What is the application of modern biology and agricultural technology in the research and production of medicinal plants?

1902, G.Haberlandt, who was the first to do plant tissue culture experiments, predicted that in vitro cells of higher plants could grow into plants. This hypothesis becomes the theoretical basis of plant tissue culture in the future.

1937, White established a comprehensive culture medium for tissue culture, and together with Gautheret and others, successfully used tobacco stem cambium cells and small carrot roots to proliferate and induce callus under artificial culture conditions for the first time. The basic method of plant tissue culture established by them has become the technical basis of various plant tissue cultures in the future.

In 1948, F.Skoog and Cui Pei found that adding proper proportion of adenine and auxin to the culture medium for tobacco stems and pith can control plant tissues from growing seedlings or roots, that is, when the ratio of adenine and auxin is high, buds will be produced, and when the ratio is low, roots will be formed. Later, C.D.Miller et al. (1956) found that the effect of replacing adenine with kinetin was better, and established a hormone model that the ratio of kinetin to auxin controlled organ differentiation. The theory of hormone "cybernetics" still plays a guiding role in plant tissue culture research.

In 1950s, tissue culture technology developed rapidly, from static solid culture to liquid culture, including suspension culture, micro-chamber culture, conservation culture and plate culture. In 1958, Steward et al. used liquid suspension culture to culture carrot somatic cells into complete plants through embryoid development, and they blossomed and bore fruit. This breakthrough not only confirmed Haberlandt's thought of "cell totipotency", but also opened up a new field for studying organ formation and embryogenesis in tissue culture.

In the early 1960s, E.C.Cocking and others successfully separated plant protoplasts with fungal cellulase, which created conditions for the rapid development of genetic engineering such as protoplast fusion technology and somatic hybridization.

In recent 20 years, due to the totipotency of cultured plant cells under certain conditions, the application of tissue culture technology in production has been paid attention to and developed, and it has gradually become an important research means in agriculture, forestry, horticulture and medicine, which has promoted the progress of a series of disciplines such as morphology, cytology, physiology, biochemistry, genetics and breeding.

With the development of molecular biology and bioengineering, plant tissue culture technology is becoming more and more perfect. After the 1960s, plant tissue culture began to be applied to production, which led to the industrialization of plant production. Flowers and herbs, rapid propagation of plants through clonal lines, commercialization of test-tube varieties produced by factories; The method of producing drugs through a large number of cell culture techniques has been successful in Japan, the Federal Republic of Germany and other countries. In a word, plant tissue and cell culture is a new biotechnology with great potential, which will enter the ranks of the new industrial revolution in the world with a brand-new look and show bright prospects.

At present, the practical application of plant tissue and cell culture technology in botany and other disciplines is mainly in two aspects: (1) plant breeding and rapid propagation; (2) The production of physiologically active substances.

I. Experimental equipment, sterilization method and basic culture medium for plant tissue culture

(1) experimental equipment for plant tissue culture

1. laboratory settings

(1) The aseptic operation room is equipped with a purification workbench for inoculation, a workbench for placing inoculation tools and cultures, and an ultraviolet lamp installed on the ceiling or wall for sterilization. It is best to have ventilation to filter bacteria.

(2) culture medium preparation room

To prepare all kinds of culture media, there must be a large flat workbench and a cabinet rack for placing all kinds of chemicals and utensils in the room. Refrigerator for storing prepared mother liquor of culture medium, and equipment for preparing and storing distilled water.

(3) Constant temperature culture room

The culture room needs air conditioning, constant temperature control and lighting. Generally, the temperature of the incubator should be kept at about 25℃, and the temperature difference should not exceed 65438 0℃.

The culture room must have culture devices and equipment such as culture rack, shaking table and rotating table.

(4) Cytology laboratory

Various microscopes and dissecting mirrors were placed to observe the culture results. If conditions permit, photographic equipment can be set up to shoot the experimental results.

(5) Chemistry laboratory

Prepare all kinds of conventional and advanced instruments and equipment for chemical analysis and determination, and carry out all kinds of chemical analysis and determination.

2. Instruments and appliances

(1) glassware

Instruments and utensils made of hard glass with low alkali solubility, especially long-term culture, will easily have adverse effects if non-quality glass products are selected.

Commonly used glassware include: triangular flask, nipple flask, T-shaped tube, angular flask, round flask, L-shaped test tube, parallel angular test tube, round flat flask, Petri dish, glass ring, glass slide, cover glass, funnel, subpackager, syringe, round bottom flask, separating funnel, glass plate, glass column, condenser, extractor and dyeing.

(2) Appliances and instruments

Choose medical instruments and instruments used in microbiology laboratory, such as knives, scissors, tweezers, scalpels and inoculation needles.

(3) Instruments and equipment

Generally, balance, acidity meter, centrifuge, microscope, dissecting mirror, incubator, oven, refrigerator, shaker, rotating bed, rotating culture rack, spectrophotometer, thin-layer scanner, high-pressure liquid chromatograph and so on are needed.

(2) Sterilization method of plant tissue culture

1. Sterilization of containers and culture media

Petri dishes, work clothes, masks, hats, etc. It can be sterilized at high temperature, generally using 1.2 atmospheric pressure for 20-30 minutes; The medium disinfection time should not be too long, otherwise some substances are easy to decompose and destroy, 1.2 atmospheric pressure, 15 minutes is enough. The disinfection of metal instruments is generally soaked in 70% ethanol before use, and then ignited on a flame for disinfection and cooling before use.

2. Sterilization of plant materials

The sterilization of plant materials is mainly external sterilization, and the type and concentration of disinfectants and the length of treatment time should be selected according to the sensitivity of materials to disinfectants. Firstly, the experimental materials were carefully scrubbed in soap powder solution and washed with running water, and then sterilized according to the methods and sequences listed in Table 13- 1 and 13-2.

Table 13- 1 Comparison of the effects of common sterilizers

Table 13-2 Sterilization sequence of different organs of plants (III) Basic medium for plant tissue culture

1. medium composition

Plant tissue culture medium usually consists of the following substances (Table 13-3):

Table 13-3 Components of Several Common Culture Media (unit: mg/L)

(1) inorganic nutrients

Inorganic nutrients include macroelements and microelements. In addition to carbon (C), hydrogen (H) and oxygen (O), a large number of elements are nitrogen (N). Nitrate nitrogen or ammonium nitrogen is usually used as nitrogen, but nitrate nitrogen is often used in culture, and nitrate nitrogen and ammonium nitrogen are also mixed. Phosphate is commonly used for phosphorus, and sulfate is commonly used for sulfur. Potassium is the main cation, and the requirements of calcium (Ca), sodium (Na) and magnesium (Mg) are less. The proportion of major elements is usually modified according to the formula of Knop solution for cultivating the whole plant. Generally speaking, the nutrient medium should contain at least 25 mmol of nitrate and 25 mmol of potassium. When the content of ammonium exceeds 8mmol, it is often toxic to the culture. But for conventional callus culture and cell suspension culture, the concentration of nitrate+ammonium can be increased to 60mmol. The concentrations of calcium, sulfur and magnesium are in the range of 1-3 mmol. The required sodium chloride is provided by calcium salt, phosphate or micronutrients. Trace elements include iodine (I), boron (b), manganese (Mn), zinc (Zn), molybdenum (Mo), copper (Cu), cobalt (Co) and iron (Fe), among which iodine may be unnecessary.

(2) Carbon sources and energy sources

Most plant cells need 2-4% sucrose, and in some plant tissue cultures, the concentration of sucrose is as high as 7% or even 15%. Sugar source may have other functions besides carbon source and energy source in culture medium. Sucrose can also be replaced by glucose and fructose, and other sugars are not ideal. Inositol may not be necessary, but it is widely used in general culture medium, which may be related to its role in promoting callus growth.

(3) vitamins

Among various vitamins, thiamine hydrochloride (B 1) may be necessary, while nicotinic acid and pyridoxine hydrochloride (B6) can only promote growth.

(4) Growth regulating substances

Plant hormones are indispensable components in the culture medium, which have a direct impact on the induction of plant callus, the growth of culture, the differentiation of organs and the metabolism of secondary products. Two hormones are usually added to the culture medium: one is auxin, and commonly used are 2,4-D, NAA, IAA and IBA. The other is cytokinin, commonly used are kinetin (Kt), zeatin (Zt) and 6- benzylpurine (6-BA). The suitable concentration of 2,4-D is 10-7- 10-5 mol, the suitable concentration of IAA is 10- 10-5 mol, and the highest concentration is1-10 mg. Under normal circumstances, only 2,4-D (10-5-10-7mol) can successfully induce callus. If auxin such as 2,4-D is combined with cytokinin, the effect will be better. When inducing explants to differentiate plants, it may be better to use NAA combined with cytokinin. Among cytokinins, kinetin and 6- benzylpurine are widely used, which can promote the growth of callus. The suitable concentration is 10-7- 10-6 mol.

(5) amino acids

The culture medium should include certain amino acids, such as glycine. In addition, hydrolyzed casein is often used in tissue culture, which is a mixture of various amino acids. Especially, adding a certain amount of hydrolyzed casein to the differentiation medium can promote embryogenesis and polyembryony. Some amino acids are precursors of some secondary substances (such as phenylalanine and ornithine, which are precursors of scopolamine alkaloid biosynthesis). When they are added to the culture medium, the content and yield of such secondary substances in tissue culture will increase obviously.

(6) Organic additives

Adding some natural products to the culture medium is beneficial to the induction and maintenance of callus, and also to the promotion of growth and the formation and accumulation of secondary substances. Among them, coconut milk, yeast extract, tomato juice and soybean powder are the most commonly used. Its concentration range: coconut milk 10% (volume/volume), yeast extract 0.5%, tomato juice 5- 10%, and soybean powder 0. 1-0.5%. These natural additives tend to choose fully known synthetic compounds in the preparation of culture medium, because their components are complex and it is difficult to ensure consistency.

2. Preparation of culture medium

(1) water and medicine

It is best to use distilled water distilled from demineralized salt in a glass container to prepare the culture medium. The chemicals used should be pure. The growth regulator is preferably recrystallized before use. It is best to hydrolyze protein hydrolysate with enzymes, so that amino acids can be better preserved in the natural state.

(2) Mother liquor (stock solution)

A simple method for preparing culture medium is to prepare a series of mother liquor first. A large number of mineral salts (a large number of elements) can be prepared to be 10 times thicker than the concentration used. When dissolving mineral salts, try to stagger Ca2+ with SO2-4- and PO3-4 to avoid forming insoluble substances of calcium sulfate and calcium phosphate. It is best to dissolve mineral salts with a certain amount of distilled water, then add them in turn, and finally add water to a certain volume. Trace elements can be made into concentrated solution with the concentration of 100- 1000 times, and stored in the refrigerator together with a large number of elements. Each vitamin can be prepared separately and stored in a volumetric flask with a concentration of 0.2-1mg/L. Iron salts need to be prepared separately (see above). Plant hormones have different requirements in preparation. NAA, 2,4-D, IAA and other auxins are weighed, dissolved in a small amount of 95% ethanol, and then diluted to a certain concentration with distilled water; Cytokinin should be dissolved with a small amount of 0.5 or 1N hydrochloric acid, and then distilled water should be added to the required amount; Folic acid should be dissolved in a small amount of dilute ammonia water, and then distilled water should be added to the required amount; Biotin can be directly dissolved in water when it is prepared.

(3) autoclaving and preservation

When preparing the culture medium, take out all kinds of mother liquor, mix them together according to the required volume, temporarily add additional ingredients such as sucrose and hormones, mix them with agar that has been heated and melted in advance (agar is not added in liquid culture), then adjust the pH value to a required value (between 5.5 and 5.8) with sodium hydroxide solution or 1N hydrochloric acid, and then put them into culture containers respectively. Sterilize the prepared culture medium in an autoclave at 120℃ and 1. 1kg/cm2 15-20min. The sterilized culture medium can be stored at room temperature (the optimum temperature is 65438 00℃) and should be used within two weeks.

3. Characteristics of several common culture media

In different media, inorganic salts generally change greatly, and sometimes they form their own characteristics by adding different nitrogen sources. MS is a culture medium designed by Murashige, T. and Skoog, F. in 1962. Remarkable achievements have been made in callus induction by solid culture, cell suspension culture by liquid culture, embryo, stem tip, stem segment and anther culture and morphogenesis research. The amount and proportion of inorganic nutrients in MS medium are appropriate, which can meet the nutritional and physiological needs of many plant cells. Therefore, in general, there is no need to add organic additives such as casein hydrolysate, yeast hydrolyzate or coconut milk to the culture medium. Compared with other media, the content of nitrate, potassium and ammonium in MS media is higher, which is its remarkable feature. Similar to MS medium, there are LS(linsmailer, E.M. and Skoog, F. 1965) and RM (Tanaka, 1964) medium, and their basic components are the same as MS medium, except that glycine, vitamin B6 and nicotinic acid are removed from the former, while the dosage of NH4NO3 is increased from the latter to 4950. Compared with MS medium, White( 1963) medium has a lower concentration of inorganic salts, but it is also widely used, and has a good effect on embryo culture and general tissue culture. B5 medium was designed by Gamborg et al. (1968). Its main feature is low ammonium content, which may inhibit the growth of some plants. N6 medium is designed by Institute of Botany, Chinese Academy of Sciences and Heilongjiang Academy of Agricultural Sciences, and it is also a very suitable medium. It has been widely used in anther culture and tissue culture of some plants in China. Its composition is similar to B5, but it does not contain molybdenum. Heller( 1953) culture medium is widely used in Europe, which has low inorganic salt content and lacks molybdenum salt, but contains compounds such as nickel and aluminum.

From the general trend, modern culture media tend to use high concentrations of inorganic salts. In nitrogen application, many people use a mixture of nitrate nitrogen and ammonium nitrogen, or just nitrate nitrogen. There is little research on the function of trace elements, and most of the formulas are basically close to MS medium. These trace elements have met the needs of callus and cell growth in tissue culture. For iron salts, a mixture of FeSO4 and Na2-EDTA (chelating agent) is usually used.

Second, the application of plant tissue and cell culture in drug production

In recent years, due to man-made destruction of the natural environment, indiscriminate collection, rising labor costs and technical and/or economic difficulties in introducing wild plants, plant resources have been drastically reduced, and it is increasingly difficult to ensure adequate supply of medicinal plants. Since 1960s, people began to apply plant tissue culture technology to study the production of plant drugs. In recent years, with the development of modern biotechnology, a large number of plant cells have been successfully cultivated, which has opened up a new way for the industrialized production of plant drugs.

Compared with the general whole plant culture, the cell culture system has many advantages: (1) the production of useful substances is carried out under controllable conditions, independent of climate and soil conditions, saving land; (2) Cell culture is free of microorganisms and pests; (3) The growth cycle is short, which shortens the long process of plant introduction, domestication and propagation; (4) specific biotransformation reactions can be carried out, new synthetic routes can be explored, and new useful substances can be obtained; (5) By screening new cell lines and changing culture conditions, productivity can be improved and production costs can be reduced.

(A) plant tissue and cell culture technology

1. Callus induction and culture

Callus is the basic material of plant tissue and cell culture, so callus induction and culture is one of the basic work of plant tissue and cell culture.

The working procedure of callus induction is roughly as follows:

(1) Selection of plant materials (including plant species and parts); (2) Preparation and disinfection of materials; (3) Selection and preparation of culture medium; (4) inoculation and culture; (5) Sub-culture.

At present, many plants have successfully induced callus, among which dicotyledons are the most and monocotyledons are the least. In addition, gymnosperms, ferns and mosses also have successful examples. Therefore, it can be said that all multicellular plants have the potential to successfully induce callus. Dicotyledonous plants have a wide range of practical values, and they can quickly grow callus. Various tissues of plants, such as vascular cambium, parenchyma of storage organs, stele sheath of roots, endosperm, cotyledons, mesophyll tissues and vascular bundle tissues, can form callus under suitable conditions.

Callus induction is mostly carried out on solid medium. Solid culture is generally carried out at 25-28℃, and subculture is carried out every 4-6 weeks.

Callus culture methods can generally be divided into solid culture and liquid culture. Solid culture refers to adding a certain amount of coagulant (0.6- 1.0% agar, etc. ) are added into a culture medium, heated and dissolved, respectively placed in culture containers, and cooled to obtain a solid culture medium. While those without coagulant are liquid culture medium.

Solid culture is static culture, which has the advantages of simplicity and convenience. It only needs ordinary glassware, unlike liquid oscillation culture, it does not need complicated mechanical equipment such as shaking table and rotating bed, and occupies less land. A small culture room can accommodate many culture containers. However, solid culture also has the following disadvantages: only a part of the callus surface is in contact with the culture medium, which leads to uneven growth of callus; The gas exchange of the bottom tissue in contact with the culture medium is poor, and the harmful substances discharged during the growth process are piled up; When standing still, due to the effect of gravity or uneven light irradiation, the tissue is polarized, and it is difficult to have a fairly consistent group.

Liquid culture can be divided into two types: standing and shaking. Liquid static culture is as simple as solid culture, and there will be no difference in nutrient concentration in the culture solution. However, due to the limitations of its use, it has been rarely used at present. Oscillatory culture is to make plant tissues rotate continuously in liquid culture medium, thus eliminating the shortcomings in static culture. Oscillatory culture can be divided into two types: (1) continuous immersion, which suspends tissues in the culture medium by stirring or vibrating the culture solution, and is usually cultivated by shaking table; (2) For those submerged regularly, T-tubes and nipple bottles are used as culture containers and cultured on a rotating bed. In the process of rotation, the culture appears repeatedly in liquid phase and gas phase.

2. Cell suspension culture

Plant cell suspension culture technology is a new culture technology based on callus liquid culture technology. In recent twenty years, from suspension culture in vitro to large-scale fermentor culture, from discontinuous culture to semi-continuous and continuous culture, until the latest turbidity constant method and chemical constant method realize automatic control in large-scale continuous culture in recent years. The characteristics of cell suspension culture are: (1) can provide a large number of uniform plant cells; (2) Cell proliferation is faster than callus; (3) Suitable for large-scale culture. Therefore, we can cultivate plant cells like microorganisms and apply them to fermentation industry to produce some unique products of plants, thus opening up a new way for the industrial production of plant products. Cell suspension culture is to cultivate free plant cells in liquid culture medium, but because of the aggregation characteristics of plant cells, so far, only free single cells can not be produced in suspension, but often some small cell clusters.

(1) Equipment and device for suspension culture

① Shaker is widely used in plant cell suspension culture. Fragile callus can get dispersed cell suspension by continuous oscillation of shaking table. It can also be used for subculture of suspended cells.

(2) Turn the bed once every minute. T-tube or nipple bottle is used as culture container.

③ The rotary culture rack can use larger bottles as culture containers.

④ Continuous culture equipment usually relies on introducing sterile compressed air or air and continuous stirring to keep cells suspended. In this stirring device, since the container for holding the culture solution is fixed, the container for storing fresh culture solution, air supply device and the like can be easily connected with the culture container. Some snake-shaped tubes can be installed in the culture container, and electric heating wires are put in the tubes for heating, and cold water is introduced for cooling. Such devices do not need to be installed in a constant temperature room. Generally, this kind of device can simply control the temperature, stirring speed, ventilation speed, light intensity, nutrient solution inflow speed and so on. , and often connected with oxygen electrode, pH electrode, cell density tester, etc. And a set of continuous culture device with preliminary automatic control is formed. Several plant cell mass culture devices include stirred fermentation devices, fermentation devices using oscillators, fermentation devices using conduits and rotating impellers, bubble stirred fermentation devices and airlift fermentation devices.

(2) Culture medium for suspending cells

The commonly used medium suitable for callus is not necessarily the most suitable for cell suspension culture. Usually, the medium for callus induction can be used as the starting point to determine the most suitable medium. Special attention should be paid to the influence of the dosage of auxin and cytokinin on the aggregation of suspension culture cells, and the medium that makes cells easily dissociated should be selected.

In suspension culture, the pH value often changes greatly, so chelating agents such as EDTA must be added to make iron and other ions available for a long time. Adjusting the ratio of nitrate nitrogen to ammonium nitrogen can also be used as a method to stabilize pH. Adding some solid buffers, such as slightly soluble calcium hydrogen phosphate, insoluble calcium phosphate and calcium carbonate, is also a method to stabilize pH..

(3) subculture of suspension cells

During suspension culture, the change of cell number growth is shown in figure13-1. It's basically an s-shaped curve. At first, it is a delayed period, and cells rarely divide; Followed by logarithmic growth period, the number of cells increased rapidly and the growth rate remained unchanged; After that, it will enter a static period of gradual slowdown; Finally, the growth period was completely stopped. In culture, cells are usually subcultured at the beginning of dormancy period, some need to be subcultured before dormancy period when proliferation slows down, and some even need to be subcultured immediately at the end of logarithmic growth period in order to accelerate cell proliferation.

Figure13 ——1is a schematic diagram of the increase of cell number in the first generation of culture.

In recent years, it has been studied that under certain conditions, cells in culture enter a certain period of cell division cycle (such as G phase) by using DNA synthesis inhibitors and plant hormones, and then most or all cells divide synchronously from the next period, which is called synchronous culture. In this way, we can not only know the real process of cell cycle in detail, but also know the factors that control the biochemical change order from mother cell to daughter cell. Because synchronous culture can be frequently sampled for biochemical and cytological analysis, the sampling amount can be large, and it will not affect the continued growth of the remaining population, which provides necessary technical means for studying important processes such as cell cycle, cell differentiation and secondary metabolism, and is another important progress in the development of plant cell culture technology.

To sum up, the current plant tissue and cell culture technology has developed from static solid culture to liquid culture, which is characterized by improving the nutrition and oxygen supply of cultured tissues and cells. The scale and method are developing from small quantity to large quantity, from test tube to large tank and synchronous culture in application, and from manual operation to automation in operation. These advances and developments have had a great impact on the research and production application of plant tissue and cell culture.

(2) Pharmaceutical ingredients produced by plant tissue and cell culture.

Since Bonner et al. studied the production of natural rubber from Echinacea callus in 1950, many scientific workers have done a lot of work on what useful substances can be produced from callus and how to improve the yield of effective components, and achieved some results. More and more people think that it is possible to use plant tissues and cultured cells to produce alkaloids, terpenoids, quinones, sterols, enzymes and so on for treating various diseases, and to extract peptides and protein substances from the culture. Nicole (1980) concluded that plant tissue culture can produce more than 50 compounds and 28 potential products. Zheng (1980) listed more than 60 kinds of medicinal components, source plants and drug contents. Misawa, M., 1980 of Japan Concord Fermentation Company introduced the research results of alkaloids, sterols, terpenoids, quinones and other physiologically active substances including enzymes produced by industrial laboratories or the government through plant tissue culture. According to the statistics of the World Health Organization, the most common and basic drugs extracted from higher plants are 17. Among them, 1 1 The plant from which the drug originated has been subjected to tissue culture (Table 13-4).

Table 13-4 Common and Essential Drugs from Higher Plants

1. Alkaloids

It is particularly difficult to produce alkaloids by plant tissue and cell culture, but the tissue culture of medicinal plants that produce alkaloids is one of the most studied aspects (table 13-5). But its content is usually lower than that of the original plant.

Table 13-5 Alkaloids in Plant Callus

Table 13-5 Alkaloids in Plant Callus (Continued)-1

(1) scopolamine alkaloid

Moses, West, Chen, Mace, Masao Mujima, bhandari, Thomas, Stoss, Salam, tabata, Hiraoka, Cleokhi, etc. The roots, calli and suspension cells of Datura stramonium, Belladonna, hyacinthus orientalis and centipede were studied extensively. , but no or little contains medicinal substances. West( 1957) first confirmed the existence of scopolamine alkaloids in belladonna root callus. The content of atropine in root callus is 0.47-0.53%. However, it was not detected in the callus of stems and leaves. Chan et al. (1956) carried out static and shaking culture on the callus induced by different organs of Datura stramonium, Datura stramonium and Datura stramonium, and determined the alkaloid content in the callus. Datura stramonium and Datura stramonium have the highest content in seed callus, reaching 0.0 16-0.056%, 0.0 12-0.0 15% in root, 0.004-0.0 14% in stem and 0.5% in leaf. The results showed that the amount of alkaloids produced was different because of the different organs that induced callus. West and others also pointed out that there are differences in the ability to synthesize alkaloids among Datura stramonium callus strains from the same source. The contents of scopolamine and scopolamine were 0.025% and 0.009% respectively, which were lower than the original plants (0. 127% and 0.0 16% respectively). However, due to the study of improving culture conditions, increasing nutrition, replacing growth regulators and supplementing precursors, the total content of two alkaloids reached 0.554% (0. 139% in the original plant), and the highest content of scopolamine reached 0.495% (0.0 16% in the stem), which was 4% higher than that in the original callus. The research results of Cheng Kedi et al. (1987) show that cells such as anisodamine can not only produce tropine alkaloids, but also convert scopolamine into anisodamine and scopolamine.

(2) Pyridine alkaloids

In the research of producing pyridine alkaloids by tissue culture, tobacco is the earliest and most studied. As early as 1948, Dawson studied the biosynthesis of alkaloids in tobacco. His 1960 reported that in the tissue culture of viscose tobacco, the nicotine content dropped sharply in the early stage, and by the time of typical callus, there was no nicotine. However, Speake et al. (1964) proved the roots, stems and leaves of tobacco (Virginia variety).