(Zhu Weihua)
In 1902, G. Haberlandt, who made the earliest experiments on plant tissue culture, predicted that isolated cells of higher plants could grow into plant bodies. This hypothesis became the theoretical basis of plant tissue culture in the future.
In 1937, White established a comprehensive medium for tissue culture, and together with Gautheret et al. succeeded for the first time in using stem-forming layer cells of tobacco and small pieces of carrot root to proliferate cells and induce healing tissues under artificial culture conditions. The basic method of plant tissue culture established by them became the technical basis for subsequent tissue culture of various plants.
In 1948, F. Skoog and Cui found that attaching an appropriate ratio of adenine and growth hormone to the medium used for cultivating tobacco stem segments and pith could control the plant's tissue growth of seedlings or roots, that is to say, shoots were produced when the ratio of adenine/growth hormone was high, and roots were formed when the ratio was low. Later, C.D. Miller et al. (1956) found that agonists were more effective in place of adenine, and established a hormonal model of agonist/growth hormone ratios for controlling organ differentiation. This theory of hormonal "cybernetics" still plays a guiding role in plant tissue culture research.
In the 1950s, the rapid development of tissue culture technology, from static solid culture to liquid culture, which is divided into suspension culture, microchamber culture, caretaker culture, plate culture, etc. In 1958, Steward et al. used liquid suspension culture to culture the somatic cells of carrots through the embryoid pathway, and then cultivated them into a complete plant and flowering and fruiting. This breakthrough not only confirmed Haberlandt's concept of "cellular totipotency", but also opened up a new field for the study of organogenesis and embryogenesis in tissue culture.
E.C. Cocking and others succeeded in separating plant protoplasts with fungal cellulase in the early 60's, which created conditions for the rapid development of genetic engineering such as protoplast fusion and somatic cell hybridization in recent years.
In the past two decades, due to the cultured plant cells under certain conditions of the totipotency law, so that the application of tissue culture technology in production has been emphasized and development, has gradually become an important means of research in agriculture, forestry, horticulture, medicine and other aspects, and led to the morphology, cells, physiology, biochemistry, genetic breeding and a series of disciplines such as the progress of the progress.
Due to the development of molecular biology and bioengineering, plant tissue culture technology has become more and more perfect. 60 years later, plant tissue culture began to be applied in the production, so that plant production to the factory application period. Flowers and herbaceous plants, with asexual reproduction line rapid propagation of plants, factory production of test tube varieties of commercialization; application of cell mass culture technology to produce drugs, has been successful in Japan, Germany and other countries. In short, plant tissue and cell culture is an emerging biotechnology with great potential, will be a new look into the ranks of the world's new industrial revolution, and show a better future.
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 for plant tissue culture, sterilization methods and basic media
(I) Experimental equipment for plant tissue culture
1. Laboratory setup
(1) aseptic operation room indoor purification workbench for inoculation, placed inoculation tools and cultures on the side of the work table, the ceiling or the wall is installed with ultraviolet light tubes for sterilization. If there is a bacterial filtration ventilation device is better.
(2) medium preparation room
For the preparation of various culture media, the room must have a large area of flat workbench and place a variety of chemicals and utensils of the cabinet shelf. Store the prepared medium mother liquor refrigerator, distilled water preparation and storage equipment.
(3) constant temperature culture room
The culture room needs to have a constant temperature control air regulator, and lighting. The temperature of the constant temperature room is generally maintained at about 25 ℃, and the temperature difference should preferably not exceed ± 1 ℃.
There must be culture racks, shaking beds, rotary beds and other culture devices and equipment in the culture room.
(4) cytology laboratory
Place a variety of microscopes and dissecting scopes, mainly to observe the results of culture. When there are conditions can be established photographic equipment, filming the results of experiments.
(5) Chemical Laboratory
Prepared with a variety of conventional and advanced chemical analysis and determination of the instrumentation, chemical analysis and determination of a variety of work.
2. Instruments and appliances
(1) glassware
Application of alkaline solubility of small hard glass made of instruments and utensils, especially for long-term cultivation, such as the choice of non-quality glass products, is prone to adverse effects.
Commonly used glassware are: triangular bottles, nipple bottles, T-tubes, angular culture bottles, round culture bottles, L-shaped test tubes, parallel angular test tubes, round flat bottles, Petri dishes, glass rings, slides, coverslips, funnels, dispensers, syringes, round-bottomed flasks, dispensing funnels, slides, glass columns, condensers, extraction devices, staining cylinders, alcohol lamps.
(2) utensils and instruments
The selection of medical equipment and instruments used in microbiology laboratories, such as knives, scissors, a variety of tweezers, scalpels, inoculation needles.
(3) Instruments and equipment
Generally need to have a balance, acidometer, centrifuge, microscope, dissecting mirror, temperature box, oven, refrigerator, shaking bed, rotary bed, spinning culture rack, spectrophotometer, thin-layer scanner, high-pressure liquid chromatograph and so on.
(B) plant tissue culture sterilization methods
1. Sterilization of vessels and media
Dishes, overalls, masks, hats, etc. can be sterilized at high temperatures, generally 1.2 atmospheric pressure, 20-30 minutes; media sterilization time should not be too long, otherwise some substances are easy to decompose and destroy, 1.2 atmospheric pressure, 15 minutes is sufficient. The sterilization time of culture medium should not be too long, otherwise some substances are easy to decompose and destroy, 1.2 atmospheric pressure, 15 minutes is enough. Disinfection of metal instruments, generally soaked in 70% ethanol before use, when used on the flame ignited disinfection cooling after use.
2. Sterilization of plant materials
The sterilization of plant materials is mainly external sterilization, according to the type of material to the sensitivity of the fungicide to select the type of disinfectant and concentration and length of treatment time. First, the experimental material is carefully brushed in a soap powder solution and rinsed with running water, and then sterilized by referring to the methods and sequences listed in Table 13-1 and Table 13-2.
Table 13-1 Comparison of the effects of commonly used sterilizing agents
Table 13-2 Order of sterilizing different organs of plants (C) Basic media for plant tissue culture
1. Composition of culture media
Plant tissue culture media is usually made up of the following categories of substances (Table 13-3):
Table 13-3 Composition of several commonly used culture media (unit: mg/l)
(1) Inorganic nutrients
The inorganic nutrients include macroelements and trace elements. In addition to carbon (C), hydrogen (H), oxygen (O), large elements is nitrogen (N). Nitrogen is usually used nitrate nitrogen or ammonium nitrogen, but in the culture of nitrate nitrogen is mostly used, but also nitrate nitrogen and ammonium nitrogen mixed use. Phosphorus is commonly used as a phosphate and sulfur as a sulfate. Potassium is the main cation, calcium (Ca), sodium (Na), magnesium (Mg) needs less. The matching ratios of the bulk elements are generally modified along the lines of the formulation of Knop's solution for cultivating holophytes. In general, nutrient media should contain at least 25 mmol each of nitrate and potassium. Ammonium levels above 8 mmol are often toxic to the culture; however, for routine healing tissue culture and cell suspension culture, the concentration of nitrate plus ammonium can be increased to 60 mmol. calcium, sulfur, and magnesium are more appropriately concentrated in the range of 1-3 mmol. In contrast, the required sodium chloride is provided by calcium salts, phosphates, or micronutrients. Micronutrients include iodine (I), boron (B), manganese (Mn), zinc (Zn), molybdenum (Mo), copper (Cu), cobalt (Co), and iron (Fe), where iodine may not be essential.
(2) Carbon and energy sources
The sucrose requirement of most plant cells ranges from 2-4%, and in some plant tissue cultures, sucrose concentrations are as high as 7% or even 15%. Sugar sources may have other roles in the culture medium besides being a carbon and energy source. Sucrose can also be replaced by glucose and fructose; none of the other sugars are ideal. Inositol may not be essential, but it is used in large concentrations in the general medium, which may be related to its role in promoting the growth of healing tissue.
(3) Vitamins
Among the various vitamins, thiamine hydrochloride (B1) may be essential, whereas niacin and pyridoxine hydrochloride (B6) have only growth-promoting effects.
(4) Growth-regulating substances
Plant hormones are indispensable components of the medium, which have a direct effect on the induction of plant healing tissues, culture growth, organ differentiation and the metabolism of secondary products. There are two types of hormones usually added to the culture medium: one is growth hormone, commonly used 2,4-D, naphthalene acetic acid (NAA), indole acetic acid (IAA), indole butyric acid (IBA), etc.; the other is cytokinin, commonly used agonist (Kt), zeatin (Zt), 6-benzyl purine (6-BA). 2,4-D is suitable for concentrations of 10-7-10-5mol, IAA, 6-benzyl purine (6-BA), 2,4-D is suitable for concentrations of 10-7-10-5mol, IAA, 6-BA and 2-BA. -10-5mol for 2,4-D and 10-10-10-5mol for IAA, with 1-10mg/l being the most versatile. the range of suitable concentrations for NAA is higher than both of the former two. In general, the production of healing tissues can be successfully induced with only 2,4-D (10-5-10-7 mol). The results are even better when growth hormone substances such as 2,4-D are used in combination with a cytokinin. It may be better to use naphthalene acetic acid in combination with one cytokinin when inducing explants to differentiate plants. Among cytokinins, both agonist and 6-benzyl purine are more commonly used, and both promote the growth of healing tissues, with a suitable concentration of 10-7-10-6 mol.
(5) Amino Acids
The medium should include certain amino acids, such as glycine. Also like hydrolyzed casein is often used in tissue culture, which is a mixture with a variety of amino acids, especially in the differentiation medium to add a certain amount of hydrolyzed casein, can promote embryogenesis and polyembryonic emergence. Some amino acids are precursors of certain secondary substances (e.g., phenylalanine, ornithine, etc., are precursors for the biosynthesis of scopoletin-like alkaloids), and when they are added to the medium, they markedly increase the content and yield of such secondary substances in tissue cultures.
(6) Organic additives
Some natural products are added to the culture medium, they are beneficial to the induction and maintenance of healing tissues, as well as to promote the growth and the formation and accumulation of secondary substances. Some of the most commonly used are coconut milk, yeast extract, tomato juice and soybean meal. The range of concentrations used is 10% (v/v) for coconut milk, 0.5% for yeast extract, 5-10% for tomato juice, and 0.1-0.5% for soybean powder. These natural additives, due to the complexity of their composition and the difficulty of ensuring repeatable consistency, are preferred to fully known synthetic compounds in the preparation of culture media.
2. Preparation of the medium
(1) Water and drugs
The medium is best prepared using distilled water with demineralized salts distilled in a glass vessel. The chemicals used should be pure. Growth regulating substances should be recrystallized before use. Protein hydrolysates should preferably be enzymatically hydrolyzed so that the amino acids can be better preserved in their natural state.
(2) mother liquor (reservoir)
The convenient way to prepare the medium is to prepare a series of mother liquors first. A large number of mineral salts (large amounts of elements) can be formulated to be 10 times more concentrated than the concentration used. When dissolving mineral salts, strive to stagger Ca2+ with SO2-4- and PO3-4, so as to avoid the formation of insoluble calcium sulfate and calcium phosphate, it is best to use a certain amount of distilled water to dissolve the mineral salts separately, and then add them in order, and finally add water to a certain volume. Trace elements due to the small amount, can be formulated to use the concentration of 100-1000 times the concentrated solution, and large elements, etc. stored in the refrigerator. Vitamins are prepared separately and can be stored in volumetric flasks at concentrations of 0.2-1mg/l. Iron salts need to be prepared separately (see previous). Plant hormone substances, the preparation of different requirements. NAA, 2,4-D and IAA and other growth hormone substances, weighed after the drug first with a small amount of 95% ethanol solubilization, and then add distilled water to dilute to a certain concentration; cytokinin substances, it is appropriate to use a small amount of hydrochloric acid 0.5 or 1N to dissolve, and then add distilled water to the required amount; folic acid should be a small amount of diluted ammonia to dissolve, and then added to distilled water to the required amount. Folic acid should be dissolved in a small amount of dilute ammonia, and then add distilled water to the required amount; biotin can be dissolved directly in water when preparing.
(3) autoclaving and preservation
When preparing the culture medium, firstly, take out all kinds of mother liquor according to the required volume and mix them together, temporarily add sucrose, hormone and other additional ingredients, and mix with agar which has been heated and dissolved beforehand (no agar is added in the case of liquid culture), and then adjust the pH to a certain value (between 5.5-5.8) by using sodium hydroxide or 1N hydrochloric acid, and then adjust the pH to a certain value (between 5.5 and 5.8). 5.5-5.8), and then packed into culture containers separately. The prepared medium was sterilized in autoclave at 120℃, 1.1kg/cm2 under high pressure for 15-20min. the sterilized medium can be stored at room temperature (the optimum temperature is 10℃) and should be applied within two weeks.
3. Characteristics of several commonly used culture media
In different culture media, the general inorganic salts are more varied, and sometimes also due to the addition of different nitrogen sources to form their own characteristics. It has been used for the induction of healing tissue under solid culture conditions, cell suspension culture under liquid culture conditions, and for the culture and morphogenesis of embryos, stem tips, stem segments, and anthers, etc. The amount and proportion of inorganic nutrients in the MS medium are appropriate and sufficient to 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 hydrolysate or coconut milk to the medium. The high content of nitrate, potassium and ammonium in MS medium is a remarkable feature compared to other media. Similar to MS medium, there are LS (Linsmaier, E.M. and Skoog, F. 1965) and RM (Tanaka, 1964) mediums, which have the same basic composition as MS medium, except that glycine, vitamin B6 and niacin have been removed from the former, and the latter has increased the dosage of NH4NO3 to 4950 mg/l and KH2PO4 to 510 mg/l. White (1963) medium is a medium with a lower concentration of inorganic salts than MS medium, but it is also widely used and gives good results both in embryo culture and in tissue culture in general.The B5 medium was designed by Gamborg et al. (1968), and its main feature is that it is low in ammonium, a nutrient that may have an inhibitory effect on the growth of some growth organisms. N6 medium was designed by the Institute of Botany of the Chinese Academy of Sciences and the Heilongjiang Academy of Agricultural Sciences, and is also a very suitable medium, which has been widely used in China for anther culture and tissue culture of certain plants, with a similar composition to that of B5 but without molybdenum.Heller's (1953) medium, which is a medium more commonly used in Europe, is low in inorganic salts and also contains molybdenum. low content of inorganic salts and also lacks molybdenum salts, but contains compounds such as nickel-aluminum.
The general trend is that recent media have tended to use high concentrations of inorganic salts. In the use of nitrogen, many of them use a mixture of nitrate and ammonium nitrogen, or just nitrate nitrogen. The role of trace elements has been less well studied, and most formulations are basically close to those of MS media, while the inclusion of these trace element components has met the needs of healing tissue and cell growth in tissue culture. For iron salts a mixture of FeSO4 and Na2-EDTA (chelating agent) is usually used.
The application of plant tissue and cell culture in the production of drugs
In recent years, due to the man-made destruction of the natural environment, indiscriminate logging, rising labor costs and the introduction of wild plants in the technical and/or economic difficulties, resulting in a drastic decrease in plant resources, and to ensure an adequate supply of medicinal plants is increasingly difficult. Since the sixties, people have begun to apply plant tissue culture technology to study the production of botanical drugs. In recent years, with the development of modern biotechnology, plant cell mass culture has been successful, opening up a new way of industrialized production for plant drugs.
Cell culture system than the general overall plant cultivation, has a variety of superiority: (1) the production of useful substances is carried out under controlled conditions, without having to rely on climatic and soil conditions, and save land; (2) cell culture is free of microorganisms and insect pests; (3) the growth cycle is short, to shorten the introduction of the plant to domesticate and expand the propagation of the lengthy process; (4) it can be carried out in a specific biotransformation reactions, which can explore new synthetic routes and obtain new useful substances; (5) it is possible to change the culture conditions by screening new cell lines in order to increase productivity and reduce production costs.
(I) Plant Tissue and Cell Culture Technology
1. Induction and Cultivation of Healing Tissues
Healing tissues are the basic materials for plant tissue and cell culture, so induction of healing tissues and culturing them is one of the basic work of plant tissue and cell culture.
The working procedure of inducing healing tissues 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 cultivation; and (5) Successional culture.
Healing tissues have been successfully induced from many plants, with dicotyledons being the most numerous and monocotyledons less so. There have also been successes from gymnosperms, ferns and mosses. Thus it can be said that all multicellular plants have the potential to induce successful healing tissues. Dicotyledonous plants, in addition to their broad utility, are capable of rapidly growing healing tissue. Various plant tissues, such as the forming layer of vascular bundles, the thin-walled tissues of storage organs, the mid-column sheaths of roots, the endosperm, cotyledons, chloroplasts, and vascular tissues, are capable of forming healing tissues under the right conditions.
The induction of healing tissue is mostly carried out on solid medium. Solid culture is usually carried out at 25-28℃, and succession culture is carried out every 4-6 weeks.
The culture of healing tissues can generally be divided into two types: solid culture and liquid culture. Solid culture is to add a certain amount of coagulant (0.6-1.0% agar, etc.) to the culture medium, heat and dissolve, respectively, into the culture container, and then cooled as solid culture medium. And those without coagulant are liquid culture medium.
Solid culture for static culture, the advantage of its simplicity, only general glassware can be, unlike the liquid oscillation culture that requires shaking beds, rotary beds and other complex mechanical equipment, and occupies a small area, a small culture room can be placed in a lot of culture vessels. However, solid culture also has the following shortcomings: only part of the surface of the healing tissue and medium contact, resulting in uneven growth of the healing tissue; contact with the medium of the underlying tissue gas exchange is poor, but also make the growth process of the accumulation of hazardous substances discharged; static placement, due to the effect of gravity or uneven exposure to light, and make the tissue polarization phenomenon, and it is difficult to obtain a fairly consistent group.
Liquid culture is divided into two kinds of static and oscillation. Liquid static culture and solid culture is also a simple method, and the culture medium will not appear in the phenomenon of nutrient concentration differences; but because of the limitations of the use of larger, so it has been rarely used. Oscillatory culture is to make the plant tissue in the liquid medium constantly rotating, so that can eliminate the shortcomings of static culture. Oscillatory culture can be divided into two categories: (1) continuous submerged, by stirring or vibration of the culture medium to make the tissue suspended in the medium, commonly used shaking bed for culture; (2) regular submerged, with T-tube, nipple bottle as a culture container, on the rotary bed for culture. The culture material is repeated in the liquid and vapor phases during rotation.
2. Cell suspension culture
Plant cell suspension culture technology is a new culture technology developed on the basis of liquid culture technology of healing tissue. In the past twenty years, from the test tube suspension culture to large volume fermenter culture, from discontinuous culture to semi-continuous and continuous culture, until the latest type of turbidity constant method and chemical constant method in recent years, such as automatic control of the larger scale of continuous culture. The characteristics of cell suspension culture are (1) the ability to provide uniform plant cells in large quantities, (2) the rate of cell proliferation is faster than that of healing tissues, and (3) it is suitable for large-scale culture. Therefore, it is possible to cultivate plant cells like microorganisms and apply them to the fermentation industry to produce some plant-specific products, thus opening up a new way of industrialized production of plant products. Cell suspension culture is to make the free plant cells in the liquid medium for culture, but because the plant cells have the characteristics of aggregation together, so far, can not make the suspension only free single cells, but often some small cell clusters.
(1) equipment and devices for suspension culture
①Shaker widely used in plant cell suspension culture. Easy to break up the healing tissue block, the continuous oscillation of the shaker can be dispersed cell suspension. It can also be used for succession culture of suspended cells.
②Turning bed One turn per minute. Use T-tube or nipple bottle as the culture vessel.
③Spinning culture rack Larger bottles can be used as culture vessels.
④Continuous culture equipment Usually relies on the passage of sterile compressed air or both the passage of air and constant stirring to keep the cells in suspension. In such a stirring device, because the container holding the culture fluid is static, so it can be easily connected to the storage of fresh culture fluid container, air supply device and culture container. Culture container can be installed in some snake-shaped tube, tube into the electric wire can be heated, through the cold water can be cooled down, so this kind of device does not have to be installed in the constant temperature room. These devices generally have the ability to easily control the temperature, stirring speed, aeration speed, lighting intensity, nutrient flow rate, etc., and often with oxygen electrodes, pH electrodes, cell density meter, etc., connected to become a set of preliminary automatic control of the continuous culture device. There are several kinds of plant cell mass culture devices such as fermentation device with stirring, fermentation device with oscillator, fermentation device with conduit and rotating impeller, fermentation device with bubble stirring, and air-lift fermentation device.
(2) Medium for cell suspension
The medium commonly used for healing tissue is not necessarily the most suitable for cell suspension culture. Often the medium that induces healing tissue can be used as a starting point for determining the most suitable medium. Particular attention should be paid to the effect of the amount of growth factors and cytokinins on the aggregation of cells in suspension culture, and it is important to use a medium that makes it easy for the cells to free themselves singly.
The pH often changes considerably in suspension culture, so it is necessary to add chelating agents such as EDTA to keep iron and other ions available for a long time. Adjustment of the ratio between nitrate and ammonium nitrogen can also be used as a way to stabilize pH. Adding some solid buffers such as slightly soluble calcium hydrogen phosphate, insoluble calcium phosphate, calcium carbonate is also a way to stabilize pH.
(3) Succession culture of suspension cells
The change of cell number growth during suspension culture is shown in Fig. 13-1. it is basically an S-shaped curve. At the beginning of the delayed period, the cell rarely divides; followed by the logarithmic growth period, the number of cells grows rapidly, the growth rate remains the same; later enter the gradually slowing down of the stationary period; and finally reach the growth of the complete cessation of the period. Cells in culture, generally at the beginning of the stationary phase of the successor culture, some in the stationary phase before the slowdown in proliferation, some even in the logarithmic growth phase at the end of the immediate generation, in order to accelerate cell proliferation.
Figure 13-1 Schematic diagram of the growth of cells in suspension culture in one culture generation
In recent years, researchers have used DNA synthesis inhibitors and phytohormone treatments to enable cells in culture to enter a certain period of the cell division cycle (e.g., the G-phase) under certain conditions, and then start from the next period to synchronize cell or cell division, i.e., to synchronize the cell or cell division of all cells. cells or all cells divide synchronously, which is called synchronized culture. This not only provides a detailed understanding of the true course of the cell cycle, but also allows us to recognize the factors that control the sequence of biochemical changes that take place from the parental cells to the daughter cells. Because synchronous culture allows frequent sampling for biochemical and cytological analysis, the sampling volume can be large and will not affect the remaining population to continue to grow, which provides the necessary technical means for the study of the cell cycle, cell differentiation, secondary material metabolism and other important processes, is another important progress in the development of plant cell culture technology.
In summary, the current plant tissue and cell culture technology has evolved from static solid culture to liquid culture, which is characterized by the possibility of improving the nutrient and oxygen supply to tissues and cells in culture. The development is proceeding from small to large quantities in terms of scale and methodology, from test tubes to large tanks and synchronized cultures in terms of application, and from manual to automated in terms of operation. These advances and developments are having a significant impact on the research and production applications of plant tissue and cell cultures.
(II) Pharmaceutical ingredients produced by plant tissue and cell culture
Since Bonner et al. conducted a study on the production of natural rubber from Ginkgo biloba healing tissue in 1950, many scientific workers have worked around the question of what useful substances can be produced from healing tissue and how to improve the rate of active ingredient yield, and some results have been achieved. More and more people believe that the use of plant tissues and cultured cells has the potential to produce alkaloids, terpenoids, quinones, steroids, enzymes, etc. for the treatment of various diseases, as well as to extract peptides and proteinaceous substances from cultures.Nickell (1980) outlined the fact that plant tissue cultures can produce more than 50 classes of compounds and 28 classes of potential products thereof. Jung Kwang-sik (1980) synthesized a list of more than 60 medicinal ingredients, source plants and drug content ****. Misawa, M. (1980) of the Concord Fermentation Company of Japan describes the results of research conducted by industrial laboratories or the government to produce alkaloids, steroids, terpenoids, quinones, and other physiologically active substances, including enzymes, by plant tissue culture. According to the World Health Organization, there are 17 of the most common and essential drugs derived from higher plants. Tissue cultures are available for the source plants of 11 of these drugs (Table 13-4).
Table 13-4 Common but essential drugs derived from higher plants
1. Alkaloids
The production of alkaloids using plant tissues and cell cultures is extraordinarily difficult, yet the tissue culture of alkaloid-producing medicinal plants has been one of the most researched aspects of the process (Table 13-5 ). However, their content is usually lower than that of the original plant.
Table 13-5 Alkaloids in Plant Healing Tissues
Table 13-5 Alkaloids in Plant Healing Tissues (Continued)-1
(1) Scopoletane Alkaloids
Mothes, West, Chan, Metz, and Masao Kimishima, Bhandary, Thomas, Stohs, Sairam, Tabata, Hiraoka, Cliokshi, etc. have successively carried out a lot of studies on the roots, healing tissues, and suspension cells of mandarins, belladonna, aspergillus, scopoletta, etc., but the medicinal purposes were not available or were in low levels.West (1957) was the first to affirm that in the healing tissues of belladonna roots presence of scopolamine alkaloids. The atropine content of the root healing tissue was examined to be 0.47-0.53%. However, it was not examined in the healing tissues of stems and leaves.Chan et al. (1956) determined the alkaloid content of the healing tissues of various types of organs of Mandragora, Cypress-leaved Mandragora and Spineless Mandragora by subjecting them to static versus oscillatory culture. The highest content of 0.016-0.056% was found in the seed healing tissues of Mandragora and Cypress-leaf Mandragora, 0.012-0.015% in roots, 0.004-0.014% in stems and 0.007-0.010% in leaves . This result indicated that the amount of alkaloids produced varied depending on the organ from which the healing tissue was induced.West et al. also pointed out that there were differences in the ability to synthesize alkaloids between healing tissue strains of mandarins of the same origin. Jung Kwang-sik et al. (1976) initially determined the content of scopolamine to be 0.025% and that of scopolamine to be 0.009% in a three-quarter healing tissue culture, which were lower than those of the original plants (0.127% and 0.016%, respectively). However, due to the study of various aspects such as improving culture conditions, increasing nutrition, changing growth regulators, and supplementing precursors, the total **** of the two alkaloids amounted to 0.554% (0.139% for the original plant), with the highest content of scopolamine amounting to 0.495% (0.016% for the stems), which was 4-4.5 times higher than that of the initial healing tissues. The results of Cheng K-Di et al. (1987) showed that cells such as scopolamine not only produce tropane-like alkaloids, but also convert scopolamine into scopolamine and scopolamine.
(2) Pyridine alkaloids
In the tissue culture studies on the production of pyridine alkaloids, tobacco has been studied the earliest and the most. The biosynthesis of alkaloids in tobacco was studied as early as 1948 by Dawson, whose 1960 report of tissue culture of sticky-haired tobacco showed that the content of nicotine was drastically reduced at the initial stage, and when it became a typical healing tissue, there was no nicotine left. However, Speake et al. (1964) demonstrated that roots, stems, and leaves of tobacco (Virginia variety)