Therefore, it is proved that each somatic cell of a plant has the potential to form a complete plant. For example, plant cells are totipotent and can be induced to differentiate organs and regenerate plants under certain conditions of in vitro culture. Since 1970s, the research on plant protoplast culture and somatic hybridization has made great progress. For example, tobacco, Datura stramonium, belladonna, carrot and rape can be regenerated from their protoplasts and differentiated into embryos or whole plants. Hybrid cells of Nicotiana and Petunia can proliferate through protoplast fusion and differentiate into hybrid plants.
Therefore, using tissue culture method, we can study the reproduction, growth and differentiation of cells, tissues or organs and the influence of various external factors under relatively simple and easy-to-observe conditions, thus opening up broad prospects for solving some problems in agricultural production and drug production. At present, several important achievements have been applied to production practice. One is the rapid propagation of clonal lines, such as sugarcane, which used to use 0.5 ~ 1 ton of sugarcane seeds per mu. Another example is that the reproduction rate of Fritillaria is very low, and the bulbs differentiated by tissue culture take about three months, which is equivalent to the biennial bulbs of seed reproduction. The other is the industrial production of drugs and biological products. The effective components of medicinal plants are generally extracted from plants, and their yield and quality are inevitably affected by plant genetics, growth conditions, harvest time, storage and transportation and other factors. These shortcomings can be overcome if effective ingredients can be produced by similar methods of cultivating microorganisms to produce antibiotics. For example, in recent years, a large number of ginseng tissues have been cultivated and effective components have been extracted. Therefore, it is a new direction of drug production to use tissue culture to produce medicinal components and explore the industrialization road of natural drug production. With the success of large-scale artificial culture technology, it is possible to extract effective components by tissue culture instead of whole grass, which will be one of the central topics of plant medicine research in the future.
I. Composition and preparation method of culture medium
The chemical synthesis medium used in recent years consists of six components: (1) sugars, more than two inorganic salts, (3) trace elements, (4) amino acids, amides and purines, (5) vitamins and (5) vitamins. 3 auxin. In addition, some media can also add natural fruit juice, such as coconut milk, yeast extract, hydrolyzed casein, malt extract and so on. If 0.5 ~ 1% agar is added to the medium, it is a solid medium for static culture, otherwise it is a liquid medium for suspension culture. Different plant materials often need to change the formula, such as maintaining growth and inducing cell division and differentiation, so there are many kinds of formulas. At present, Ms? (village weight? And then what? Skoog) medium formula is the most commonly used basic medium, which is beneficial to the rapid growth of general plant tissues and cells.
In short, the composition of culture medium should be determined according to the research purpose and the type of cultivated plants. Besides nutrition and induction, attention should also be paid to ion balance and toxicity. For example, distilled water is generally used for water, and chemically pure drugs are generally used for inorganic salts. Can the pH be 1N? KoH (or NaOH) solution and 2N? HCI adjustment. Sometimes ordinary drugs can be used instead, but it should be noted that these drugs not only have nutritional value, but also are non-toxic. If cells or tissues are cultured in large tanks to produce effective components and biological products in industry, the amount of culture medium will be measured by tonnage, so it is necessary to carefully consider what substitutes are more economical and practical.
Second, cultural conditions.
(1) temperature: for most plant tissues, 20 ~ 28℃ can meet the growth needs, and 26 ~ 27℃ is the most suitable.
(2) Illumination: Tissue culture is usually carried out under scattered light. The influence of light will lead to different results. Some plant tissues grow better in the dark, while others grow better in the light. When callus differentiates into organs, it needs a certain amount of light every day to form buds and roots. The formation of some secondary substances is determined by only three factors.
(3) Osmotic pressure: Osmotic pressure is closely related to the growth and differentiation of plant tissues. Osmotic pressure can be adjusted by adding salt, sucrose, mannitol and ethylene glycol to the culture medium. Generally 1 ~ 2 atmospheric pressure can promote the growth of plant tissues. When the atmospheric pressure is above 2, growth obstacles appear, and plant tissues cannot survive at 6 atmospheres.
(4) pH: Generally, the most suitable pH for plant tissue growth is 5 ~ 6.5. In the process of culture, the pH value can be changed, and adding hydrogen phosphate or dihydrogen salt can play a stabilizing role.
(5) Ventilation: The vigorous growth of cells in suspension culture must have good ventilation conditions. When a small amount of suspension culture is used, it often rotates or oscillates, which can play the role of aeration and stirring. Special aeration and stirring devices can be used in mass culture.
Three. Materials and methods
All kinds and parts from lower algae to higher plants such as moss, ferns and seed plants can be used as tissue culture materials. Generally gymnosperms use seedlings, buds and phloem cells, while angiosperms use embryos, endosperm, cotyledons, seedlings, shoot tips, roots, stems, leaves, anthers, pollen, ovaries and ovules.
Because the surface of plants is often polluted by mold and bacteria under natural conditions, the materials must be disinfected. Generally, bleaching powder solution (1 ~ 10%), sodium hypochlorite solution (0.5 ~ 10%), mercuric chloride solution (0.0 1%), ethanol (70%) or hydrogen peroxide (3 ~/kloc-0) are used. Under suitable conditions, a dedifferentiated tissue block, called callus, can quickly grow on the surface of the injured tissue incision, and can be induced to grow into a complete plant after a certain period of time on the appropriate culture medium. Therefore, callus is not only the source of some plant metabolites, but also one of the main ways to induce plants.
Under suitable culture conditions, callus can be subcultured for a long time, which is called subculture. However, in subculture, with the increase of subculture generations, the differentiation ability of many plant tissues or cells is gradually reduced or even lost, which may be caused by the gradual consumption of special substances related to organ formation in the original matrix during the culture process, so it can be restored by hormones or improving nutritional conditions. Some people think that it is the hereditary change of tissues and cells in long-term culture, mainly the change of chromosomes, and a large number of polyploid or aneuploid cells appear, which may be restored. Different media can make callus grow at different rates, and the structure can be loose or tight. Using these characteristics, it can be dispersed into single cells or small cell clusters. To form single cells, we should cultivate them in a culture medium with high salt, high auxin and high casein hydrolysis, then move them into liquid and disperse them into single cells through stirring. Adding some pectinase is also useful, but generally speaking, pure single cells are rarely obtained.
When selecting materials for cultivating medicinal plants, we should also consider the synthesis site of the required secondary substances in plants. If the materials and culture methods are appropriate, the main metabolites produced in the original plants can be obtained by biochemical transformation through cell or tissue culture.
Effective components can be obtained through tissue culture, but in fact, only a large number of successful cultures have economic value. Therefore, suspension culture is often used instead of solid medium containing agar in production. Callus in suspension culture usually grows faster than that in static culture, because nutrients can penetrate into cells faster, metabolic wastes that inhibit growth can be removed faster, and oxygen supply is better. In this culture process, attention should be paid to ventilation and periodic renewal of nutrient solution, which is one of the keys to ensure stable growth and high secondary biomass yield.
Fourth, the formation of effective components.
Some effective components synthesized by tissue culture are listed.
Using tissue culture to produce medicinal components has gradually become one of the new directions of drug production. Since 1960s, some countries have conducted tissue culture on Dioscorea zingiberensis and other related families and genera to study the formation of diosgenin and explore its biosynthesis mechanism. It is known that seven species of Dioscorea plants can obtain diosgenin or other steroidal compounds after tissue culture, including Euphorbia humifusa? Delto Ides? The content of diosgenin obtained by Wal 1. tissue culture is 0.3 ~ 2.5%, in addition to rotenone, glycyrrhizin and nicotine. For example, 2.9% (dry weight) nicotine can be produced by suspension culture of tobacco root tip cells.
Adding growth hormone, vitamins or other chemicals to the commonly used basic culture medium can sometimes increase metabolites. For example, when 0. 1% tyrosine is added to the culture medium of Datura stramonium, the yield of atropine can be increased by more than 7 times, and when Rutaceae tissue is cultured, adding 4- hydroxy -2- quinine to the culture medium can promote the synthesis and accumulation of dictamnine. In both cases, additives are considered as precursors of biosynthesis. If the callus was supplemented with 1my/ 1 kinetin at the late growth stage, the content of scopolamine could reach 0.495%, which was much higher than that of the original plant.
In addition to the composition of the culture medium, environmental factors also affect the production of secondary substances. For example, although celery tissue culture proliferates in the dark, it does not form flavonoids, but apigenin can be detected under light. Although the history of tissue culture applied to pharmacy is not long, it has developed rapidly. It has the following advantages:
1. Use tissue culture instead of the original plant culture to obtain the required effective components, achieve the purpose of high yield and low cost, and save land.
2. It can be used not only for producing secondary biomass, but also for biotransformation. For example, in tobacco tissue culture, thebaine can produce morphine after demethylation.
3. New compounds were found from the qualitative analysis of tissue culture. For example, in the tissue culture of Ruta, rutin is synthesized and accumulated, which is a compound that has not been detected from the original plant or other plants. Therefore, tissue culture will become the source of obtaining new bioactive compounds.
Generally speaking, tissue culture is heterotrophic, but there are also autotrophic cell lines, which have the ability of photosynthesis and do not depend on external sugar supply. This feature will make cell culture technology superior to the whole plant and more economical.
At present, the relevant units in China have successfully industrialized ergot, Ganoderma lucidum, Hericium erinaceus and other fungi, and the application of higher plant tissue culture in industrialization is also under study.
In a word, the application prospect of plant tissue culture in Chinese herbal medicine is infinite, which is not only conducive to discussing and clarifying a series of theoretical problems such as physiology, heredity and component biosynthesis of medicinal plants, but also will make great contributions to the prevention and treatment of diseases once the industrial production problems are solved.