Hexaploid Salvia miltiorrhiza and cultivation method thereof

Abstract

The present invention provides a hexaploid Salvia miltiorrhiza and a cultivation method, which relate to the technical field of hexaploid Salvia miltiorrhiza cultivation, including chromosome manipulation technology. The hexaploid Salvia miltiorrhiza and the cultivation method include the following steps: Sp1, collecting diploid Salvia miltiorrhiza germplasm resources, collecting 6 materials from Yunnan, Sichuan, Shaanxi, Shandong, Hebei, Tianjin and other places, and establishing a Salvia miltiorrhiza germplasm resource material planting nursery; Sp2, performing morphological observation, trait analysis, chromosome ploidy identification and genome analysis on the Salvia miltiorrhiza germplasm resources, the results of which show that the somatic cells of white Salvia miltiorrhiza and purple Salvia miltiorrhiza are all diploid, that is, 2n=2x=16; Sp3, based on the identification results, selecting diploid white Salvia miltiorrhiza and artificially inducing tetraploid Salvia miltiorrhiza using a chemical mutagen. Taking the mutagenized white Salvia miltiorrhiza seedlings for chromosome ploidy identification, screening homozygous tetraploid white Salvia miltiorrhiza, and then performing triploid hybridization of the tetraploid white Salvia miltiorrhiza and the diploid purple Salvia miltiorrhiza.

Description

Technical Field

[0001] The present invention relates to the technical field of hexaploid salvia miltiorrhiza cultivation, in particular to a hexaploid salvia miltiorrhiza and a cultivation method thereof. Background Art

[0002] Salvia miltiorrhiza Bunge, also known as red root and red root, belongs to the Lamiaceae family. It is a perennial herb used primarily for its roots and rhizomes. Salvia miltiorrhiza has two flower colors. Its original variants, S. miltiorrhiza Bunge var. miltiorrhiza and S. miltiorrhiza Bunge var. charbonnelli, both have purple flowers, while S. miltiorrhiza f. alba has white flowers. Both direct and reciprocal crosses of purple and white Salvia miltiorrhizas produce purple flowers in the F1 line. The F2 line has a 3:1 segregation of purple and white flowers, indicating that the purple and white flowers in Salvia miltiorrhiza are controlled by a pair of dominant and recessive genes, with the purple flower trait being dominant and the white flower being recessive.

[0003] Danshen (Salvia miltiorrhiza) is a traditional Chinese medicinal herb and one of the earliest and most widely used remedies in traditional Chinese medicine. It is a renowned blood-activating and stasis-removing herb, widely used clinically to treat conditions such as coronary heart disease, angina pectoris, and ischemic stroke. Over 100 Chinese herbal medicines are produced using Danshen as a raw material. Among them, Tasly's Compound Danshen Dropping Pills are not only popular in the domestic pharmaceutical market but also exported worldwide. Danshen Dropping Pills were approved for the US market as a traditional Chinese medicine in 1997 and have since entered the mainstream pharmaceutical market in 16 countries.

[0004] With the aging of my country's population and the younger age of cardiovascular and cerebrovascular diseases, cardiovascular and cerebrovascular diseases have become one of the major diseases that endanger human health. The incidence rate is increasing year by year, and the demand for therapeutic drugs is huge. Salvia miltiorrhiza plays an irreplaceable role in clinical treatment and prevention. At present, the number of domestic companies involved is increasing year by year, the demand for Salvia miltiorrhiza is increasing year by year, and the export volume is also increasing, which has doubled the value of Salvia miltiorrhiza and doubled the amount used. Therefore, it is very important and urgent to cultivate high-yield and high-quality stable varieties of Salvia miltiorrhiza. With the rapid development and progress of biotechnology, people's understanding and utilization of polyploids are also deepening. Using polyploid technology to create new plant varieties (lines) is one of the effective methods of plant genetic improvement and germplasm innovation.

[0005] Currently, the majority of Salvia miltiorrhiza cultivated in my country is triploid. Plant polyploidization is a key evolutionary pathway for higher plants. Triploids have the lowest ploidy among polyploids, but they possess the unique dual advantages of being both polyploid and hybrid, manifesting in their enormous size and irreplaceability. Triploid Salvia miltiorrhiza, with its "chromosome number of 2n = 3x = 24," not only boasts high yield and quality, but also contains medicinal ingredients that meet pharmacopoeial standards. However, triploid Salvia miltiorrhiza is highly sterile, flowering but not fruiting, and reproducing asexually through rhizomes. Long-term asexual reproduction and viral infection have led to severe germplasm degradation, weakened resistance, and a decline in medicinal yield and commercial quality. These phenomena have severely hampered the further production and development of triploid Salvia miltiorrhiza. Furthermore, there is currently no polyploid Salvia miltiorrhiza to replace the triploid varieties, emphasizing the need to develop a new hexaploid Salvia miltiorrhiza variety and its cultivation methods. Summary of the Invention

[0006] Technical problems solved

[0007] In response to the deficiencies in the prior art, the present invention provides a hexaploid Salvia miltiorrhiza and a cultivation method. Hexaploid Salvia miltiorrhiza not only maintains the dual advantages of triploid Salvia miltiorrhiza, but also solves the problem that triploid Salvia miltiorrhiza is highly sterile, only blooms but does not produce fruit, and relies on rhizomes for asexual reproduction to reproduce offspring. Long-term asexual reproduction and viral infection lead to severe germplasm degeneration, weakened resistance, and reduced medicinal material yield and commercial quality, which seriously affect the further production and development of triploid Salvia miltiorrhiza. Therefore, hexaploid Salvia miltiorrhiza has important value in maintaining and maintaining the dual advantages of triploid Salvia miltiorrhiza.

[0008] Technical Solution

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions: a hexaploid Salvia miltiorrhiza and a cultivation method, the hexaploid Salvia miltiorrhiza and the cultivation method comprising the following parts:

[0010] Sp1, collect diploid Salvia miltiorrhiza germplasm resources, collect 6 materials from Yunnan, Sichuan, Shaanxi, Shandong, Hebei, Tianjin and other places, and establish Salvia miltiorrhiza germplasm resource material planting nursery;

[0011] Sp2, morphological observation, character analysis, chromosome ploidy identification and genome analysis of Salvia miltiorrhiza germplasm resources were carried out. The results showed that the somatic cells of Salvia miltiorrhiza and Salvia miltiorrhiza were diploid, that is, 2n=2x=16;

[0012] Sp3. Based on the identification results, diploid white-flowered Salvia miltiorrhiza was selected and artificially induced into tetraploid Salvia miltiorrhiza using chemical mutagens. Chromosome ploidy was identified from the mutagenized white-flowered Salvia miltiorrhiza seedlings, and homozygous tetraploid white-flowered Salvia miltiorrhiza was screened. The tetraploid white-flowered Salvia miltiorrhiza was then triploid-hybridized with the diploid purple-flowered Salvia miltiorrhiza to obtain triploid Salvia miltiorrhiza seeds, which served as the mutagenic material for hexaploid Salvia miltiorrhiza.

[0013] Sp4. Use mercuric chloride disinfection method to sterilize triploid Salvia miltiorrhiza seeds.

[0014] Preferably, the triploid Salvia miltiorrhiza seeds are sterilized by using a mercuric chloride disinfection method.

[0015] Preferably, the mercuric chloride disinfection method is: disinfection with 75% alcohol for 40 to 60 seconds, rinsing with sterile water 3 times, disinfection with 0.1% mercuric chloride for 12 minutes, and rinsing with sterile water 3 times.

[0016] Preferably, the triploid Salvia miltiorrhiza needs to be artificially induced into a hexaploid Salvia miltiorrhiza by transferring the sterile triploid Salvia miltiorrhiza seeds into a nutrient solution supplemented with (containing) a mutagen, and germinating and doubling induction are carried out simultaneously for 3 to 6 days at 20°C ± 2°C in the dark.

[0017] Preferably, the additional mutagen nutrient solution is: Ca(NO3)2·4H2O 59.04g + KNO325.23g + MgSO4·7H2O 24.65g + KH2PO4 6.8g + Na2-EDTA 3.73g + FeSO4·7H2O2.78g + sucrose 20g, add water to 1000ml, and then prepare a mutagen nutrient solution with additional (containing) colchicine, and the final concentration of this mutagen-containing nutrient solution is 50ppm.

[0018] Preferably, the treated germinated seeds are transferred to 1 / 2MS solid culture medium and cultured at 23°C±2°C with an illumination of 1000 Lux for 12 hours per day.

[0019] Preferably, when the roots of the seedlings grow to more than 3 centimeters and the young leaves develop to more than 4 pieces, chromosome ploidy is identified plant by plant, hexaploid strains are screened out, and then genome analysis is performed.

[0020] Preferably, the steps for chromosome ploidy identification and genome analysis are as follows: young leaves or new roots are collected between 8:30 and 9:00 am and treated with a saturated aqueous solution of p-dichlorobenzene for 3 hours → hypotonic treatment with 0.075 M KCl for 15 minutes → cell wall removal: treatment with 1% mixed enzyme (pectinase + cellulase) for 40 minutes → hypotonic treatment with 0.075 M KCl for 15 minutes → fixation with a 3:1 (methanol:glacial acetic acid) fixative for 30 minutes → slide preparation → Giemsa staining → microscopic observation and analysis. Triploids, chimeras, and aneuploids are screened out, and hexaploid lines with 2n = 6x = 48 somatic cells are selected for propagation.

[0021] Preferably, the clonal propagation of hexaploid Salvia miltiorrhiza is carried out, and the leaves of hexaploid Salvia miltiorrhiza are cut and inoculated onto a culture medium of MS+6-BA2mg / L+sucrose 25g / L+activated carbon 0.5g / L+carrageenan 5g / L, pH 5.6-5.8, and cultured in the dark at 23°C±2°C until differentiation, and after clustered buds are differentiated, transferred to light culture, with a light intensity of 1000Lux, 12 hours / day.

[0022] Preferably, the hexaploid Salvia miltiorrhiza clonal propagation is carried out by dividing and expanding the propagation, and the clustered buds are differentiated into clustered seedlings and then expanded. The clustered seedlings are divided and inoculated into a culture medium of MS+6-BA 1mg / L+sucrose 20g / L+activated carbon 0.5g / L+carrageenan 6g / L, pH 5.6-5.8, and cultured at 23°C±2°C, with an illumination of 1000Lux for 12 hours / day.

[0023] Preferably, the cultivation is for rooting, and when the clustered seedlings grow to 2-4 leaves, they are transferred to a rooting medium consisting of 1 / 2MS + I BA 0.4 mg / L + sucrose 20 g / L + activated carbon 1 g / L + carrageenan 7 g / L, pH 5.8 for rooting culture.

[0024] Preferably, the seedlings are transplanted after the rooting is complete. When the new roots of the seedlings grow to 3-5 cm, the seedlings are transplanted. Before transplanting, the seedlings are hardened for 3-5 days by opening the bottle cap, and then planted one by one in a 3:1 (nutrient soil: sandy loam) mixed substrate.

[0025] Preferably, the transplanted seedlings should be protected from direct sunlight, be well ventilated, maintain a humidity of 50%-70%, and have a survival rate of over 95%.

[0026] Beneficial effects

[0027] The present invention provides a hexaploid Salvia miltiorrhiza and a cultivation method thereof, which has the following beneficial effects:

[0028] The present invention aims to provide a method for cultivating hexaploid Salvia miltiorrhiza. This method uses a chemical mutagen to first induce a diploid white Salvia miltiorrhiza into a tetraploid state. Then, utilizing a combination of polyploidization and hybridization, a triploid hybridization is performed between a diploid purple Salvia miltiorrhiza and a tetraploid white Salvia miltiorrhiza to produce triploid Salvia miltiorrhiza seeds. Subsequently, seed germination and polyploidization are performed simultaneously, forcing the triploid Salvia miltiorrhiza seeds to germinate while absorbing the mutagen. Chromosome ploidy of each muted Salvia miltiorrhiza seedling is then individually identified to exclude triploids, remove chimeras, and remove aneuploids, ultimately identifying hexaploid Salvia miltiorrhiza strains. The results showed that the mutagenesis rate of the hexaploid Salvia miltiorrhiza reached over 60%, with no chimeras or aneuploids detected. This represents the first new hexaploid Salvia miltiorrhiza germplasm to be obtained. The next step will be to analyze the characteristics of the new hexaploid Salvia miltiorrhiza germplasm. To shorten the reproductive cycle, clonal propagation of the hexaploid Salvia miltiorrhiza was used to expand the propagation of the hexaploid Salvia miltiorrhiza, increasing the propagation rate and providing sufficient material for large-scale planting and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a diagram showing chromosomes of somatic cells of the triploid Salvia miltiorrhiza of the present invention;

[0030] Figure 2 This is a diagram showing chromosomes of somatic cells of the hexaploid Salvia miltiorrhiza of the present invention;

[0031] Figure 3 This is a diagram showing the hexaploid Salvia miltiorrhiza tissue culture seedlings of the present invention;

[0032] Figure 4 A comparison of the triploid (left) and hexaploid (right) Salvia miltiorrhiza transplanted seedlings (30 days) of the present invention. DETAILED DESCRIPTION

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. Specific embodiment one:

[0035] like Figure 1-4 As shown, a hexaploid Salvia miltiorrhiza and a cultivation method thereof, the hexaploid Salvia miltiorrhiza and the cultivation method thereof include the following parts:

[0036] Sp1. Collect diploid Salvia miltiorrhiza germplasm resources, collect 6 materials from Yunnan, Sichuan, Shaanxi, Shandong, Hebei, Tianjin and other places, and establish a planting nursery for Salvia miltiorrhiza germplasm resources materials;

[0037] Sp2, morphological observation, character analysis, chromosome ploidy identification and genome analysis of Salvia miltiorrhiza germplasm resources were carried out. The results showed that the somatic cells of Salvia miltiorrhiza and Salvia miltiorrhiza were diploid, that is, 2n=2x=16;

[0038] Sp3. Based on the identification results, diploid white-flowered Salvia miltiorrhiza was selected and artificially induced into tetraploid Salvia miltiorrhiza using chemical mutagens. Chromosome ploidy was identified from the mutagenized white-flowered Salvia miltiorrhiza seedlings, and homozygous tetraploid white-flowered Salvia miltiorrhiza was screened. The tetraploid white-flowered Salvia miltiorrhiza was then triploid-hybridized with the diploid purple-flowered Salvia miltiorrhiza to obtain triploid Salvia miltiorrhiza seeds, which served as the mutagenic material for hexaploid Salvia miltiorrhiza.

[0039] Sp4. Use mercuric chloride disinfection method to sterilize triploid Salvia miltiorrhiza seeds.

[0040] The triploid Salvia miltiorrhiza seeds were sterilized using mercuric chloride disinfection method.

[0041] The disinfection method of mercuric chloride is: disinfect with 75% alcohol for 40 to 60 seconds, rinse with sterile water 3 times, disinfect with 0.1% mercuric chloride for 12 minutes, and rinse with sterile water 3 times.

[0042] Triploid Salvia miltiorrhiza needs to be artificially induced into hexaploid Salvia miltiorrhiza. Sterile triploid Salvia miltiorrhiza seeds are transferred into a nutrient solution with (or containing) a mutagen, and germination and doubling induction are carried out simultaneously for 3 to 6 days at 20°C ± 2°C in the dark.

[0043] The additional mutagen nutrient solution is: Ca(NO3)2·4H2O 59.04g + KNO3 25.23g + MgSO4·7H2O24.65g + KH2PO4 6.8g + Na2-EDTA 3.73g + FeSO4·7H2O2.78g + sucrose 20g, add water to 1000ml, and then prepare the mutagen nutrient solution with additional (containing) colchicine. The final concentration of this mutagen-containing nutrient solution is 50ppm.

[0044] The treated germinated seeds were transferred to 1 / 2MS solid culture medium and cultured at 23°C ± 2°C with an illumination of 1000 Lux for 12 hours per day.

[0045] When the roots of the seedlings grow to more than 3 centimeters and the young leaves develop to more than 4 pieces, the chromosome ploidy of each plant is identified, the hexaploid strains are screened out, and then the genome analysis is performed.

[0046] The steps for chromosome ploidy identification and genomic analysis were as follows: young leaves or roots were collected between 8:30 and 9:00 AM and treated with a saturated aqueous solution of p-dichlorobenzene for 3 hours → hypotonic treatment with 0.075 M KCl for 15 minutes → cell wall removal: treatment with a 1% enzyme mix (pectinase + cellulase) for 40 minutes → hypotonic treatment with 0.075 M KCl for 15 minutes → fixation with a 3:1 (methanol:glacial acetic acid) fixative for 30 minutes → slide preparation → Giemsa staining → microscopic observation and analysis. Triploids, mosaics, and aneuploids were eliminated, and hexaploid lines with a somatic cell ratio of 2n = 6x = 48 were selected for propagation.

[0047] To carry out clonal propagation of hexaploid Salvia miltiorrhiza, cut the leaves of hexaploid Salvia miltiorrhiza and inoculate them onto the culture medium of MS+6-BA2mg / L+sucrose 25g / L+activated carbon 0.5g / L+carrageenan 5g / L, pH 5.6-5.8, and culture them in the dark at 23℃±2℃ until differentiation. After the clustered buds are differentiated, transfer them to light culture with a light intensity of 1000Lux for 12 hours / day.

[0048] The hexaploid Salvia miltiorrhiza was cloned and propagated by dividing into bottles. After the clustered buds differentiated into clustered seedlings, they were propagated. The clustered seedlings were divided into bottles and inoculated into MS+6-BA 1mg / L+sucrose 20g / L+activated carbon 0.5g / L+carrageenan 6g / L, pH 5.6-5.8 culture medium. The culture was continued at 23℃±2℃ and 1000Lux of light for 12 hours / day.

[0049] The cultivation is for rooting. When the cluster seedlings grow to 2-4 leaves, they are transferred to a rooting medium of 1 / 2MS + IBA 0.4 mg / L + sucrose 20 g / L + activated carbon 1 g / L + carrageenan 7 g / L, pH 5.8 for rooting culture.

[0050] After the roots have grown, transplant the seedlings. When the new roots grow to 3-5 cm, transplant the seedlings. Open the bottle cap and harden the seedlings for 3-5 days before transplanting. Then plant the seedlings one by one in a 3:1 (nutrient soil: sandy loam) mixed substrate.

[0051] Transplanted seedlings should be protected from direct sunlight, and ventilation should be ensured. The humidity should be maintained at 50%-70%, and the survival rate should be above 95%. Specific embodiment two:

[0053] like Figure 1-4As shown, during the entire cultivation process, from May to October 2020, 1 sample of white-flowered Salvia miltiorrhiza and 5 samples of purple-flowered Salvia miltiorrhiza were collected from Yunnan, Sichuan, Shaanxi, Shandong, Hebei, and Tianjin, and then chromosome ploidy was identified to determine that their somatic cell chromosomes were all diploid, that is: 2n=2x=16; in December 2020, diploid white-flowered Salvia miltiorrhiza was selected for artificial tetraploid induction, and the method steps adopted the steps in Sp3 of claim 1 above; in May 2021, the artificially induced materials were transplanted, and then the ploidy of the transplanted seedlings was identified, that is: Pretreatment: At 8:30 in the morning, young leaves or new roots were taken and placed in a saturated aqueous solution of p-dichlorobenzene, and treated at 20℃±2℃ for 3 hours → Hypotonicity: 0.075M KCl for 15 minutes → Enzymatic wall removal: 1% mixed enzyme (pectinase + cellulase) treated at 25℃ for 40 minutes → Hypotonicity: 0.075M KCl 15 minutes → Fixation: 3:1 (methanol: glacial acetic acid) fixative for more than 30 minutes → specimen preparation → staining → microscopic observation and ploidy identification, screen out homozygous tetraploid plants, remove chimeras and aneuploids; from May to June 2021, carry out field transplantation of tetraploid white flower Salvia miltiorrhiza, and cultivate and manage according to conventional Salvia miltiorrhiza cultivation methods; from November to December 2021, harvest tetraploid Salvia miltiorrhiza seed roots; from March to April 2022, carry out field planting of tetraploid Salvia miltiorrhiza and diploid Salvia miltiorrhiza; from June to August 2022, carry out hybridization and breeding of triploid Salvia miltiorrhiza, and harvest triploid Salvia miltiorrhiza seeds in September; from October to December 2022, carry out artificial induction of hexaploid Salvia miltiorrhiza, take 42 triploid Salvia miltiorrhiza seeds, and carry out hexaploid Salvia miltiorrhiza Artificial induction, the method steps adopt the above-mentioned claim 1.Sp4,2-5, transfer the treated germinated seeds into 1 / 2MS solid culture medium, continue to culture at 23℃±2℃, light 1000Lux, 12 hours / day, and wait until the root system of the seedlings grows to more than 3 cm and the young leaves develop to more than 4 pieces, perform chromosome ploidy identification on each plant, screen out hexaploid strains, and then perform genome analysis; in March 2023, continue to identify hexaploid Salvia miltiorrhiza, screen out hexaploid Salvia miltiorrhiza; in April 2023, expand the hexaploid Salvia miltiorrhiza, adopt the cloning propagation method, the method steps adopt the above-mentioned claim 9, after differentiation into cluster seedlings, perform rooting culture, and transfer the cluster seedlings into 1 / 2MS+I when they grow to 2-4 leaves. BA 0.4mg / L + sucrose 20g / L + activated carbon 1g / L + carrageenan 7g / L, pH 5.8 rooting medium for rooting culture, after the rooting culture is completed, the bottle seedlings are transplanted. When the new roots of the bottle seedlings grow to 3-5 cm, the bottle seedlings are transplanted. Before transplanting, open the bottle cap to harden the seedlings for 3-5 days, and then plant the seedlings one by one in a 3:1 (nutrient soil: sandy loam) mixed substrate. Transplanted seedlings should be protected from direct sunlight, pay attention to ventilation, and maintain 50%-70% humidity. The survival rate is above 95%. At present, cloned seedlings have begun to take shape, such as Figure 3As shown, hexaploid Salvia miltiorrhiza field planting and trait analysis will be carried out in 2024 to provide excellent new hexaploid Salvia miltiorrhiza germplasm for the next step of promotion.

[0054] This method uses a nutrient solution with added (containing) mutagens to synchronize seed germination and polyploidy induction, forcing the embryo cells to absorb the mutagen in the early stages of germination, thereby interrupting the mitotic spindle and significantly increasing the polyploidy mutation rate. This is the first time that a new hexaploid Salvia miltiorrhiza germplasm has been obtained in my country. Specific embodiment three:

[0056] like Figure 1-4 As shown, when comparing the new germplasm hexaploid Salvia miltiorrhiza with the triploid Salvia miltiorrhiza, the leaf size, thickness and root system of the hexaploid Salvia miltiorrhiza reached and exceeded those of the triploid Salvia miltiorrhiza. The results showed that the hexaploid Salvia miltiorrhiza maintained and fixed the dual advantages of the triploid Salvia miltiorrhiza.

[0057] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further restrictions, an element defined by the statement "comprising a reference structure" does not exclude the presence of additional identical elements in the process, method, article, or device comprising the element.

[0058] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for cultivating hexaploid Salvia miltiorrhiza, characterized by: The cultivation method comprises the following steps: Sp1, collect diploid Salvia miltiorrhiza germplasm resources, collect 6 materials from Yunnan, Sichuan, Shaanxi, Shandong, Hebei and Tianjin, and establish a planting nursery for Salvia miltiorrhiza germplasm resources; Sp2, morphological observation, character analysis, chromosome ploidy identification and genome analysis of Salvia miltiorrhiza germplasm resources were carried out. The results showed that the somatic cells of Salvia miltiorrhiza and Salvia miltiorrhiza purpurogenum were diploid, that is, 2n=2x=16; Sp3. Based on the identification results, diploid white flower Salvia miltiorrhiza was selected and artificially induced into tetraploid Salvia miltiorrhiza using chemical mutagens; chromosome ploidy was identified on the induced white flower Salvia miltiorrhiza seedlings to screen out homozygous tetraploid white flower Salvia miltiorrhiza; then, the tetraploid white flower Salvia miltiorrhiza was triploid hybridized with the diploid purple flower Salvia miltiorrhiza to obtain triploid Salvia miltiorrhiza seeds as the mutagenic material for hexaploid Salvia miltiorrhiza; Sp4, using mercuric chloride disinfection method to sterilize triploid Salvia miltiorrhiza seeds; the mercuric chloride disinfection method is: disinfection with 75% alcohol for 40 to 60 seconds, rinse with sterile water 3 times, disinfection with 0.1% mercuric chloride for 12 minutes, and rinse with sterile water 3 times; The process of artificially inducing the triploid Salvia miltiorrhiza into hexaploid Salvia miltiorrhiza is as follows: sterile triploid Salvia miltiorrhiza seeds are transferred into a nutrient solution supplemented with a mutagen, and germination and doubling induction are carried out synchronously at 20°C±2°C in the dark for 3 to 6 days; the nutrient solution supplemented with the mutagen is: Ca(NO3)2·4H2O 59.04g+KNO325.23g+MgSO4·7H2O 24.65g+KH2PO46.8g+Na2-EDTA 3.73g+FeSO4·7H2O 2.78g+sucrose 20g, water is added to 1000ml, and then a mutagen nutrient solution supplemented with colchicine is prepared, wherein the final concentration of the mutagen nutrient solution supplemented with the mutagen is 50ppm.

2. The cultivation method according to claim 1, wherein: The germinated seeds artificially induced by hexaploid Salvia miltiorrhiza were transferred into 1 / 2MS solid culture medium and cultured at 23℃±2℃ and 1000Lux for 12 hours / day.

3. The cultivation method according to claim 2, wherein: When the roots of the seedlings grow to more than 3 centimeters and the young leaves develop to more than 4 pieces, the chromosome ploidy of each plant is identified, the hexaploid strains are screened out, and then the genome analysis is performed.

4. The cultivation method according to claim 3, wherein: The steps of chromosome ploidy identification and genome analysis are as follows: young leaves or new roots are taken between 8:30 and 9:00 in the morning, treated with a saturated aqueous solution of p-dichlorobenzene for 3 hours → hypotonic treatment with 0.075M KCl for 15 minutes → cell wall removal: treatment with 1% mixed enzyme for 40 minutes → hypotonic treatment with 0.075M KCl for 15 minutes → fixation: fixation with fixative for 30 minutes → preparation → Giemsa staining → microscopic observation and analysis; triploids, chimeras and aneuploids are screened out, and a hexaploid strain with somatic cells of 2n=6x=48 is selected for reproduction; the mixed enzyme is pectinase + cellulase; the fixative is composed of methanol and glacial acetic acid, and the methanol:glacial acetic acid ratio is 3:

1.

5. The cultivation method according to claim 4, wherein: The hexaploid Salvia miltiorrhiza is cloned and propagated by cutting the leaves of the hexaploid Salvia miltiorrhiza and inoculating them on a culture medium containing MS+6-BA 2mg / L+sucrose 25g / L+activated carbon 0.5g / L+carrageenan 5g / L, pH 5.6-5.8, and culturing them in the dark at 23°C±2°C until differentiation occurs. After clustered buds are differentiated, the culture medium is transferred to light culture, with a light intensity of 1000 Lux for 12 hours per day.

6. The cultivation method according to claim 5, wherein: The hexaploid Salvia miltiorrhiza was cloned and propagated by dividing into bottles. After the clustered buds differentiated into clustered seedlings, they were propagated. The clustered seedlings were divided into bottles and inoculated into MS+6-BA 1mg / L+sucrose 20g / L+activated carbon 0.5g / L+carrageenan 6g / L, pH 5.6-5.8 culture medium. The culture was continued at 23℃±2℃ and light 1000Lux for 12 hours / day.

7. The cultivation method according to claim 6, wherein: When the clustered seedlings grow to 2-4 leaves, they are transferred to a rooting medium consisting of 1 / 2MS + 0.4 mg / L IBA + 20 g / L sucrose + 1 g / L activated carbon + 7 g / L carrageenan, pH 5.8, for rooting culture.

8. The cultivation method according to claim 7, wherein: After the rooting and cultivation is completed, the seedlings are transplanted into the bottle. When the new roots of the seedlings grow to 3-5 centimeters, the seedlings are transplanted into the bottle. Before transplanting, the bottle caps are opened to harden the seedlings for 3-5 days, and then the seedlings are planted one by one into a mixed matrix of nutrient soil and sandy loam at a ratio of 3:

1.

9. The cultivation method according to claim 8, wherein: The transplanted seedlings should be protected from direct sunlight, ventilated, and kept at a humidity of 50%-70%, with a survival rate of over 95%.

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