I. Significance of Plant Regeneration Plant biotechnology takes plant tissue culture as the main means, including five branches, namely, improving plant varieties by in vitro techniques, rapid propagation of economic plants, cryopreservation of germplasm, mass culture of plant cells and plant genetic engineering. In vitro culture of medicinal plants, except a large number of plant cells, the rest involve the culture of regenerated plants. Different from ordinary plants, it is necessary to pay attention to the changes of medicinal components when cultivating regenerated plants of medicinal plants.
(1) rapid propagation
The purpose of rapid propagation is achieved by a large number of regenerated plants cultured in vitro, which are mainly used for: (1) plants with low natural reproduction rate; (2) heterozygous plants with inconsistent characters, (3) endangered plants with scarce resources; (4) Newly cultivated fine varieties; (5) the first choice of varieties with excellent germination and excellent characters; (6) A small amount of plant materials imported from abroad; (7) Some inbred lines, original materials and hybrid offspring that need rapid propagation in the breeding process; (8) Triploid and polyploid plants obtained by tissue culture or natural conditions; (9) Plants that remove pathogenic bacteria (fungi, bacteria and viruses). Whether the rapid propagation technology can be applied depends on: (1) the culture technology is simple, stable and perfect; (2) The cost is lower than that of conventional methods; (3) Have certain equipment conditions and technical strength; (4) The market needs and the price is appropriate. Such as Siraitia grosvenorii and Dendrobium nobile.
(2) Virus-free plants
Virus-free plants with main viruses removed can be obtained by shoot tip culture, or by petal culture and callus culture for a long time. A large number of virus-free seedlings can be obtained by using them as female parents for field production, which can improve yield and quality and prevent variety degradation. Such as rehmannia glutinosa and chrysanthemum.
(3) Germplasm preservation
The viability of plant tissues and cells can be maintained for a long time at a low temperature of 4℃ or in liquid nitrogen. Plant materials that can be used for cryopreservation are: (1) shoot tip and lateral meristem; (2) cultured plant cells and embryoids; (3) protoplast; (4) Plant organs, such as embryo, endosperm, ovary, anther, pollen and seeds. Freezing plant materials can save manpower, soil and material resources. It is convenient for regional and international germplasm exchange, and provides breeding materials and virus-free seedlings through in vitro propagation.
(4) Variety improvement
The methods of improving plant varieties by in vitro techniques mainly include: (1) obtaining haploid plants by culturing anthers, pollen, unfertilized ovaries and ovules, and conducting haploid breeding; (2) obtaining triploid plants by endosperm culture, or obtaining polyploid plants by colchicine or other factors in plant tissue culture; (3) Mutants and mutants with excellent characters are obtained through somatic cell clone variation and cell mutant screening; (4) Protoplast culture and cell hybridization. No matter what breeding technology is adopted, it is necessary to induce plant regeneration and identify its offspring. Such as ginseng and medlar.
(5) Plant genetic engineering
Plant genetic engineering includes gene isolation, vector system and receptor system. It directly targets the genes that control the inheritance of traits, and can improve varieties more accurately. At present, more than 20 genes have been isolated and cloned. The vector system mainly studies the Ti plasmid system (Agrobacterium T-DNA) of Pseudomonas aeruginosa. As the receptor system, protoplasts, cells cultured in suspension, in vitro plant organs from callus or whole plants can be used. No matter what receptor system is used, transformed cells must be regenerated into complete plants, and plant regeneration is a major link in plant genetic engineering. Such as Solanum nigrum and Isatis indigotica.
Second, the basic principle of plant regeneration
There is a widespread phenomenon of regeneration in biology, and biological regeneration is the result of natural selection and artificial selection. Generally, lower organisms have strong regenerative ability, while higher animals with more developed nervous system have weak regenerative ability. In plants, cells, tissues and organs can grow and develop in vitro to form complete plants. However, different kinds of plants, or different varieties of the same plant, or even cells of different tissues and organs of the same plant have different regeneration abilities. Generally, wild plants have stronger regeneration ability than cultivated plants, and asexual plants are stronger than seed-propagating plants.
The basis of plant regeneration lies in the genetic totipotency of cells, that is, each cell contains all the genetic information to produce a complete organism. Under appropriate conditions, the development and differentiation of a totipotent living cell is controlled by the activation or inhibition of some genes in its DNA, and a cell can form a brand-new organism. Plant regeneration in vitro is the result of dedifferentiation and redifferentiation of cell culture in vitro under the stimulation and action of various environmental factors. It may be because the differentiation of plant cells is only relatively stable. In vitro culture, specialized cells are more likely to recover totipotency through dedifferentiation, and totipotency dedifferentiated cells are redifferentiated to produce complete plantlets. Differentiation is the central problem of plant regeneration, which is related to many factors. Plant hormones are chemical messengers. As "effectors" of gene expression, they have a direct impact on the synthesis of RNA and protein, and play an important and sometimes decisive role in the process of cell dedifferentiation and redifferentiation.
Although many problems are still unclear, it is believed that the basic principles of plant regeneration are: (1) totipotency of plant cells; (2) dedifferentiation and redifferentiation of plant cells; (3) the balance of plant hormones. These basic principles are not only the general guiding principles of organizational training, but also some basic theoretical topics to be further studied.
Third, the basic ways of plant regeneration
There are two basic ways of morphogenesis of regenerated plants in vitro, namely organogenesis and embryogenesis.
Some of these two morphogenesis go through the callus stage, while others are directly produced in the maternal tissue in vitro.
organogenesis
The way of organogenesis is to form complete plants through organ differentiation, such as Fritillaria, Crocus sativus, Gynura bicolor, Aloe Vera, etc. Organ differentiation in vitro culture includes the formation of adventitious buds, adventitious roots, bulbs, bulbs, tubers, protocorms and flowers. There are three ways for adventitious buds and adventitious roots to form regenerated plants: (1) forming buds first, and then generating roots on the buds; (2) forming roots first, and then producing buds on the roots; (3) buds and roots were produced on the callus at the same time, and then a vascular system was formed between the buds and roots, which were connected into a unified axial structure. In most cases, it is the first way. There are also different cases of differentiation through bulbs, corms, tubers, protocorms and other organs.
In plant tissue culture, although there are many reports on plant regeneration through organogenesis, there is still little information about basic research. Most studies continue to focus on the empirical operation of explants, culture media and environmental conditions adjustment. In most cases, people can only observe the differentiation state that has been shown, but it is difficult to track the process of differentiation. The factors related to the origin and direction of morphogenesis are still unclear. Among these factors, there are the changes and interactions of various substances in the in vitro culture system, which come from the culture medium, explants and their metabolites during the culture process.
In vitro culture, only a few cells were activated, and the activation was asynchronous. Torrey( 1966) put forward the meristem hypothesis, and morphogenesis started from the meristem mass formed in callus, that is, meristem. Under the influence of various factors in the culture system, meristem produces different primordia, which either form roots and buds or develop into embryoids due to different properties of factors. Meristem may occur at the interface between tissue and culture medium, and its formation site may be determined by physiological gradient caused by substances diffusing from culture medium to tissue.
Skoog and Miller put forward the hypothesis of hormone balance, which holds that organ differentiation is related to the ratio of cytokinin to auxin. In most cases, the high ratio of auxin to cytokinin is beneficial to the formation of roots, and vice versa. Due to the existence of endogenous hormones and the accumulation of explant inhibitors, many contrary situations have appeared. In the practical application of this principle, it is sometimes necessary to add or omit some plant hormones, and the following factors should be considered: (1) the types and combinations of plant hormones; (2) the absolute concentration of plant hormones; (3) Plant hormones, mainly the ratio of cytokinin to auxin; (4) The sequential effect of plant hormones; (5) Other factors, such as explants, nutritional conditions, types and concentrations of carbon sources and other physical and chemical factors.
(2) Embryogenesis
Embryogenic plant regeneration is to form an embryoid first, and then form a complete plant through embryoid germination. Embryogenesis is a common phenomenon in higher plants. It is observed that more than 40 families 150 plants have the ability of embryogenesis. With regard to the concept of embryoid, Zhu Kun proposed (1978) that embryoid is an embryoid structure which originated from non-zygotic cells in plant tissue culture and was formed through embryogenesis and embryonic development. This definition includes the following meanings: (1) embryoid is the product of tissue culture, which is only used in tissue culture and is different from apomixis embryo; (2) Embryoids originate from non-zygotic cells, which are different from zygotic embryos; (3) The formation of embryoids goes through the process of embryonic development, which is different from the differentiated buds in tissue culture.
The sources of embryoids can be divided into five categories: (1) tissues and organs; (2) Callus; (3) Free single cell; (4) Microspores; (5) Protoplast. Some originated from single cells, while others originated from multi-cells. There are two ways to form embryoids (Sharp et al, 1980): (1) Directly: the embryoids are formed directly from a pre-determined embryo cell culture tissue without callus; (2) Indirect occurrence: Embryogenesis passes through the callus, and develops from the callus into an embryo-inducing determinant cell. Under the influence of related factors in the culture system, embryonic cells continue to divide and proliferate, forming embryonic cell clusters, and then developing into spherical embryos. Usually, embryonic cells are small in size, large in nucleus, dense in cytoplasm and large in starch granules. Spherical embryo tissue can be separated from surrounding tissue, which is similar to the development process of zygote embryo. Spherical embryos develop and differentiate into heart-shaped embryos, torpedo-shaped embryos and mature embryos with cotyledons. Mature embryoid is similar to zygotic embryo in morphology, with radicle, embryo and cotyledon. On the differentiation medium, embryoids germinate like seeds, hypocotyls elongate, leaves unfold, roots develop and form chlorophyll, and various organs further grow to form plantlets. The formation of embryoids led to the development of artificial seeds.
Embryogenesis to plant formation is a continuous process, which can be divided into three continuous stages: (1) the origin of embryoid; (2) embryoid differentiation; (3) Embryoids germinate and grow to form plantlets. In practice, the development process of embryonic cell induction and embryoid formation may be formed on the same medium, or it may need to be cultured twice respectively. When embryoids form green plants, they need to be transferred to differentiation medium. If the chemicals in the culture medium, mainly plant hormones, are unbalanced. The differentiation of embryonic cells and the development of embryoids will be inhibited, and abnormal embryonic tissue blocks will appear. These malformed structures may also have the potential for embryogenesis.
The relationship between embryogenesis and plant hormones varies from plant to plant and can be divided into several types:
1. Specific auxin types
Embryogenesis must have auxin, and the existence of other plant hormones plays an inhibitory role. There are two types of this type: (1) auxin is necessary to induce embryonic cells, and auxin, such as carrot, needs to be reduced or removed for embryoid formation and development; (2) The induction and development of embryoids can be carried out on the same auxin medium. Such as ginseng.
2. Nonspecific auxin types
Embryogenesis requires auxin, and other plant hormones such as cytokinin, gibberellin or abscisic acid have synergistic effects in a certain proportion, such as jujube.
3. Non-auxin type
Embryogenesis does not need auxin and can be formed on cytokinin medium, such as sandalwood.
4. Non-hormonal type
Embryogenesis does not require plant hormones, such as Datura flower culture.
Embryoids usually need to reduce or remove auxin to differentiate into green seedlings, and cytokinins and gibberellins play a major role, such as ginseng and American ginseng.
Reducing nitrides play an important role in somatic embryogenesis. The reduced nitride includes inorganic nitrogen, such as NH+4; Organic nitrides, such as amino acids, amides, hydrolyzed casein, etc. Under normal circumstances, reduced nitrides are beneficial to the formation of embryoids, but the mechanism of action is not clear, and different plants react differently. Many studies have shown that the concentration ratio of auxin and reduced nitride may play an important role.
Different plants and different in vitro cultures may have different ways to regenerate plants, some are mainly through organogenesis, such as tobacco, and some are easy to produce embryoids, such as carrots. However, many plants can regenerate plants through organogenesis and embryogenesis, and plant hormones can be regulated, such as ginseng and American ginseng. Cytokinins are generally needed to induce bud differentiation, and auxin plays an important role in inducing embryoid formation.
In vitro culture, bud and embryoid are differentiated, and their main differences are as follows: bud is unipolar, which is related to vascular tissue in vitro culture, and roots and buds are not produced at the same time; Embryoids are bipolar, that is, there are root tips (radicles) and stem tips (embryos), which are not directly related to in vitro culture, so they are complete plant embryo morphology. The development of somatic embryogenesis leads to the development of artificial seeds.
Fourthly, the limiting factors of plant regeneration.
(A) the limiting factors of plant regeneration
Regenerated plants cultured in vitro can be divided into three types of regulatory factors: explants, culture media and environmental conditions. Although all of these three factors play an important role, comparatively speaking, the limiting factor for the success of plant regeneration is not the culture medium, but the culture material itself, that is, the expression degree of cell totipotency of the culture material.
The effect of 1. genotype
The regeneration ability of different plant species varies greatly, and some plants have strong regeneration ability, such as Solanaceae, Umbelliferae, Cruciferae and so on. Some are difficult to regenerate, such as legumes. Different varieties of the same plant have different regeneration abilities. Due to the influence of genetic types, the difficulty and application prospect of plant regeneration are determined. When we studied the embryo culture of Araliaceae medicinal plants Panax ginseng, Panax quinquefolium, Panax notoginseng and Acanthopanax senticosus, we got the following results: (1) On the medium supplemented with BA2mg/l+NAA0.5mg/l, the embryo culture of Panax ginseng and Panax quinquefolium can directly start bud differentiation, and when transferred to the medium containing gibberellin, adventitious buds will be formed, while Panax notoginseng and Acanthopanax senticosus have no bud differentiation. (2) In the medium containing auxin, embryoids exist in the embryo cultures of these four medicinal plants, but they have different responses to the types and concentrations of auxin. When 2,4-D0.5-1.0 mg/L was added, the embryogenesis ability was American ginseng > ginseng > acanthopanax senticosus, but no embryogenesis was observed in Panax notoginseng. On the medium containing IAA, high concentration IAA promoted somatic embryogenesis of ginseng seeds, while low concentration IAA was suitable for Panax notoginseng and Acanthopanax senticosus. (3) Under the above auxin conditions, Panax ginseng and Panax quinquefolium are indirect embryogenesis, while Panax notoginseng and Acanthopanax senticosus are direct embryogenesis.
2. The source of explants
In vitro culture, there are many materials available for culture, including organs, tissues and even single cells. Although plant cells are totipotent, their expression may be limited to some special cells, which may exist in explants in advance or may be produced during culture. Many experiments show that the characteristics of culture materials not only affect the possibility of regeneration, but also affect the growth speed and quality of plants. When selecting culture materials, it can be observed that meristem is easy to differentiate, cotyledon base is strongly regenerated, leaves of adult trees may be poorly reversible, and cell totipotency is difficult to express. When we studied the regenerated plants of American ginseng in vitro, the calli from different explants showed different degrees of cell totipotency (table13-10).
Table13 ——10 Expression degree of cell totipotency in callus of different explants of Panax quinquefolium L.
3. The state of explants
The development stage and age of explants, the endogenous hormones and physiological state of explants (sampling season), the size of explants, the pretreatment of explants and the quality of mother plants all affect the success of plant regeneration. When selecting explants, it is necessary to understand the biological characteristics of plants.
(2) Types and selection of explants
In vitro culture, explants have a variety of materials, which can be selected according to the research purpose, regeneration pathway and experimental experience.
1. explants with buds
Including stem heads, lateral buds, protocorms, bulbs, etc. These explants have high success rate, small variability and easy to maintain the excellent characteristics of materials, and are usually used for rapid propagation and culture of virus-free plants (shoot tip culture). In culture, auxin and gibberellin are often added to the culture medium in order to induce the elongation of stem axis; If axillary buds are induced to grow and produce clustered buds, there are often a lot of cytokinins in the culture medium.
2. Explants composed of differentiated tissues
Including stem segments, leaves, roots and other vegetative organs; Flowering stems, petals, calyx, anther, ovary, ovule, fruit and other reproductive organs. This kind of explants usually form callus during culture and regenerate plants through organogenesis and embryogenesis. Plants with organ regeneration usually choose organs and tissues that can produce adventitious buds under natural conditions, which should be selected according to the characteristics of different plants, such as aloe as stem segment, chrysanthemum as petal and lily as scale. Embryos, inflorescences and meristems are often used as explants through embryogenesis. This kind of explants can be used for clonal variation and mutant screening, or for haploid breeding, such as pollen plants.
3. Explants composed of genetically transformed cells
Mainly refers to the crown gall transformation system and DNA direct transformation system used in plant genetic engineering.
Verb (abbreviation of verb) cultivation method of regenerated plants
(1) culture steps of regenerated plants
1. Establish sterile culture.
This step includes the selection of explants, surface sterilization of materials and inoculation on culture medium. Whether you can get sterile culture depends on the situation of plant materials. Try to use healthy and vigorous materials grown in culture rooms or greenhouses as much as possible. Disinfection of field materials is difficult, so the following methods are often used: (1) Pretreatment of plants with antibiotic solutions and fungicides; (2) wrapping branches with plastic bags; (3) collecting branches for cutting and utilizing new buds; (4) sterilizing the culture material for many times. Some plant explants turn brown after inoculation and during culture. Some antioxidants, antidotes or adsorbents, such as citric acid, ascorbic acid, cysteine, polyvinylpyrrolidone, dithiothreitol, growth promoter, chloramphenicol, diethyl dithiocarbamate and activated carbon, can be added to the culture medium. To improve the browning.
2. Callus culture
Callus is the most common stage in plant tissue culture. In order to rapidly propagate excellent lines with consistent heredity and make the variation less than 3%, it is necessary to avoid callus as much as possible. However, it is beneficial to induce callus in variety improvement.
Callus is usually produced by surface cells of explant wounds. The growth characteristics of callus depend on plant materials, culture medium and environmental conditions. From explant to callus establishment, it usually goes through several stages: induction, cell division and differentiation. Under the action of external conditions, especially plant hormones, a few cells start up, their metabolism is enhanced, and they enter active cell division, and explant cells reverse to meristem or dedifferentiate into callus. The growth of callus has no obvious polarity, only the internal and external growth steepness, and the peripheral cells divide and grow faster than the internal cells. In the third stage, the callus differentiated and produced some secondary metabolites. The most important feature of callus is that it can form roots, buds and embryoids through organogenesis or embryogenesis, and then form small plants.
Calluses are usually pale yellow, white and green, and some contain the color formed by anthocyanins. Callus has loose type and dense type. Callus from the same source can be artificially selected in subculture to obtain different cell lines. They differ greatly in cell differentiation, color, growth rate, requirements for nutrition and hormone conditions, and the ability to synthesize special substances. Variation is another important feature of callus. These variations include phenotypic variation and genotypic variation. Genotypic variation may include ploidy change, chromosome aberration and gene sequence change. The selection of beneficial variation can be used for variety improvement.
The relationship between callus formation from explants and plant hormones is as follows: (1) Only auxin is needed; (2) only cytokinin is needed; (3) auxin and cytokinin are needed; (4) Plant hormones are not needed. In most cases, auxin is the main factor to induce callus.
In the process of culture, due to the gradual depletion of nutrients, agar dehydration gradually dried, metabolites accumulated, increasing toxicity. Callus culture needs to be transferred to fresh medium for subculture for a period of time. In solid medium, subculture is usually carried out every 4-6 weeks. The transferred culture is about 5- 10 mm and weighs about 20- 100 mg (street, 1969). If the volume is too small, it may grow slowly or not, but when screening for resistance, the volume of the culture is smaller. The composition and hormone conditions of callus subculture medium and dedifferentiation medium can be the same or different, which varies from plant to plant. Because long-term subculture has increased the content of endogenous hormones, it is necessary to adjust the types and contents of hormones in subculture medium appropriately.
3. Establishment of clone
Cloning from in vitro culture often encounters problems such as variation, loss of cell differentiation ability, glass seedling and so on. In vitro culture of each plant requires a lot of experiments to determine the best culture conditions and make them programmed in order to achieve the purpose of application. The method of establishing clones can be roughly represented by figure 13-2.
Fig. 13-2 schematic diagram of clone establishment 4. rooted
The difficulty of bud and bud-induced rooting depends on the genetic type of plants first. Herbs are generally easier than woody plants. Buds and branches produced by adult trees are more difficult to take root than young trees. In order to induce rooting, an appropriate amount of auxin, such as naphthylacetic acid, indolebutyric acid and indolebutyric acid, is usually added to the rooting medium. Some plants can take root on hormone-free media. The rooting medium can be the same as the former medium, and commonly used is a high-salt medium with the concentration of 1/2, 1/3 or 1/4 or a medium with low total salt concentration. Iron salt is beneficial to the formation of roots, and generally maintains the original concentration. Sucrose is necessary for rooting, and different plants may need different amounts.
Some plants that are difficult to take root can try some other methods: (1) pretreat with high concentration auxin solution for several hours, then rinse with sterile water and inoculate into hormone-free medium; (2) using filter paper bridge or vermiculite instead of agar as carrier to induce rooting; (3) Adding some additives to the rooting medium, such as activated carbon, B9, dimethyl sulfoxide, phloroglucin, phloroglucinol, etc. (4) inducing dormant organs such as small tubers, small bulbs and underground stems in test tubes; (5) Cutting twigs, directly cutting or hydroponics.
transplant
Some plants are easy to transplant, for example, the survival rate of aloe plantlets transplanted directly into soil can reach 100%, but the transplantation of ginseng plantlets in vitro has not been reported successfully. For many plants, it is necessary to study their special transplanting techniques.
(1) Cultivate strong seedlings and exercise before transplanting.
(2) gradual transition: firstly, artificial matrix, such as vermiculite, sand, perlite, cinder, core soil, etc. Is introduced into the soil. For plants susceptible to diseases, artificial substrates can be sterilized by high temperature, chemical sterilization or chemical disinfection.
(3) Select the appropriate transplanting period. Many woody plants and some herbs have the highest survival rate when the root primordium protrudes or forms short roots of several millimeters. Some herbs can be induced by secondary rooting, and new roots can be induced to transplant after the first rooting and aging.
(4) Control the environmental conditions of transplanting, maintain high humidity and good ventilation, and avoid direct sunlight and excessive temperature fluctuation. Some plants need to simulate their ecological environment.
(5) timely prevention and control of diseases. The common disease is damping-off, and pesticides such as carbendazim can be used to spray seedlings and substrates regularly.
(6) Grafting test-tube seedlings.
(2) Culture medium and environmental conditions
1. Medium
According to the culture materials and purposes, plant tissue culture can be divided into many types, such as organ culture, shoot tip culture, anther and pollen culture, ovary and ovule culture, embryo culture, endosperm culture, fruit culture, cell suspension culture, meristem culture, callus culture, protoplast culture and cell fusion. Different plants and different kinds of tissue culture need different media. At present, there are about 250 kinds of media that can be used for plant tissue culture. For general in vitro propagation, in order to reduce the production cost, some people adopt simplified culture medium, replace reagents and sucrose with edible sugar, and prepare culture medium with ordinary water instead of distilled water. Static shallow liquid culture can reduce the cost by 70-80% compared with agar solid culture, and has been used for rapid propagation of some plants.
2. Environmental conditions
Including pH value of culture medium, culture temperature, light condition and ventilation condition.
Generally, the optimum pH for plant tissue growth is between 5 and 6.5, and the solid medium is usually pH5.8 and the liquid medium is usually pH5.0.
The culture temperature is generally around 20-28℃. High temperature (about 27℃) is suitable for tropical varieties, and low temperature (about 20℃) is beneficial to the in vitro culture of alpine plants. When dormant organs such as bulbs and tubers are formed in test tubes, their germination often needs low temperature treatment.
Callus culture can generally be carried out in the dark. Photoautotrophic culture in vitro, organ development and plant formation all need light. The range of light intensity is 300-1000-3000lx, generally1000-3000lx. General culture room can be designed with natural light instead of fluorescent lamp. Studies have shown that different wavelengths of colored light have certain effects on callus formation and organ differentiation, and vary with plant species and organs. The illumination time depends on plant characteristics and experimental purposes, and can be continuous illumination or periodic illumination 10- 16 hours. The formation of tubers and other storage organs may take a long dark period. When studying floral organogenesis, it is necessary to control the illumination time according to plant characteristics.
Generally, air components cannot be considered in solid culture medium, although ethylene will be produced during the culture process. Liquid culture needs ventilation. Cell suspension culture is generally ventilated by shaking method. In organ differentiation, rotating bed can be used to make tissues grow at intervals in liquid culture medium. Small volume static shallow liquid culture needs to shorten the culture time and subculture once every four weeks. In order to preserve propagation materials, solid culture should still be used.
A survey of in vitro regeneration of Chinese medicinal plants with intransitive verbs
In the cultivation and production of medicinal plants, many precious medicinal plants have a long growth cycle, and it takes a long time to adopt conventional breeding, such as ginseng and coptis root. Some medicinal plants, such as Fritillaria, Crocus sativus, etc. The propagation coefficient is small and the seed consumption is large. Some are degraded by viruses, which affect the yield and quality, such as Rehmannia glutinosa and Pseudostellaria heterophylla. Some expensive wild medicinal plants, such as Polygonum nigrum and Dendrobium huoshanense, have few resources and slow growth. Some imported southern medicinal materials, such as frankincense and dragon's blood, were all affected by seedling shortage. In order to apply plant biotechnology to variety improvement, rapid propagation, germplasm preservation and genetic engineering research of medicinal plants, the research and application of in vitro regeneration of medicinal plants have attracted increasing attention.
At present, there are more than 100 kinds of medicinal plants in vitro, such as the nine lions of Acanthaceae. Actinidiaceae, Macaca. Actinidia chinensis var. pilosa. Hispide, Catharanthus roseus of Apocynaceae, Apocynum indicum, Rauvolfia, Acanthopanax senticosus, Ginseng, Panax japonicus, Panax quinquefolium, Panax quinquefolium, Panax notoginseng, Panax notoginseng, Ascidium indicum.