What is the application prospect of modern biotechnology in medicinal plant industry?

Medicinal plants have attracted worldwide attention because of their unique curative effects and less toxic and side effects, and their demand is increasing day by day. The effective components of traditional Chinese medicine are the material basis of its exact clinical efficacy. The existence (authenticity) and quantity (quality) of effective substances are the core parts of their quality. However, the development of medicinal plant industry is hindered by the complex components, unclear effective substances, different sources, different preparation techniques, difficult quality control, and the problem of counterfeiting of plant drugs is also very prominent. At the same time, due to the destruction of the natural environment and people's long-term over-exploitation and abuse, many original medicinal plant resources have been threatened with exhaustion, and wild resources are far from meeting people's needs.

Therefore, in order to meet the country's demand for ensuring and improving the quality of important medicinal plants, as well as the severe situation of shortage and serious quality degradation of wild resources of traditional Chinese medicine, it is necessary to better develop and utilize medicinal plant resources, improve and improve their quality, increase industrial productivity, increase the output of medicinal substances to meet market demand, and at the same time increase the protection of wild resources to make them better and sustainable for human use.

There are many problems in the development and utilization of medicinal plants, such as unclear species and quantity, difficult preservation of germplasm resources, serious destruction of wild resources, and declining quality of artificially cultivated varieties, which seriously restrict industrial development. How to effectively classify and identify medicinal plant resources, protect endangered and scarce resources, repair and regenerate, prevent degradation and extinction, thus ensuring the sustainable supply of medicinal materials and improving the quality of medicinal materials is the most urgent task in the field of modern medicinal plant development, and also the key measure to realize the modernization and internationalization of Chinese medicine industry.

The traditional classification and identification methods of medicinal plants are mainly based on the sensory characteristics of medicinal materials, such as color, shape, smell, taste, texture and so on. Their shortcomings are that the grasp of these characteristics varies from person to person, which is subjective, emphasizes the accumulation of experience and is not accurate, and has not been widely recognized by international peers. Therefore, how to reveal the differences between germplasm at the molecular level has become a very concerned issue for researchers. Modern biotechnology has opened up a new way for germplasm identification of medicinal plants.

Based on DNA molecular differences, DNA molecular markers generally have many advantages, such as rapidity, trace, strong specificity, good stability, intuitive and reliable results, and are not affected by growth stages, testing sites, environmental conditions, storage and other factors [1].

The application of DNA molecular markers in the research of medicinal plants first began in Japan. The earliest and most widely used is the identification of authenticity and variety classification of medicinal materials. Early DNA molecular marker techniques include restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD). With the development of biotechnology, more efficient and rapid DNA molecular markers have appeared one after another, such as amplified restriction fragment polymorphism (AFLP), simple sequence repeat (SSR), sequence characteristic amplification region (SCAR), simple sequence repeat (ISSR), sequence-related amplification polymorphism (SRAP), single strand conformation polymorphism (SSCP) and so on.

Zhongxing University in Taiwan Province Province used RFLP technology to accurately identify Sophora flavescens and its adulterants [2], and the study of Pueraria lobata by Ji Baoyu and others [3] showed that RAPD could be used as a key technology for screening and identifying germplasm resources; Hao Gangping [4] successfully applied AFLP technology to the authentic identification of Salvia miltiorrhiza; Pan Qingping and others [5] used ISSR technology to provide a molecular basis for the identification of commercial medicinal materials of Polygonatum odoratum. Therefore, DNA molecular marker technology is an effective method to identify medicinal plants.

Table 1 compares several commonly used DNA molecular marker technologies, and each method has its own advantages and limitations. In practical application, it can be selected according to the experimental purpose, materials and experimental conditions.

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DNA barcode uses one or several standard DNA sequences as markers to identify species, which is similar to barcode scanning in supermarkets to distinguish different commodities. It has the advantages of rapidity, simplicity, accuracy, reliability and automation.

Chen et al. [6] studied 6 600 samples of 4 800 kinds of medicinal plants and their relatives, which proved that it played a key role in the identification of medicinal plants. Liu Meizi and others [7] found that ITS2 sequence has the highest success rate in identifying nine common Artemisia plants collected in different regions, and can be used as a potential barcode for identifying Artemisia plants; Cui Zhiwei and others [8] used ITS2 and psbA-tmH to effectively distinguish different varieties of honeysuckle, indicating that ITS2 and psbA-tmH can be used as the dominant bar code combination to identify different varieties of honeysuckle; The identification of Rubiaceae plants in Hainan by Li et al. [9] showed that the sequence can be used for rapid identification of Rubiaceae plants in Hainan.

In recent years, the newly developed single nucleotide polymorphism (SNP) marker technology can only detect individual base differences or only detect tiny nucleotide differences such as insertions and deletions between different alleles, thus distinguishing the genetic material differences between two individuals [10]. Chen et al. [1 1] successfully identified Korean ginseng and American ginseng by SNP labeling technique combined with standard barcode sequences of matK and psbA-trnH. It is proved that SNP labeling technology based on DNA barcode can be used as an effective means to identify ginseng. SNP can be directly labeled by sequence variation, and its detection and analysis method replaces traditional gel electrophoresis with cutting-edge DNA chip technology, which is considered to be the most promising genetic marker.

DNA barcode technology can identify species quickly and effectively, and has become the mainstream method for classification and identification of medicinal plant germplasm resources.

Traditional medicinal plant germplasm resources are generally preserved in seed bank, which has some shortcomings, such as large space occupation, limited preservation types, troublesome management, easy infection and mildew, and short preservation time. Using biotechnology for in vitro preservation can solve the above problems well. After being revived, the preserved materials can rapidly propagate a large number of seedlings in a short time, which is not affected by the natural environment, saves time and labor, reduces the deterioration frequency, and achieves the purpose of being used at any time and preserving high-quality germplasm resources for a long time [12].

According to the totipotency of plant cells, explants were inoculated on MS semi-solid medium or liquid medium filter paper, then cultured at room temperature or low temperature, and subcultured in time [13]. Tissue culture preservation methods are divided into room temperature subculture preservation method and slow growth preservation method. Tissue culture preservation method can effectively expand the reproduction of medicinal plants, alleviate the situation that wild resources can not meet the market demand, and is also an effective means to protect endangered rare medicinal plants.

(1) room temperature subculture method: at room temperature, explants are subcultured at regular intervals to achieve the purpose of preserving germplasm, and can be propagated at any time if necessary [14]. Using this method to preserve the germplasm resources of Dendrobium candidum has achieved certain results, and the rapid propagation system of Dendrobium candidum has been successfully established [13]. This method has a short interval and needs continuous subculture.

(2) Slow growth preservation method: by adjusting the culture conditions, the growth of explants can be inhibited without causing death, and the consumption of nutrients can be reduced as much as possible, thus prolonging the subculture time as much as possible. The main measures are reducing temperature, adjusting osmotic pressure, controlling nutrition level, using growth inhibitors or retarders, controlling the proportion of nutrients in culture medium and adjusting illumination [13]. In vitro culture of honeysuckle. The optimum conditions for in vitro preservation of Lonicera japonica Thunb were studied. Germplasm resources exploration [15].

This method can preserve plant germplasm for a long time without subculture, so the genetic variation is relatively small. At present, the most mature cryopreservation method is vitrification. The plant culture is treated with high concentration compound protective agent for a certain period of time, and then quickly frozen with liquid nitrogen, so that the solution inside and outside the plant cells can be solidified into an amorphous vitrified state, and the mechanical damage to the cells caused by ice crystals in the process of formation and melting can be avoided. In this state, the metabolism and growth activities in plant cells almost completely stop, while the morphogenetic potential of biomaterials is maintained [16], which is an effective method to preserve germplasm.

The exploratory study on cryopreservation of American ginseng suspension cells proved the feasibility of this method [17]; In vitro preservation of yam germplasm can be realized by embedding vitrification cryopreservation technology [18]; By vitrifying the endangered plant cornflower, the freezing procedure of its stem tip was successfully realized [19].

The drop vitrification method developed on the basis of drop freezing method and vitrification method has the advantages of high survival rate, high regeneration rate, wide adaptability, large capacity and simple operation [20]. There are few reports on the application of droplet vitrification in the preservation of medicinal plant germplasm, but its application in other plants can be used as a reference.

Artificial seeds are embryoids produced by tissue culture, wrapped in capsules that can provide nutrition, and then coated with a protective film to form a structure similar to natural seeds. Artificial seeds have the advantages of no seasonal restriction, good nutrition supply and disease resistance, maintaining the genetic characteristics of excellent varieties, and being convenient for storage and transportation. This is of great significance to the preservation of endangered medicinal plant germplasm resources.

For a long time, many rare medicinal plants have been in short supply because of their unique therapeutic, health care and beauty effects, and the prices of raw materials and medicinal materials have continued to rise, which has greatly stimulated people's predatory exploitation and acquisition of wild rare medicinal plant resources and caused devastating damage to resources. In addition, changes in the natural environment such as global warming also make many areas no longer suitable for the growth of native medicinal plants. Due to various reasons, many rare medicinal plant resources are on the verge of extinction.

Artificial seed technology is of great significance for the preservation of endangered plant germplasm resources. However, this technique depends on plant tissue culture and is not suitable for plants that are difficult to cultivate.

The use of organ culture, plant stem cell culture and other biotechnology methods [30] can also realize the sustainable utilization of medicinal plant resources. In addition, the application of DNA molecular marker technology in the identification of germplasm resources, the determination of protected objects and in-situ protected units, the sampling strategy and effect evaluation of ex-situ protection, and the scientific explanation of endangered reasons can also provide reference for the formulation of rare and endangered medicinal plant resources protection strategies and measures.

The application of biotechnology can not only better develop and utilize medicinal plant resources, but also protect them to the maximum extent. Biotechnology will play a great role in promoting Chinese medicine, a cultural treasure of China, to the world.

There are some problems in the cultivation of medicinal plants, such as virus infection leading to quality decline and lack of scientific quality evaluation system. Therefore, cultivating virus-free high-quality medicinal plants, establishing a scientific quality evaluation system and creating new varieties of medicinal plants with better quality than natural varieties are the hot topics in the research and development of medicinal plants.

Plant virus is called "plant cancer" because it interferes with the metabolism of the host, reduces the yield and quality, and even leads to death. Especially asexual crops, it is easy to accumulate a variety of viruses after planting for many years, thus causing quality decline [3 1]. Plant virus has become one of the main factors that reduce the yield and quality of crops.

At present, there are nearly a thousand kinds of plant viruses discovered by human beings. Viruses infected by medicinal plants mainly include cucumber mosaic virus, taro mosaic virus, soybean mosaic virus and tobacco mosaic virus [32]. The global economic loss caused by plant viruses is about $60 billion every year. Therefore, strengthening the research on virus-free technology of medicinal plants and taking scientific and effective control measures are the key and difficult points to enhance and improve the quality of medicinal plants at present and in the future [33]. Table 2 summarizes the application progress of several detoxification technologies in recent years.

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In addition to the common detoxification methods listed in Table 2, anther or pollen culture and nucellar embryo culture can also play a role in detoxification to some extent.

New plant varieties refer to those developed from wild plants discovered by artificial cultivation, introduction and domestication, or those transformed by biotechnology, which have novelty, specificity, consistency, stability and certainty in their names [45].

New varieties of traditional medicinal plants are usually created by cross breeding and other methods. Such as Platycodon grandiflorum [46] and Salvia miltiorrhiza [47], have been studied in cross breeding or heterosis utilization, and new varieties have been created. However, there are some shortcomings in this method, such as the inability to produce new genes, the separation of traits in hybrid offspring, the slow breeding process and the complicated process. Modern biotechnology has opened up new ways to create new varieties.

Mutation breeding refers to the breeding technology that uses various physical, chemical and biological factors to induce gene mutation in plants, promote gene recombination, expand genetic variation, and then select new varieties according to breeding objectives [48].

Ion beam implantation mutation technology is a technology that uses charged ion beams with controllable, clustered and directional implantation range to obtain higher mutation rate and wider mutation spectrum under the condition of less damage to cells, thus breeding new varieties. After the seeds of perilla frutescens were irradiated with different doses of 12C6+ ion beam, some chromosome aberrations were produced, which provided more possibilities for screening excellent mutant varieties [49]. It shows that the low dose 12C6+ heavy ion beam has great potential in the irradiation mutation of new types and the cultivation of excellent new varieties.

Space breeding is a new breeding technology that uses special space environment to mutate biological genes and select new varieties and materials. Its greatest advantage is that it can obtain rare gene resources that are difficult to obtain by conventional breeding and conventional mutation breeding methods in a short time, so that plants can obtain new genes, new types and new characters [50]. It is reported that the "Tian Dan" space salvia miltiorrhiza was successfully cultivated by Tasly Group. In 2008, the group carried Salvia miltiorrhiza seeds into space and returned to the ground to cultivate and breed "Tiandan No.1" space Salvia miltiorrhiza, and its effective components were significantly higher than the control.

Mutation breeding can improve the mutation rate and obtain more mutation types in a short time, but the direction of induced mutation is difficult to control, and most mutations are harmful. To obtain more excellent traits, it is necessary to increase the number of mutations. So the workload of screening is quite large.

Ploidy breeding includes haploid breeding and polyploid breeding. Haploid breeding is a new breeding method formed by combining haploid culture technology with breeding practice, which has the advantages of overcoming the sterility of distant hybrids, improving breeding efficiency and selection efficiency, and quickly obtaining pure lines [48]. Using the developing anther of Isatis indigotica as explant, haploid plants were obtained by culture and haploid induction. After chromosome doubling, homozygous diploids can appear in the first generation, and the characters are not separated, and the phenotype is neat and consistent, which can significantly shorten the breeding cycle [5 1].

Polyploidy refers to individuals, populations and species with 3n or more chromosomes. Polyploid plants have stronger adaptability and plasticity. Polyploidy of medicinal plants has the characteristics of strong stress resistance, high biological yield, low fertility and increasing the content of some medicinal components. The most commonly used polyploid inducer is colchicine. Colchicine induction method can be divided into in vivo treatment multiplication method and in vitro treatment multiplication method [52]. The methods of doubling the living body include drop method, immersion method, agar method, spray method, injection method and so on. In vitro doubling method, that is, tissue culture mutagenesis method, is a method of treating in vitro parts of plants with colchicine and then conducting tissue culture, or doubling chromosomes during tissue culture. Colchicine and agar are mixed to make semi-solid, and then applied to the terminal buds or axillary buds of plants to induce polyploidy. This method has been successful in medicinal plants such as Platycodon grandiflorum [53] and Lonicera japonica [54]. Dipping the shoots and stems of Rehmannia glutinosa with appropriate concentration of colchicine solution can also induce tetraploid plants, but the induction rate is not high [55]. Dendrobium protocorms were inoculated on the medium containing 0.075% colchicine, and a high induction rate was obtained [56]. The method of in vitro culture has also achieved success in inducing chromosome doubling of Echinacea purpurea [57]. In addition, physical factors such as temperature mutation, mechanical trauma, ionizing radiation, non-ionizing radiation, centrifugal force, and biological methods such as sexual hybridization, endosperm culture, somatic hybridization, somatic cell clonal variation, etc. can also be used to induce chromosome doubling.

Although artificially induced polyploid has high frequency, quick effect and simple method, it can produce huge economic benefits in production and breeding practice. But at the same time, there are also some problems, such as toxicity, serious chimera, reduced fertility, long time-consuming stabilization and high breeding cost [58]. Therefore, more and more extensive research is needed in polyploid breeding of medicinal plants.

Transgenic breeding, also known as genetic engineering breeding, is to recombine foreign genes into the genome of recipient cells, make them express specifically according to people's wishes, and obtain a new genetically engineered variety with stable expression through screening. Its main advantage is that it can overcome the incompatibility obstacle of distant hybridization of plants, expand the hybridization range of species, accelerate the mutation speed, and provide the possibility for the directional creation of organisms [59]. It can play an important role in creating new varieties and developing high-quality, high-yield and efficient crops with various resistances. At present, the main methods of plant transgene are Agrobacterium-mediated method, polyethylene glycol-mediated method, gene gun method, pollen tube passage method, electric stimulation perforation method, microinjection method and ultrasonic introduction method.

Agrobacterium-mediated gene transformation is the most widely used, mature and ideal method. Firstly, the plant expression vector connected with the target gene was transformed into Agrobacterium tumefaciens, then the plant was infected with Agrobacterium tumefaciens, and the target gene on the vector was introduced and integrated into the plant genome, so as to complete the transformation of the target gene and obtain transgenic plants. It can be used to transform large DNA fragments, with stable heredity and good repeatability, and it is not easy to produce gene silencing, but it is only sensitive to dicotyledonous plants. This method has been successfully applied to Salvia miltiorrhiza [60], Orychophragmus violaceus and Isatis indigotica [6 1], Astragalus membranaceus [62], Artemisia annua [63] and other materials.

Gene bombardment is another widely used genetic transformation technology after Agrobacterium-mediated transformation. Using gunpowder explosion or other driving force, metal particles loaded with exogenous DNA are injected into target cells or tissues in a vacuum chamber, thus introducing exogenous genes. This method has no host restriction, simple operation and short transformation time, but the transformation rate is relatively low, and the integration mechanism of foreign DNA is not clear. In recent years, new achievements have been made in medicinal plants such as garlic [64] and white clover [65].

Pollen tube pathway method is to introduce exogenous DNA into fertilized eggs by pollen tube pathway that germinates when pollinated plants bloom, and then integrate the target gene into the genome of recipient plants, so that they naturally develop into seeds and form transgenic plants. This method is simple and the breeding time is short. A new transgenic variety was obtained by transforming Dendrobium candidum [66] and Ricinus communis [67] with this method. Table 3 compares the characteristics of several main plant gene transformation methods.

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Although the application of transgene in medicinal plants has achieved quite good results, its safety has always been a hot topic of debate. Therefore, we should be cautious about transgenic medicinal plants and must conduct more systematic and in-depth research.

Secondary metabolic engineering is to transform the biochemical reaction pathway of secondary metabolites or introduce new biochemical reactions through DNA recombination technology, so as to directly improve or inhibit the synthesis of one or some specific secondary metabolites and improve cell performance. With the discovery of biosynthetic pathway of secondary metabolites in medicinal plants, it has become a research hotspot to apply metabolic engineering technology to genetically improve the secondary metabolic pathway of plants in order to greatly increase the amount of target products.

Since American scholar Bailey put forward the concept of secondary metabolic engineering in 199 1, the application of secondary metabolic engineering technology has been reported a lot. The most classic research in the early days was to realize provitamin A (β-carotene) in rice endosperm from scratch with this technology [68]. In recent years, reports on the application of this technology in medicinal plants have emerged endlessly. The contents of various effective substances in medicinal plants are often very low, which can not meet people's needs. Their number in plants can be steadily increased by secondary metabolic engineering. The application progress of secondary metabolic engineering in several important medicinal substances in medicinal plants was briefly introduced.

Phenylpropanoid is an important natural organic compound produced by plants in the long-term natural selection process. They generally have many biological activities such as antibacterial, antiviral, antitumor, anti-free radical, anti-inflammatory and analgesic, liver protection, cardiovascular system protection and so on, and are very important natural medicinal substances.