. Wang Zheng et al. measured artemisinin content at different growth stages of Artemisia annua introduced to Shandong using Thin Layer Chromatography Scanning (TLCS) and found that the budding stage has the highest artemisinin content, making it the best time for harvesting. Jin Ling et al. used column chromatography to measure artemisinin content in Artemisia annua introduced from Yunnan, Guangxi, and Gaoyou, indicating the highest content during the budding stage. Singh and Laughlin also believe that artemisinin content is highest during the budding stage. Huang Li et al. conducted field experiments using Artemisia annua extracts at different stages to assess their antipyretic and heat-resistant properties, finding that the antipyretic active components are highest during the flowering and budding stages, significantly better than the budding stage. Xie Dezhi concluded that Artemisia annua should be harvested from the late growth stage to the budding stage. Fu Desen used TLCS to measure artemisinin content in different forms of Artemisia annua at harvest and found that dried products are best. Charles experimented with various drying methods, including sun drying, shade drying, indoor drying, and artificial drying at 30℃, 50℃, and 80℃ for 12h, 24h, 30h, and 48h. The results showed that indoor drying for 24h yielded the highest artemisinin content, and natural drying was superior to artificial drying. Ferreira measured artemisinin content in the upper, middle, and lower parts of Artemisia annua plants, finding variations from 0.023% to 0.046%, and discovered that indoor drying yielded 0.13% artemisinin content, while freeze drying and oven drying resulted in 0.02% and 0.10%, respectively. Charles's experiments showed that artemisinin content in one-third of Artemisia annua leaves was about 89%, with the upper and middle parts having twice the content of the lower part. Laughlin believed that artemisinin is evenly distributed throughout the Artemisia annua plant.

4.2 Requirements for Standardized Cultivation Environment

The National Medical Products Administration has formulated the "Good Agricultural Practices (GAP) for Medicinal Plants" and the "GAP Guidelines for Medicinal Plant Production." Artemisia annua cultivation must strictly follow these regulations and guidelines to ensure that artemisinin content meets various national and environmental standards for production, thereby achieving the goal of stable quantity and reliable quality and gaining international recognition.

4.2.1 Soil Environment

The soil environment of high-quality Artemisia annua planting bases must meet the secondary standards of soil quality GB15618-1995, mainly monitoring the residues of mercury, lead, cadmium, copper, zinc, hexachlorocyclohexane, and DDT.

4.2.2 Irrigation Water Quality

Water quality must meet the requirements of GB5084-92 for farmland irrigation water, strictly monitoring the contents of mercury, cadmium, lead, chromium, cyanide, and chloride.

4.2.3 Atmospheric Environment

The air quality in Artemisia annua production areas must meet or exceed the secondary standards of GB3095-82, with no harmful gases, smoke, dust, or chlorides nearby. Additionally, the production area must be free of urban waste and hospital discharge that may carry various pathogens.

4.3 Standardized Cultivation Techniques

4.3.1 Sowing Time

Since Artemisia annua seeds do not have a dormancy period, they can be sown in both spring and autumn. Artemisia annua is a short-day plant, sensitive to short daylight for flowering. Autumn sowing may result in early flowering and reduced yield due to the short vegetative growth period and proximity to autumn short days. Therefore, spring sowing is preferred for better control. To stabilize artemisinin content, Artemisia annua seedlings are usually propagated through tissue culture, where light duration must be strictly controlled during the culture and acclimatization stages. If seedlings are exposed to short daylight prematurely, even spring planting under long daylight conditions will lead to early flowering. Seedlings may flower at about 10 cm in height, completing their lifecycle and resulting in low yield.

4.3.2 Propagation Methods

Artemisia annua is propagated by seeding and tissue culture. Different types, sowing methods, and seed selection techniques significantly affect seed germination. The highest germination rate of 93.38% is achieved with early March sowing, using a 7:7 mix of soil and fertilizer. Seeds stored in bottles under low or ambient temperatures for six months maintain a higher germination rate compared to bag storage. Seeds harvested after mid-October should be sun-dried or shade-dried, then cleaned and sieved through a 1mm sieve, and stored in a ventilated, dry place or at low temperatures (3-6℃). Seedbeds should be located in elevated, well-drained, and sunny areas, with sandy soil or peat as the substrate, and adequately fertilized with organic manure. The seedbed should be 100 cm wide and 20 cm high, leveled, and prepared with a mix of soil and fine sand. The sowing amount is 60-75 g/ha, mixed evenly with fine sand and lightly covered with soil. Straw mulch may be used to cover the seeds, which should be watered timely to keep the seedbed moist.

In addition to seeding, tissue culture is a significant method for large-scale propagation of Artemisia annua seedlings. Various explants such as seedlings, stem segments, and flower heads can induce callus formation, with leaf explants forming callus more easily. Within 5-10 days of culture, green or light green callus appears, while stem and flower head segments take 2-4 weeks to form callus. The optimal medium for callus induction and proliferation is MS+KT 4.0 mg/L+IAA 4.0 mg/L. Transferring buds to 1/2MS medium containing NAA 0.5-2.0 mg/L induces rooting, with a rooting rate of 98% at 1.0 mg/L NAA. Rooted seedlings can be transplanted to nutrient bags with turf and soil after a 2-day acclimatization period, achieving a survival rate of 95%.

4.3.3 Land Selection and Preparation

Artemisia annua is highly adaptable and can be cultivated in most soils. For high-yield cultivation, choose sunny areas with loose, fertile soil, sandy loam, or clay loam with good water retention and fertility. For seedling nurseries, select well-drained, sunny locations with fertile soil. General farmland can be used for standardized Artemisia annua cultivation.

Before seedling cultivation, plow the soil 20-30 cm deep, remove weeds, level the soil, and prepare raised beds 100 cm wide and 20 cm high with 20 cm wide furrows. Apply basal fertilizer at 37.5-52.5 t/ha of well-rotted organic manure such as plant ash or 375-525 kg/ha of potassium dihydrogen phosphate or ammonium phosphate. Chicken manure is preferable as an organic fertilizer. For field cultivation, ensure good drainage, deep plowing, weed removal, bed preparation, and fertilizer application, primarily using organic manure and plant ash with moderate amounts of superphosphate.

4.3.4 Seedling Cultivation

 

1. Prepare the Seedbed: Choose well-drained, sunny loam soil, deeply plow, apply soil manure, and prepare raised beds 120 cm wide with unlimited length, aiming for flat bed surfaces. The furrows between beds should be 40 cm wide and 20-25 cm deep.

2. Select Good Seeds: Artemisia annua has a complex genotype, with significant differences in artemisinin content among different ecotypes. Select seeds tested for ecological adaptability or specifically bred for high artemisinin content.

3. Determine Sowing Time: Sow when the temperature stabilizes around 12℃. In Chongqing, the optimal sowing period is late February to early March.

4. Choose Sowing Method: Wet the seedbed with a watering can, spread fine soil or sand on top, and press lightly with a wooden board. Mix seeds with sand (10:1 ratio) or plant ash, and sow evenly in rows. Do not cover with soil after sowing; press the seedbed with a wooden board to ensure seeds contact the soil closely. Cover the seedbed with 5 cm of straw and water thoroughly with a watering can. Build a small plastic arch over the seedbed.

5. Post-Sowing Procedures: After sowing, do not cover the seeds with soil. Instead, use a wooden board to press down the seedbed surface, ensuring the seeds are in close contact with the soil. Cover the seedbed with a 5 cm layer of straw and water thoroughly with a watering can. Finally, construct a small plastic arch over the seedbed.

6. Seedling Management: Keep the seedbed moist after sowing and check frequently. After the seeds germinate, remove the straw cover and adjust the plastic cover for ventilation and light exposure based on temperature changes to reduce the air temperature inside the plastic tunnel. When seedlings reach 3-5 cm in height, apply diluted manure or 0.2% ammonium phosphate solution every 10 days. Ensure good ventilation and gradually harden the seedlings to reduce the incidence of diseases, weak seedlings, and dense growth. Transplant the seedlings when they are about 10 cm tall.

7. Thinning and Transplanting: If temperature and humidity are suitable, seedlings will emerge in 10-20 days. After emergence, if the seedlings are crowded, thin them out to expand the nutritional area and improve air circulation and sunlight exposure, promoting robust growth. Otherwise, dense seedlings may lead to poor development. Thinning should be done after the cotyledon stage, but not too late, as overcrowded seedlings can lead to excessive growth, which is not conducive to cultivating strong seedlings. During thinning, keep the healthiest seedlings at the desired spacing, removing weak, diseased, overly tall, or slightly damaged seedlings. Also, remove any mixed varieties and weeds.

8. Frequent Transplanting: Before transplanting, deeply plow the soil and apply 1000 kg/ha of decomposed compost and 60 kg/ha of phosphate fertilizer. Prepare raised beds 150 cm wide and plant seedlings at a spacing of 30 cm between plants and 30-40 cm between rows, with a density of 1200-1500 plants per hectare. Transplanting should be done from late March to early April. Water the seedlings thoroughly the day before transplanting to ease uprooting. Transplant on rainy or cloudy days and water the roots immediately after planting.

 

4.3.5 Growth Period Management

During field management, water management is crucial during the seedling stage (before the six-true-leaf stage). Both drought and excess water can affect normal growth, so timely irrigation and drainage are essential. Before the rows close, cultivate and weed to loosen the soil, usually before topdressing. When seedlings reach about 100 cm in height, pinch the tops to promote branching and increase yield.

Cultivate and weed in time after seedling establishment. Even without weeds, cultivation after rain or irrigation can help. Cultivation should be shallow during the seedling stage and deeper as the plants grow.

Weeding methods include manual, mechanical, and chemical. Before transplanting, chemical weeding can be chosen, but after transplanting, manual or mechanical weeding is preferred to avoid herbicide effects on Artemisia annua. Chemical weeding is efficient, timely, labor-saving, and economical. Herbicides and machinery are available for field use. The impact of previous crops on Artemisia annua growth is significant. Herbicides should be quick-acting and short-lived, applied before sowing or transplanting to kill weeds. Options include non-selective herbicides like paraquat, glyphosate, and sodium pentachlorophenate, used under expert guidance to avoid adverse effects. Choose herbicides based on weed species and apply before sowing or transplanting. Follow safety guidelines for herbicide concentration, method, and dosage.

Apply 2-3 additional fertilizers as needed. During the seedling stage, the goal is to promote growth and increase branching. Apply more nitrogen at this stage, then gradually increase phosphorus and potassium during later growth stages. Apply 10 kg/ha of compound fertilizer (N, P, K) each time. Quick-acting fertilizers like decomposed human manure, urea, ammonium sulfate, ammonium water, and superphosphate can be used. Application methods depend on fertilizer type and plant growth. Apply chemical fertilizers in shallow furrows between rows or use human manure during irrigation. Foliar feeding with phosphorus and potassium or trace element solutions is also effective, usually during late growth stages.

Common foliar feeding concentrations: potassium dihydrogen phosphate (0.1%-0.3%), superphosphate (1%-3%), urea (0.5%-1%). Multiple foliar feedings can increase yield by at least 30%. Pinch the tops when seedlings are about 1m tall to promote side branches and increase yield.

Studies on Artemisia annua nutrition and fertilizer absorption show sensitivity to nitrogen and potassium. Excess nitrogen reduces artemisinin content, while too little affects leaf yield. Adequate potassium supports artemisinin synthesis and accumulation. The leaf yield and artemisinin content in cultivated Artemisia annua depend on meeting nitrogen, phosphorus, and potassium requirements. Special fertilizers for Artemisia annua, tailored to its nutritional needs, can improve leaf yield and artemisinin content.

Currently, no special fertilizers for Artemisia annua are available on the market. Common fertilizers have unsuitable nutrient ratios, with high nitrogen and low potassium, adversely affecting artemisinin synthesis and accumulation. General organic fertilizers do not meet Artemisia annua's organic matter needs. Fertilizer type and amount directly impact leaf yield and artemisinin content, making it challenging for growers. Professor Ding Derong from Southwest University proposed a special fertilizer application plan for Artemisia annua.

Special fertilizers for Artemisia annua include: urea (10%-20%), potassium phosphate (12%-25%), potassium sulfate (20%-30%), special organic matter (15%-32%), special additives (0.5%-2.0%), and zeolite powder (7%-18%). Special additives, containing Mg, Mo, B, Zn, and Mn, can be added based on regional deficiencies. Zeolite powder acts as a binder and filler for fertilizer granulation and slow release. The special organic matter is prepared from Artemisia annua residue or cow manure, matured with dilute H₂SO₄ at 40-60℃, mixed with lime powder, balanced for over 24h, pH adjusted to 6-8, and then dried.

Example:

Table 4-1 Special Fertilizer Formulation for Artemisia annua

Component	Weight Ratio Scheme 1	Weight Ratio Scheme 2	Weight Ratio Scheme 3
Urea	11%	20%	15%
Ammonium Phosphate	12%	25%	15%
Potassium Sulfate	25%	30%	30%
Special Organic Matter	32%	15%	15%
Special Additives	2%	1%	0.5%
Zeolite Powder	18%	9%	14.5%

 

4.3.6 Pest and Disease Control

Root rot is a common disease in Artemisia annua cultivation, initially causing the fibrous and lateral roots to turn brown and rot, gradually spreading to the main root, eventually leading to the whole plant wilting and dying. This disease is associated with damage from soil nematodes and root mites. Artemisia annua thrives in warm, humid climates and well-managed environments, but it suffers severe disease in heavy soils and waterlogged fields. Control methods include soil disinfection before sowing, using insecticides to control soil pests, and applying organic fertilizers to enhance disease resistance. In the early stages of disease, use 800-1000 times diluted solutions of thiophanate-methyl or carbendazim for root irrigation and drain the field promptly after rain.

Wild Artemisia annua has few pests and diseases, but artificial cultivation can attract pests such as aphids, cutworms, armyworms, and beet armyworm larvae. Aphids cluster on young stems and leaves, sucking sap. Control aphids with 1000 times diluted solutions of 25% dimethoate or 1500 times diluted solutions of 50% imidacloprid. Cutworm larvae feed on leaves and stems; control them in the 1st-2nd instar stages with 800-1000 times diluted solutions of 90% trichlorfon. For cutworms, armyworms, and beet armyworms, which harm stems and leaves, use 1000-1500 times diluted solutions of 90% trichlorfon or capture them using their feigning death behavior. Control methods include spraying 1000-1500 times diluted solutions of 90% trichlorfon.

4.3.7 Harvesting and Processing

Artemisia annua leaves are the primary harvest product, with the optimal harvest time being the early budding stage when artemisinin content reaches 0.986%-1%, ensuring the best yield and artemisinin content. Harvest Artemisia annua after July 20, depending on its growth, and select sunny days for harvesting. Cut down the main stems, sun-dry for one day, and continue drying at the field edge until the leaves are dry to the touch. Harvest must be completed by the end of September. Collect clean leaves or young branches with leaves, and dry them in the shade or sun, ensuring no impurities, mold, or decay, and a strong fragrance. The ideal harvest quality includes no withered or moldy leaves, green-brown color, and high artemisinin content. Typical yield is 1500-2000 kg of fresh Artemisia annua per hectare, with a dry matter yield of 10%-20%, resulting in 150-200 kg of dried leaves per hectare. The harvested material is processed by specialized pharmaceutical companies to extract artemisinin, which is then processed into anti-malarial drugs recognized by the World Health Organization.

4.4 Seed Preservation Techniques

Artemisia annua is generally harvested from August to the end of September. In standardized Artemisia annua cultivation bases, it is crucial to select superior plants during harvest and preserve them for seed production. This ensures the continuation of quality planting materials for the next season.

Specialized breeding institutions, such as companies or research units, should adhere to specific standards for selecting seed plants. Farmers involved in Artemisia annua cultivation can follow these guidelines for selecting and preserving seed plants:

1. Selection During Harvest: Choose vigorous plants with broad leaves and high economic yield for seed preservation. Mark these plants and allow them to flower and set seeds for harvest.

2. Dedicated Seed Production Fields: Select fertile, sunny plots for dedicated seed production. Cultivate Artemisia annua with a row spacing of 25 cm and a plant spacing of 15-20 cm, ensuring robust growth and high seed yield. Seed yield can reach 16-50 kg per hectare.

By following these seed preservation techniques, growers can ensure the availability of high-quality seeds for the next planting season, supporting sustainable Artemisia annua cultivation and production.

 

 

 

第四章 黄花蒿的高效栽培技术

中药材规范化栽培技术的研究与应用，是保证中药材质量可靠、疗效稳定、用药安全的前提与基础。所谓的中药材栽培技术研究，就是在系统掌握中药材生长发育规律的基础上，人为的、合理的对中药材种植生产的各个环节加以控制与管理，从而提高中药材的质量与产量。
自青蒿素问世以来的30多年間，国内专家尤其是我的科研工作者在青蒿的资源分布状况、生长发育规律、栽培管理技术、优良品种筛选等方面，做了大量的科学研究，取得了显著的成果。这些基础方面的系统研究，为我国的青蒿种植业的健康发展，提供了有力的保证。
 

4.1 青蒿栽培的基础研究

4.1.1 生长发育性研究
青等通过在广西北地区人工栽培青蒿的观察结果说明，青蒿10月下旬到次年5月中旬前可以播种，青蒿种子无休眠期。青蒿为浅根系植物。青蒿从发芽到侧枝出現前生长较为缓慢，5-7月为高速生长期，到9月上旬花蕾期停止生长，青蒿整个生育期约为240天。青蒿适应性强，抗旱抗涝能力较强，但6片真叶前的幼苗抗旱能力较弱。Ferreira试验发现青蒿存在自交不亲和性，同一株青蒿的花粉粒落在雌蕊柱头上，自花受精率低于5%；而且是一种严格的短日照植物，当日小于13.5h的临界日长连续处理两周，即可诱导黄花蒿花芽分化并进入花期。McVaugh试验得出青蒿是典型的异花授粉植物，主要是风媒和虫媒，与高原的其他植物不同。Wallaart发现，当青蒿含量较低时青蒿经过一次茎浸后，青蒿素含量会有所增加。陈霄天等通过遗传学表明，青蒿素可能属于细胞质中的代谢产物，也可能是储藏在某一细胞器中的代谢产物。

4.1.2 品种选育研究
虽然在20世纪80年代青蒿素的人工合成、生物合成就已成功，但其合成途径复杂，产率低，成本高，因而难以投入工业化生产。目前从黄花蒿中提取青蒿素仍然是当前青蒿素类药物来源的主要形式，而提取青蒿素所用的原材料，主要依靠人工栽培和野生资源。培育高青蒿素含量的优质青蒿新品种显得尤为重要。目前还没有真正意义上的高青蒿素品种。近年来，人们在高含量黄花蒿品种选育上作了一些基础性工作。Ferreira等测在温室和田间条件下对青蒿素产生的广泛遗传性状进行了分析，发现青蒿素的含量高低是受遗传性状决定的。Wallaart等利用秋水仙素诱导，叶片性格接近大麦，发现植株株高青蒿素含量比二倍体植株高38%，叶片形态仍保大麦，且植株对疾病的疑人量低。Magalhaes等报道了黄花蒿交种与青蒿素量可增加5 kg/ha。印度研究者利用分子标记，发现不同基因型黄花蒿之间存在较高的多态性，揭示了植物化学物质的差异源于遗传性状的多态性。湖州农业大学中药研究室朱卫平则对高青蒿素含量黄花蒿品种的培育和筛选育目标性状进行较深入的研究，发现通过优中选优的方法可以使青蒿素含量逐年提高，而且研究了叶的形态与青蒿素含量的相关性，发现狭裂片型叶比宽裂片型叶的含量更高，此外还对黄花蒿叶的结构进行了显微观察及青蒿素的组织化学定位，发现叶片中的毛状牙泌腺是青蒿素的储存部位，其浓度与含量呈正相关。目前，国内外改造黄花蒿的选培工作仍然进展缓慢，原因是其代谢复杂，遗传机制不甚明了。

• 1991年到1997年，重庆市药物种植所进行了“青蒿选种及栽培技术研究”，在四川省青蒿类型中选育出青蒿素含量在1.0%的青蒿样本，但产量仅为200 kg/ha。由于缺乏青蒿中青蒿素稳定技术，影响了成果的推广应用。
• 1992年陈和荣用秋水仙碱处理青蒿种子，选育出了青蒿素含量高、青蒿养殖量显著增加，植株高大健壮，生长适应性强，稳定性好的优选逆性品的青蒿新品系“京夏1号”。青蒿素含量为1.0%，但在西阳种植后含量逐年下降，也未形成产业发展。
• 1989年印度报道了印度科学院及药用植物研究所采用集团选择的方法，培育高青蒿素含量的青蒿品种，该品种叶片干物质产量为2.9 t/ha，青蒿素31.9 kg/ha的新品种。
• 1993年Delabays N在瑞士采用中国青蒿东北陵间杂交，试管内繁殖，对意大利、南斯拉夫和西班牙的青蒿投粉，获得杂交品种，该品种干物质2 t/ha，青蒿素平均含量在0.64%-0.95%之间。
• 1994年Delabays N研究了在瑞士的最佳青蒿种植密度为3株/m²，最佳种植时间为四月尾，按此规定种植，可获得青蒿素高产，广种南斯拉夫与中国青蒿类型的杂交品种（Chine X Yougoslavie 49）青蒿素产量为26.64 kg/ha。
• 1996年Debrunner N从越南两组青蒿中选择了5种青蒿素含量较高型，同中国青蒿素含量较型杂交，获得青蒿素产量为38 kg/ha的杂交品种。
• 1999年Magalhaes,P.Mde.通过从中国和越南青蒿品种选择的杂交间的杂交，获得适合巴西种植的青蒿新品种，该品种青蒿素产量最高为21.38 kg/ha。
• 1997年黄正方等研究了青蒿植物形态、茎色、叶色、叶型与青蒿素含量的关系。并用自育杆青蒿栽培于不同生态条件下，以观察其青蒿素含量变化。该研究缺乏系统性和可比性。

综上所述，近二十年来国内外不断有青蒿新品种研究成功的报道，但青蒿良种推广应用和产业化成功的报道却少见。这是因为青蒿属于异花授粉植物，自交率低，不同基因型之间又具有较多的多态性，故很难获得遗传稳定的高青蒿素含量的品种，而且青蒿素含量高的品种受种植年度、生态环境及栽培技术的影响而波动较大。因此，解决高青蒿素青蒿生产稳定问题是青蒿产业健康、快速发展的首要问题。只有将青蒿品种选育与青蒿素含量稳定技术相结合，结合起来研究，建立独特的青蒿素育体系，才能培育出具有高产业化前景的青蒿良种。

4.1.3 种子生理研究
陈琦利用都安石山类型种子分别在8-15℃、28~35℃进行催芽，发芽率分别为50.0%、46.3%、50.0%。但发芽期却分别为11天、6天、13天。又分别用水、土、火土+肥泥、肥泥、河沙四种基质进行播种，发现发芽期均为5天，但发芽率以土+肥泥基质最好，达到71.0%，其次是肥泥55.0%，水土和河沙最差，分别为37.5%和26.0%。青蒿等分别采用0.4g、0.6g、0.8g、1.0g种子在6.67m²测定植株长高和产量，表明0.4g的植株长势比较好，株高、茎粗、小枝数及产量等均显著优于其他3个播种量，但小区产量以1.0g播种量最高。

4.1.4 栽培技术研究
韦青等分别采用15x15cm、20x20cm、25x25cm、30x30cm的密度栽种青蒿，于8月中旬采收并测定植株长势及产量，结果显示30x30cm栽培面积产量最高，显著优于其他各处理。又分别采用猪粪+猪圈土以及混合处理，应用对照处理，得出猪圈土植株长势和产量以应用鸡粪效果最好，以叶绿素含量77.6%，并指出施用基肥青蒿素含量影响不显著，施用追肥对青蒿素含量影响也不显著。Simon 分别采用每亩 30×30 cm、30×60 cm、60×60 cm，同时每亩分别施 0、 67、134 kg 尿素处理测定植株生长量和产量，表明 30×30 cm、30×60 cm、60×60 cm 的平均鲜重为 275 g/株、430 g/株、750 g/株，但总产量以 30×30 cm 最高，并且以施用尿素 67 kg 的处理最好。陈裕泰通过试验认为同一天发芽的青蒿由于生长在人工控制的环境下，只要青蒿的基本生长营养条件得到满足，生长环境中营养物质的含量和生长基质对青蒿素含量没有影响。同一生长时期的不同植株，形态上有显著差异，但这种差异和青蒿素含量无相关性。高温和短日光照可使青蒿素含量成百倍增加长。路洪顺认为早播种比晚播种有高的青蒿素含量长得多，3 月播种青蒿素含量达到 0.68%，7 月播种的含量仅为 0.25%。得出青蒿在生长期间温暖、阳光充足、无霜冻、排水良好、土壤肥沃疏松的沙质或沙质性壤土生长良好，青蒿素含量高。
Charl 分别对栽培了 90 天、117 天、158 天（现蕾）的青蒿进行含量测定为 0.06%、0.15%、0.06%，并分别用 -50 kPa、-100 kPa、-150 kPa 的水压试验对青蒿的青蒿素合成的影响后得出回归方程: Y=-0138-000143X (R2=0.9997)。曾有龙也通过田间实验，证明青蒿随着生长时期的延长，青蒿素产量和青蒿素含量逐渐增大。Srivastava 报道了微量元素如硼能提高青蒿素的含量。Shukla 报道显示外施生长调节物质不仅促进营养生长，还能提高青蒿素含量。陈和崔等对不同生态型的青蒿测定青蒿素后，表明植株之间不仅互相通萌，心互相结粒性，青蒿素行向为佳。另外，青蒿粉有报道套种玉米不会影响玉米产量和青蒿素含量。
 

4.15 采收与加工

钟凤林等通过试验认为青蒿最佳采收期应在生长期至花蕾期之间，此时青蒿素含量高，营养体积积累大。Woerdenbag, El Sohly 都认为青蒿在花朵生长盛期时含量最高。但王正等用薄层扫描法（TLCS）对山东引种青蒿不同生长发育阶段青蒿素含量进行测定后表明青蒿花蕾期植株青蒿素含量最高，为最佳采收期。金玲等用柱层析分离测定引种云南、广西和高邮青蒿中青蒿素含量表明，青蒿在花蕾期含量最高。Singh、Laughlin 都认为青蒿素在青蒿花蕾期含量最高。黄黎等采用不同时期青蒿提取物对肠解热、耐高温等作田间实验结果表明青蒿的解热活性成分在花期和蕾期含量高，明显优于花蕾期。谢德志通过总结后认为青蒿的收获应该在青蒿生长末期到花蕾生长期为宜。傅德森通过正相薄层扫描法（TLCS）对不同样态、采收时对花蕾蒿中的青蒿素进行测定认为干品最好。但 Charles 分别将青蒿在室外太阳干燥、室外遮荫干燥、室内阴干、人工用 30℃、50℃、80℃的热气干燥，同时设 12h、24h、30h、48h 的处理时间后得出青蒿素含量以室内阴干 24h 最宜，自然干燥均优于人工烘干。Ferreira 分别测定青蒿植株的上、中、下各部青蒿素含量从 0.023%~0.046%变化，并且分别用冷冻干燥、烘干、室内阴干处理青蒿后发现，室内阴干青蒿素含量达到 0.13%，而冷冻干燥和烘干分别为 0.02%和 0.10%。Charles 实验表明，青蒿植株 1/3 的叶片青蒿素含量约 89%以上，上、中、下部含量上中上部含量比下部高 2 倍。Laughlin 则认为青蒿素在青蒿植株中是均匀分布的。

4.2 规范化栽培环境要求

国家药品监督管理局制定了《中药材生产质量管理规范》和《中药材生产质量管理规范和 GAP 指导原则》，进行青蒿栽培生产时必须严格按照规范和指导原则的内容进行，青蒿素的含量可符合各种国家、规范其生产的各种环保条件，才能达到可“量稳定、质量可靠”的目的，并能得到国际认可。

4.2.1 土壤环境 
优质青蒿种植基地的土壤环境要求达到土壤质量 GB15618-1995 标准中的二级标准，主要监控汞、铅、镉、铜、锌及六六六、滴滴涕等的残留量。
4.2.2 灌溉水质 
水质必须达到农田灌溉水质标准 GB5084-92 的要求，严格监控汞、镉、铅、铬、氰化物、氯化物含量。
4.2.3 大气环境 
青蒿生产地的大气环境质量要求达到 GB3095-82 标准中的二级以上标准，产区附近无有害气体、烟尘、氯化物等危害。此外，还要求青蒿生产区无带有各种病菌的城市垃圾和由医院排出的废水、废物污染。

4.3 规范化栽培技术

4.3.1 播种时间 
由于青蒿种子没有休眠期，故青蒿在春、秋两季均可播种。青蒿属短日照植物，其开花对短日照非常敏感，秋季播种会因营养生长期过短，植株矮小便接近秋季短日照而开花结实，从而引起产量下降。因此，选择春季播种容易掌握。目前，为了稳定青蒿中的青蒿素含量，通常通过组织培养培育青蒿种苗，在组培及炼苗过程中，就应严格控制光照时间，如果让种植提前接受了短日照，即使是在春季长日照的情况下栽种，也会提前开花。实际栽培育的小植株仅 10 cm 左右便开花，这样的种苗在开花结果后，就完成了生命周期，根本谈不上产量。

4.3.2 繁殖方法 
黄花蒿的繁殖采用播种和组织培养等方法。不同时类型、播种、种植和选质及种子筛选方法对种子发芽有显著的差异，以 3 月上旬播种发芽率最高，达 93.38%；播种基质以七土七肥混合最好，种子以瓶装置于低温或常温条件下贮藏 6 个月均比袋装贮藏保持较高的发芽率。
种子于 10 月中旬以后采收，晒干或阴干，除去茎、叶，然后用 1 mm 细筛过筛，置于通风干燥处或低温（3-6 ℃）贮藏。苗床宜在选地势较高、排水方便、背风向阳的地方，播种基质可选择用砂土或草炭灰，施足农家肥。苗床宽 100 cm，高 20 cm，要整细整平。
黄花蒿从采收后至次年 5 月中旬都可播种，但 2 月中旬至 3 月中旬是播种最佳时期。一般采用条播，条距 15 cm，沟深约 1-2 cm。播种量为 60-75 g/h㎡。种子与细砂混匀后，均匀撒在沟内，然后盖上一层薄土，盖土不见种子为度。有条件的可覆盖上一层稻草，种子撒播后，要及时浇水，苗期注意保持苗床湿润。
此外，组织培养法也是大规模繁殖青蒿幼苗的重要途径。黄花蒿幼苗、茎段、花序等不同外植体均能诱导形成愈伤组织，其中叶片愈伤组织更为容易，培养 5-10 天后，开始长出绿色或淡绿色的愈伤组织，而茎和花序分割需要 2 周、4 周才能形成愈伤组织。适合黄花蒿形成愈伤组织及增殖的培养基配方为 MS+KT4.0 mg/L+IAA 4.0 mg/L。芽的转入含 NAA0.5-2.0 mg/L 的 1/2MS 培养基中诱导生根，当 NAA 浓度为 1.0 mg/L 时，生根率可达 98%，生根苗再盖炼苗 2 天后移栽在草皮泥为基质的营养袋中，成活率可达 95%。

4.3.3 选地整地

黄花蒿适应性强，一般土壤都能栽培。高产栽培应选择阳光充足、土壤较疏松、质地疏松、保水保肥性能强的沙壤或粘壤土。
选地应选用土壤肥沃，灌溉方便的向阳地作为青蒿育苗地。而一般耕地均可作为青蒿规范化栽培地。
在青蒿育苗前，先将土壤翻 20-30 cm，整地，除去杂草、平沟起垄，苗床宽 100 cm，高 20 cm，沟宽 20 cm。平整苗床，施基肥，每公顷用腐熟的有机肥如草木灰 37.5-52.5 t，单用 375-525 kg 磷酸二氢钾或磷酸铵等，腐熟农家肥以鸡粪为好。
对青蒿大田栽培地的要求是排水性好，一般要求深翻，拾掉杂
草秆、建畦、施肥后，即可准备青蒿苗的移栽。施肥主要以农家有机肥、草木灰等为主，常施以适量的过磷酸钙。

4.3.4 育苗
1.    做好苗床 选择排水良好、背风向阳的壤质土，深翻，施土杂肥拌匀，做成宽 120 cm、长度不限的高厢，力求厢面平整，厢与厢之间的沟宽 40 cm，深 20-25 cm。
2.    选择良种 青蒿基因型复杂，不同生态类型的青蒿其青蒿素含量差异较大，选择经生态适应性试验或定向培养的种子做种。
3.    选准播种时间 春季当气温稳定在 12℃左右时，即可播种。如在重庆市都适宜播种期为 2 月下旬至 3 月上旬。
4.    选好播种方法 播种前，用漏壶淋湿苗床，其上撒细土或细沙，用木板稍用力压平。因种子极其细小，掺沙（种子或草木灰），种子按 10:1 的比例混合均匀撒播于苗床，行稍勿密，每亩用种量 3-5 5.播后不覆，用木板将床面压实，使种子与土壤紧密结合，再在苗床上覆盖盖稻草 5 cm，用漏壶淋透水，最后搭建塑料小拱棚。
6.苗期管理 播种后苗床要保持湿润，勤检查，种子发芽后除去覆盖的稻草，并视温度变化适时揭开塑料棚膜通气透光，降低棚内空气温度。幼苗高 3-5 cm 时，每隔 10 天施 1 次稀人畜粪水或 0.2% 磷酸铵液，并注意通风炼苗以降低病、弱、密苗，待苗高约 10 cm 时即可移栽。
7.播种后如果温湿度适宜，10-20 天即可出苗。出苗后，如果生长拥挤，应进行间苗，以扩大幼苗的营养面积，扩大其间距，使幼苗间空气流通，日照充足，生长茁壮。否则，稠苗易生长发育。间苗时应当按株距定苗，瘦弱苗、病虫苗、徒长苗或略有损伤的苗拔除。除去混杂其间的其它品种的幼苗和杂草。间苗通常在子叶发生后进行，不可过迟，因苗株拥挤易引起徒长，不利育出壮苗。
8.勤施移栽 移栽前需对土壤进行深翻，每亩撒施腐熟的土杂肥 1000 kg、磷肥 60 kg，起厢，做成宽 150 cm 的高厢，按株距 30 cm、行距 30-40 cm 规格栽植，亩栽 1200-1500 株。移栽时间为 3 月下旬至 4 月上旬，移栽前一天淋 1 次透水，以利起苗，并在雨后阴天移植，栽植后淋定根水。

4.3.5 生长期管理 
田间管理过程中，幼苗期（6 片真叶前）的水分管理尤其重要，土壤干旱或水分过多都影响正常生长，应及时灌溉和开沟排水。封行前注意中耕除草，松土工作一般在追肥前进行。当苗高为 100 cm 左右时，进行打顶摘心，一般摘去顶尖 3-4 cm 左右，以促进黄花蒿生分枝及加快分枝生长，提高产量。

• 中耕除草成活后及时中耕除草。雨后或灌溉后，在没有杂草的情况下，也要进行中耕。幼苗期中耕宜浅，植株长大后可稍深。
• 除草方法有人工除草、机械除草与化学除草。青蒿在移栽前，可选择化学除草，移栽定植后，应选择人工除草或机械除草，避免除草剂影响青蒿的生长。化学除草具有高效、及时、省工、经济等特点。田间青蒿还配有独特的除草剂和机械。前茬对青蒿的生长影响则是相当严重的。在使用除草剂时，应选择见效快、有较短的除草剂，在播种和移栽前施用，以杀死杂草，可选用灭生性除草剂，例如百草枯、草甘膦、五氯酚钠等，但必须在有实践经验的专家或技术人员指导下进行，以免造成不良后果。最好的根据田间杂草种类、针对性地用药。药剂失效后，再播种或移栽。使用除草剂要注意安全，对使用的浓度、方法和用药量，要根据说明书使用。
• 可根据需要追肥 2-3 次。幼苗时期的追肥，主要目的是促进生长、增加枝生长。追施氮肥可稍多些，但在以后的生长发育期间，磷、钾肥应逐渐增加。每次每亩追施氮、磷、钾复合肥10kg左右。速效性肥料如腐熟人粪尿、尿素、硫酸铵、氨水、过磷酸钙等均可追肥。肥料的施用方法依肥料种类及植株生长情况而定。追施化肥，可在植株行间开浅沟条施。以人粪尿做追肥，可与灌水同时进行；还可采用根外追肥，一般都在生长后期进行，在植物茎、叶上喷施一定比例的磷、钾肥或微量元素的水溶液。
• 常用的追肥液浓度：磷酸二氢钾为0.1%-0.3%，过磷酸钙为1%-3%，尿素为0.5%-1%。对青蒿进行多次根外追肥，产量至少提高30%以上。苗高1m左右时，可进行打顶，以促进侧枝萌发，提高产量。
• 我们多年来对青蒿营养生理和吸肥规律的研究结果表明：青蒿对氮素和钾素较敏感，氮过多会造成青蒿素含量的下降，过少则影响青蒿叶的产量；青蒿钾素供应充足，有利于青蒿素的合成和积累。人工种植的青蒿叶产量和青蒿素含量的高低，取决于能否最大限度的满足青蒿对氮、磷、钟营养元素的需求。针对青蒿生长发育对营养元素的特殊要求配制成青蒿专用肥，可以满足青蒿对有机质和氮磷钾的特殊要求，达到提高青蒿叶产量和青蒿素含量的目的。

目前市场上没有青蒿专用肥生产及销售，而其他复合肥养分配比和比例不符合青蒿生长发育需要，其含氮量较高，而含钾量较低，对青蒿叶青蒿素的合成与积累十分不利，一般的有机肥又不符合青蒿对有机质类的要求。普通肥料的施肥量和施肥种类又直接影响了青蒿叶的产量及青蒿叶中青蒿素的含量，种植者不易掌握。由于青蒿对氮素和钾素比较敏感，西南大学丁德蓉教授提出青蒿专用肥施用方案。
青蒿专用肥中，按重量配比包含有：尿素10%-20%，磷钾12%-25%，硫酸钾20%-30%；专用有机质15%-32%，专用添加剂0.5%-2.0%，沸石粉7%-18%；可根据地域性缺素程度加入0.5%-2.0%的专用添加剂，其专用添加剂的主要成分为Mg、Mo、B、Zn、Mn；沸石粉起到粘结剂和填充剂的作用，用于肥料造粒和缓释肥效；其专用有机质为青蒿渣或牛粪生料经稀H₂SO₄溶液在40-60℃条件下熟化，然后添加生石灰粉混匀，平衡24h以上，并调节pH值为6-8，然后干燥处理即得。

实施例：

表4-1 青蒿专用肥配比方案

成分	重量配比方案1	重量配比方案2	重量配比方案3
尿素	11%	20%	15%
磷铵	12%	25%	15%
硫酸钾	25%	30%	30%
专用有机质	32%	15%	15%
专用添加剂	2%	1%	0.5%
沸石粉	18%	9%	14.5%

 

其中，专用有机质为青蒿渣或牛粪生料经稀H₂SO₄溶液在40-60℃条件下熟化，然后添加生石灰粉混匀，平衡24h，并调节pH值为7，然后干燥处理。
大量的实验结果表明，施用本专用肥与普通肥或其他复合肥相比，对可提高青蒿叶产量30%以上，青蒿素含量提高10%以上。

4.3.6 病虫害防治
根腐病是青蒿栽培中较常见的一种病，发病时，须根、支根先变褐腐烂，逐渐向主根蔓延，最后导致全根腐烂。直至地上茎叶干枯而亡植株全株枯死。该病害与地下线虫、根螨危害有关。另外，青蒿适宜在温暖湿润的气候与良好的环境中生长，土壤粘重、田间积水过多时发病严重。防治方法：播前对土壤消毒，用杀虫药防治地下害虫，同时增加降雨热有机肥，以增强抗病力。发病初期可用甲基托布津、多菌灵等800-1000倍液灌根，雨后及时排水。
野生的青蒿病虫害较少。人工栽培后，易引起蚜虫、尺蠖、小地老虎、银纹夜蛾和斜纹夜蛾幼虫的危害：蚜虫群栖植株嫩茎、叶上，吸食液汁；发现蚜虫后，可用25%亚铵硫磷1000倍液或50%灭蚜灵1500倍液喷施防治；尺蠖(土名量步虫)幼虫咬食叶片、嫩茎、在幼虫1-2 龄期喷 90%敌百虫 800~1000 倍液防治;小地老虎、银纹夜蛾和斜纹夜蛾的幼虫，危害茎、叶，小地老虎,可用 90%敌百虫 1000~1 500 倍液浇穴毒杀;银纹夜蛾和斜纹夜蛾，利用其假死性进行捕杀；或用 90%敌百虫 1000~1500 倍液喷施防治。 

4.3.7 采收与加工
青蒿收获的产品为植株的叶片，青蒿的最佳采收时间为青蒿现蕾初期，因蕾期青蒿素含量甚为0.986%-1%，为适宜宜收获期，提前或延后采收可能影响青蒿叶的产量和青蒿素的含量。根据青蒿的长势，7月20日以后即可采收青蒿，收获时选择晴天，破倒主干，在太阳晒1天，第2天收，并到田头晒干，打落土于叶，子叶继续晒干到用手抓无潮湿感时可交售。9月底以前必须采收完毕，采收净叶或带叶的嫩枝，阴干或晒干，以手干，无杂质、不霉变、香气浓香，青蒿素含量普者为佳。收获质量要求无枯叶、无杂质、无枯叶、无霉叶。青蒿绿褐色。一般亩产1500-2000kg湿青蒿，折干率为10%-20%，亩干青150-200kg。收割料采收，由专业制药厂提取青蒿素，再由具有世界卫生组织认同的制剂机构加工成青蒿素类系列抗疟疾成药销售于市场。

4.4 留种技术
青蒿一般在8月至底月采割收获，在规范化种植的青蒿栽培基地收获时，一定要特别留意寻找一些优良植株作青蒿种株保留下来，使其开花结实收获种子，作为下一年的育源供继续生产使用。
公司或科研单位等专门的育种机构自然不必说，进行青蒿栽培生产的农户可按以下方法进行选择种株的标准和留种。一是在收获时选择植株生长旺盛、叶型宽大的品种，经济产量较高的株系保留下来并作业标记，待其开花结实后采收；二是选择土壤肥沃、向阳的地段作以为专门的制种地，进行留种黄花蒿的栽培生产。留种的种植密度以条距25cm，株距15-20cm为好，植株长势强，开花结实多，种子产量高，每公顷可达16-50kg。