Abstract Polygonatum is a traditional and precious Chinese medicine in China, as well as a traditional medicinal and food homologous material. It has a long history of cultivation and medicinal use. It not only has the effects of boosting qi and nourishing yin, nourishing the spleen and lungs, and tonifying the kidney, but also application value in food, health products, ornamental, cosmetics, etc. There are many varieties of Polygonatum resources all over China, which are easy to be mixed. There are three species of medicinal Polygonatum recorded in the 2015 edition of Pharmacopeia of the People’s Republic of China. In order to sustainably use and fully develop Polygonatum medicinal resources, we conducted textual research on relevant data and research on the distribution of Polygonatum resources, germplasm identification, genetic diversity and breeding research, so as to provide a reference for the selection and breeding of polygonatum, artificial cultivation and the development and utilization of potential Polygonatum resources and for the solution of the contradiction between supply and demand in the Polygonatum market.
　　Key words Polygonatum; Germplasm identification; New resources; Genetic diversity; Breeding
　　Received: May 30, 2021  Accepted: July 29, 2021
　　Supported by Central Major Expenditure Increase or Decrease Program (2060302); Special Fund for Guiding Local Science and Technology Development by the Central Government (2018SZYD0003); Sichuan Provincial Science and Technology Department Talent Project (2016RZ0061); Sichuan Traditional Chinese Medicine Resources Sustainable Development Strategy Research Project (2016ZR0069).
　　Kuoshu CUI (1987-), female, P. R. China, PhD, senior agronomist, devoted to research about agricultural technology extension.
　　*Corresponding author. E-mail: xiaote1986@163.com; mssiywy@sicau.edu.cn.
　　 Polygonatum sibiricum Red., P. cyrtonema Hua and P. Kingianum Coll. Et Hemsl. of Polygonatum in Liliaceae are the medicinal varieties specified in the 2015 edition of Pharmacopoeia of the People’s Republic of China. Its dry rhizomes are sweet in taste, neutral in nature, distributed to the spleen, lung and kidney meridians, and have the effects of boosting qi and nourishing yin, nourishing the spleen and lungs, and tonifying the kidney［1］. Studies have reported that Polygonatum has the effects of lowering blood sugar and blood lipids, resisting oxidation, resisting tumor, regulating immunity, inhibiting osteoporosis, improving learning and memory, inhibiting Alzheimer’s, protecting cardiomyocytes, protecting liver, and resisting bacteria, inflammation and depression. Clinically, it is used to treat atherosclerosis, myopia, diabetic complications and other diseases. Its main components include Polygonatum polysaccharides, steroidal saponins, alkaloids and flavonoids［2-4］. Meanwhile, Polygonatum, as a medicinal and food homologous product, has high medicinal value and important research significance. Its application in medicine, health care, cosmetics, food and other fields is expanding, and the demand is also increasing. Polygonatum has many varieties and strong adaptability. They are widely distributed in Yunnan, Northeast, North China, Northwest, East China, Central South, Sichuan, Guizhou and other regions of China［5］. Most of them are cultivated varieties, while the prescribed varieties of Polygonatum for medicinal purposes only account for 7.5% of the total varieties of Polygonatum, and mainly, three varieties, i.e., P. sibiricum, P. cyrtonema Hua and P. kingianum. In order to better develop and utilize Polygonatum resources, it is necessary to sort out relevant researches on Polygonatum germplasm, find out the status quo of Polygonatum germplasm resources, and provide basis for the correct use, standardized identification and evaluation, rational development and utilization and zoning planting of Polygonatum germplasm. In this study, the regional distribution and germplasm resources, trait microscopy, composition and molecular identification, genetic diversity, reproduction and planting and tissue culture of Polygonatum were investigated and analyzed. 　　Polygonatum Resource Distribution
　　Geographical distribution
　　The Flora of China records that Polygonatum plants are widely distributed in the northern temperate zone. There are 31 species in China, including four Polygonatum odoratum varieties, and the distribution of the remaining 27 Polygonatum species［6］ is shown in Table 1. The environments under shrubs or forests or hillside shades and wet grass slopes at an altitude of 500-3 600 m are suitable for the growth of wild Polygonatum. Polygonatum is especially suitable to grow in loose and fertile clay loam or sandy loam in a warm and humid damp place. Because of their strong adaptability to climate, especially cold climate (suitable soil temperature is 16-20 ℃), relatively large growth span, especially P. cirrhifolium, P. cyrtonema Hua, P. punctatum Royle ex Kunth and P. verticillatum (L.) All. which are more suitable in environments with a higher altitude, and not strict requirements for pH value (5.2-6.5 is the best), they are widely distributed and easier to be artificially cultivated［7-9］.
　　Polygonatum has many plants and is widely distributed, and new species have been discovered in recent years, while the classification of Polygonatum is a very difficult problem［10］. In 2018, Jiao’s research showed that Polygonatum plants are mainly distributed in the narrow and long region from Japan to the Himalayas. It is distributed in northern temperate regions such as North America, Northern Europe, East Asia, Eastern Europe, Southeast Asia, and Central Asia.  There are more than 80 species of Polygonatum in the world, and there are 47 species in China, which are distributed in 31 provincial administrative regions. Polygonatum in China is originated in Yunnan, Chongqing, Sichuan and other regions［11］.
　　Germplasm resources
　　During the Southern and Northern Dynasties, the Polygonatum plant group with tubercular rhizomes was the source of authentic medicinal Polygonatum, which was described as "big knots and unevenness"［12］. P. verticillatum appeared in the Tang Dynasty as a genuine medicinal product, which was described as "but those with opposite leaves are P. verticillatum medicine, while those with non-opposite leaves is Pianjing"［13］. In the Song, Yuan and Ming Dynasties, the authentic Polygonatum was described as "those growing in Songshan, Maoshan are better... pairwise opposing"［14］, "The leaves are pairwise and opposite...the ones with opposite leaves are the right Polygonatum, which is superior in efficacy"［15］, and "Those who are born with leaning leaves are Pianjing, the function of which is not as good as that of the right Polygonatum" and the mainstream medicinal species are still the group with whorled phyllotaxis. After the Qing Dynasty, in addition to the whorled-leaf group, the alternate-leaf Polygonatum group also joined the ranks of authentic medicinal products, and its medicinal resources were gradually expanded to adapt to the ever-increasing consumption of Polygonatum. During the period of the Republic of China, with the decrease of whorled-leaf Polygonatum resources, P. cyrtonema Hua with alternate leaves was the mainstream medicinal variety［17］. P. sibiricum Red., P. cyrtonema Hua and P. Kingianum Coll. Et Hemsl. were listed as a fine-quality Polygonatum varieties by the 2015 edition of Pharmacopeia of the People’s Republic of China, and the medicinal part is their dried rhizomes. In some areas, the P. verticillatum (L.) All.）, P. punctatum, P. zanlanscianense, P. filipes, P. roseum and P. cirrhifolium are also used for medicinal purposes, while it has become a habit that Polygonatum plants with bitter rhizomes are not used as medicines［7,18］. According to statistics, there are 87 species of Polygonatum in the world, including 9 varieties and 8 hybrids. There are 47 species in China, distributed all over the country［11］. 　　Polygonatum Germplasm Identification
　　Character and microstructure identification
　　At present, there are many descriptions of the properties and microstructures of the three kinds of medicinal Polygonatum. The rhizomes of P. kingianum are thick, fleshy nodular masses having a light yellow to light brown surface with wrinkles, fibrous root marks, links and disc-shaped lateral stem marks. The lateral stem marks are raised in the middle, and the surroundings are concave downwards. The texture is hard and tough, not easy to break. The cutin-like cross-section is light brown to light yellow, the outer wall of epidermal cells is thick, and the parenchyma is scattered with many large mucous cells, containing calcium oxalate needle crystal bundles. The vascular bundles are scattered, mostly amphivasal. The rhizomes of P. sibiricum are mostly branched, slightly conical or curved cylindrical and nodular, and have light yellow to yellow-gray surface, with longitudinal wrinkles and round stem marks, which are translucent, and there are many ectophloic vascular bundles. The rhizomes of P. cyrtonema Hua are in the shape of long nodules, varying in length, and consist of a constant number of connected massive nodules. The surface is gray-yellow or yellow, rough, and the stem marks are disc-shaped, and the vascular bundles are ectophloic mostly［1］. The medicinal material of Polygonatum is firm, and the cross-section is cutin-like. It has faint smell and tastes sweet, and is sticky when being chewed.
　　Cheng et al.［19］ found that the polysaccharides of Polygonatum plants and the needle crystal bundles that resemble columnar crystal bundles are distributed in mucous cells, and except that volatile oil and saponins are also distributed in the basic tissues of mucous cells, different Polygonatum species have different arrangement of vessel molecules in the degree of tightness. The vascular bundles are mainly three structural types: ectophloic, amphivasal and incompletely amphivasal. Zhou et al.［20］ used paraffin sectioning, chloral hydrate mounting and glycerol acetic acid mounting methods to identify P. kingianum and P. verticillatum. For P. verticillatum, mucous cells are numerous without endothelial cells, and the vessels are ring-shaped with marginal pits. As to P. kingianum, mucous cells are sparse, and have Casparian dots and a row of square-like endothelial cells, and the vessels are trapezoidal and threaded［20］.
　　Ingredient identification 　　The effective ingredients of Polygonatum mainly include Polygonatum polysaccharides (such as Polygonatum polysaccharide, glucomannan, etc.), Polygonatum steroidal saponins (such as diosgenin, sarsasapogenin, Polygonatum saponins, etc.), alkaloids, flavonoids and anthraquinones, triterpene saponins, phytosterols, volatile oils, amino acids and a variety of trace elements, etc., which exist in crude Polygonatum polysaccharides, Polygonatum methanol extract, Polygonatum ethanol extract, Polygonatum decoction, Polygonatum water extract, etc. Among them, Polygonatum polysaccharides are the main active ingredients and have a variety of pharmacological effects［21］. Pharmacopoeia of the People’s Republic of China uses soluble Polygonatum extract and Polygonatum polysaccharide contents as indicators to test the quality of Polygonatum medicinal materials. Specifically, the content of soluble extract of Polygonatum should not be less than 45% (solvent: dilute ethanol), and when using Polygonatum polysaccharide content as an indicator component, ultraviolet-visible spectrophotometric method is used to determine the polysaccharide content of Polygonatum, which is required to be not less than 7% (calculated by anhydrous glucose).
　　The total sugar content of Polygonatum medicinal materials is higher, all above 60%. Different production areas and different varieties have an impact on the sugar content of Polygonatum. Generally speaking, the polysaccharide content of the original medicinal material is higher, and the free sugar content of decoction pieces is higher. P. cyrtonema, P. filipes and P. verticillatum all have higher polysaccharide content; P. cirrhifolium, P. cyrtonema, P. kingianum, P. filipes and P. verticillatum have higher content of total steroidal saponins; and some bitter-tasting Polygonatum species cannot be used for medicine, but if their polysaccharide content is high, they can still be used for the extraction of Polygonatum polysaccharides. In-depth research should be carried out on Polygonatum varieties with high content of active ingredients, so as to extract their active ingredients or target ingredients and reduce the pressure of market demand. For example, P. filipes polysaccharides have an obvious immune effect, and its active ingredients can be extracted to reduce the demand for medicinal Polygonatum in the field of medicine［10］.
　　Kuoshu CUI et al. Research Progress of Polygonatum Germplasm Resources in China
　　Molecular identification 　　Due to the effects of soil, climate, moisture and other growth environment and development conditions on the growth of Polygonatum, it requires a solid foundation and rich experience to identify the characters of Polygonatum. Therefore, it is relatively difficult to identify the quality of Polygonatum through the characters of medicinal materials. With the development of science and technology, the application scope of molecular biology continues to expand, and the modernized research of traditional Chinese medicine has begun to enter a new milestone. The modernized research of Polygonatum is relatively easier, more authentic and more reliable. Among them, molecular biology methods, such as inter-simple sequence repeats (hereinafter referred to as ISSR) method, simple sequence repeats (hereinafter referred to as SSR) method, restriction fragment length polymorphism (hereinafter referred to as RFLP) method, random amplification polymorphism DNA (hereinafter referred to as RAPD) method and start codon targeted polymorphism (hereinafter referred to as SCoT, which belongs to the emerging DNA molecular marker) method have been applied to the research of germplasm identification, resource protection and genetic diversity of Polygonatum. The earliest DNA labeling technology developed into the RFLP technology. Polymerase chain reaction (hereinafter referred to as PCR), which is a molecular biology technology based on molecular labeling technology used to amplify specific DNA fragments, includes RADP technology, SSR technology, etc. ISSR is developed on the basis of SSR technology, and because of its simplicity and ease of standardized operation, it is more commonly used. SCoT method makes up for the shortcomings of the previous methods, with good repeatability, high versatility and polymorphism, more abundant information obtained and convenient operation［22-23］, and it can be used in the classification, identification and cultivation of fine varieties of Polygonatum.
　　Zhou et al.［24］ used the ISSR method to identify P. sibiricum, P. cyrtonema and P. cirrhifolium from different origins, and the results showed that the ISSR method is effective and reliable in the identification of P. sibiricum and P. cirrhifolium. Yang et al.［25］ used ISSR technology to analyze the inter-germplasm differences and inter-species genetic relationships of five Polygonatum plant samples from four regions including Wuyi Mountain. The results showed that the Polygonatum germplasm resources of Wuyi Mountain and its surrounding areas were significantly different, while the same Polygonatum species from different origins has high homology, which provides a reference for the quality control of the medicinal Polygonatum plants in this region. 　　Polygonatum Genetic Diversity
　　An important part of biological diversity is genetic diversity, and genetic diversity is the source of organism evolution and species differentiation. Intraspecific genetic diversity is the genetic diversity that scholars usually study, and the sum of the genetic information possessed by all living things on the earth is genetic diversity in a broad sense. The Bast Fiber Research Institute of Chinese Academy of Agricultural Sciences has applied for a patent on the genetic diversity research of Polygonatum germplasm resources, the identification of germplasm resources, the identification of three Polygonatum medicinal materials and the germplasm molecular breeding of Polygonatum through SSR technology［26］. Liu et al.［23］ used the SCoT technology to analyze the genetic structure of 19 Polygonatum materials from different varieties, and the results showed that Polygonatum from different origins and of different varieties had rich genetic diversity, and the genetic distance and genetic similarity of Polygonatum germplasms basically showed an inverse relationship.
　　Zhang et al.［27］ studied the wild P. sibiricum populations (produced in Dalian) and P. cyrtonema Hua using ISSR-PCR reaction system by the ISSR technology, and found that the genetic diversity of the two populations was relatively high. However, P. cyrtonema Hua had more genetic variation within species, and the degree of genetic differentiation among populations was lower than that P. sibiricum populations. Therefore, gene exchanges of P. cyrtonema Hua among populations were less restricted than P. sibiricum populations and were more moderate, and the genetic diversity of P. cyrtonema Hua populations was higher than that of P. sibiricum populations. Polygonatum has a late origin and belongs to a newly differentiated young species. Wu et al.［28］ believe that Polygonatum, which is now in the differentiation period, is vigorous and relatively active. Most of Polygonatum plants have two methods of sexual reproduction and asexual reproduction, while one of the factors that determine plant genetic diversity is the reproduction method of plants. Although the genetic diversity of sexual reproduction is higher than that of asexual reproduction, asexual reproduction can also maintain high genetic diversity. Furthermore, the distribution of Polygonatum plants is very extensive, so rich genetic diversity is the characteristic of Polygonatum germplasms.
　　Zhu et al.［29］ used the SSR technology to study 60 samples of Polygonatum of 6 species from 14 provinces. The results showed that the polymorphism information content in them was in the range of 0.031 7 to 0.731 8; when 0.26 was the genetic distance threshold, the classification showed the phenomenon of inter-species and geographical distribution crossing; both population analysis and SSR cluster analysis could classify the 60 samples into 4 categories; and Polygonatum had a large variation range, reduced inter-species differences and rich genetic diversity. It is speculated that the western region may be the origin center of Polygonatum due to its high genetic diversity. For the selection and breeding of fine Polygonatum varieties, rich genetic diversity of Polygonatum is a powerful condition. Through the selection and breeding of fine varieties, it is more likely to breed high-quality Polygonatum varieties that are easy to survive, high yielding, resistant to diseases and pests, and high in effective ingredients. Polygonatum has rich genetic diversity, so non-medicinal Polygonatum varieties may still have medicinal values similar to or different from medicinal varieties, which is worthy of in-depth study. 　　Research on Polygonatum Breeding
　　Reproduction methods
　　Polygonatum has two methods of reproduction, asexual reproduction and sexual reproduction. Rhizome asexual reproduction is the main method. Because seed reproduction requires 1 year of seedling time, some Polygonatum species (such as P. cyrtonema Hua) have a phenomenon of apomixis. Asexual reproduction is generally carried out in September-October or late March by splitting and planting rhizomes while harvesting. Rhizomes with buds were cut into several segments according to 2 to 4 nodes per segment, and the next is to wait for sprouting in spring. There is dormancy phenomenon in Polygonatum seeds during sexual reproduction. Sand storage, chemical soaking, ultrasonic treatment and other treatments are conducive to breaking dormancy. Selecting green seeds with higher living and germination rates and giving appropriate temperature and humidity facilitate seed germination［7,11,30-31］.
　　Planting method
　　For seed propagation, before sowing, wet sand and seeds are mixed in a volume ratio of 5∶1 or seeds, wood chips and plain sand were mixed in a volume ratio of 1∶1∶1, and the mixture is kept warm and wet, and watered and checked for the germination situation regularly. After being stored for about 2 months, the seeds are taken out from March to April after winter, soaked in warm water (30-40 ℃) for 20-30 min, then mixed with 2% tebuconazole or 50% carbendazim according to 0.3%-0.5% of the seed mass evenly, and finally sown［32］. During the sowing and seedling period of Polygonatum plants, the plants can be transplanted after 1 year of shading, moisturizing, heat preservation and fertilization. Polygonatum likes a humid environment and is cold-resistant, so it is generally cultivated in low-lying land or under shaded conditions. It is best to plant with loose deep soil and uniform fertility. The rhizomes or seedlings are planted according to the row spacing of 33 cm and the plant spacing of 20-30 cm, and the ditch depth is about 7 cm. Attention should be paid to drought prevention, drainage, and sufficient base fertilizer (composted fertilizer or soil and miscellaneous fertilizer) should be applied, followed by covering with a layer of fine soil. The soil should be loosened frequently while weeding (intertillage weeding), combined with pest control. Topdressing is performed once in spring and summer (mainly human and animal manure, which can be composted), and after the plants wither, soil and miscellaneous fertilizer is spread［7-8］. Liu et al.［33］ believe that the combination of hormones and layered treatments at different temperatures is beneficial to breaking the dormancy of Polygonatum seeds, shortening the breeding time, and improving the germination rate. Artificially reducing the buds during the flower bud stage of Polygonatum can reduce nutrient consumption and increase yield［34］. Artificially cultivated Polygonatum can maintain rich nutrients. Generally speaking, the output of wild Polygonatum is far less than that of cultivated Polygonatum, because the diameter of the rhizomes of cultivated Polygonatum increase significantly［35］. 　　Tissue culture
　　With the continuous increase in market demand for Polygonatum and the relative lack of wild resources, the selection and artificial cultivation of Polygonatum resources is an important way to solve the problem, and tissue culture is conducive to the realization of large-scale and standardized planting and helpful for the efficient utilization of germplasm resources, and can solve the problems of long breeding time and shortage of seedlings. However, there is little research on the tissue culture technology of Polygonatum resources, which is also not in-depth enough. Only a few high-quality Polygonatum species have been studied for tissue culture, and artificial cultivation is still mainly based on rhizome propagation or seed propagation. Rhizome-propagated varieties are easy to degenerate and have a high cost, while seeds have a dormant period during seed propagation, and the germination rate is low［35］.
　　Tai’an Taishan Academy of Forestry Sciences conducted research on sterilized Polygonatum materials (rhizomes, stem segments, single buds or leaves), which can realize rapid propagation to form rooted microrhizomes, realizing simultaneous differentiation and rooting. The developed technology has the advantages of independent establishment of a regeneration system, efficient and easy availability of propagation materials, shortened seedling time, good resistance for storage and long-distance transportation, less loss of transplanted plantlets, strong adaptability to the environment, and high survival rate, which can improve growth and reproduction efficiency［36］. Meanwhile, the academy conducted research on the induction, differentiation and culture of in-vitro ovules of Polygonatum. The in-vitro ovule tissue culture technology is shorter than conventional seed breeding and seedling raising, and has high germination rate and high harvest rate of mature seeds. It is suitable for the cultivation of fine varieties and large-scale and standardized planting［37］.
　　Conclusions and Discussion
　　Through the research on the pharmacological effects of Polygonatum plants, it has been discovered that Polygonatum polysaccharides and other ingredients have many pharmacological effects, which are of great value in the field of medicine and are worthy of continued in-depth research. The value in other fields is also constantly being developed. Polygonatum has a wide variety of plants, widely distributed and rich in genetic diversity. Polygonatum has rich potential resources. In recent years, due to changes in the natural environment and over-excavation by humans, the growth environment of wild medicinal Polygonatum has been destroyed, and the resources of wild medicinal Polygonatum are scarce, while Polygonatum is becoming more and more valuable in the fields of medicine, food, cosmetics, health care products, various additives, ornamental and research, and the market demand is increasing steadily. The selection and artificial cultivation of improved varieties is the only way to solve the problem of today’s growing market demand. In addition, protecting Polygonatum resources and finding new resources, finding alternatives to medicinal resources from other plants of Polygonatum and filling the gap of medicinal resources with high content of active ingredients can also solve the contradiction between market supply and demand. 　　References
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