High-efficiency artificial propagation method of jixiang grass

Abstract

The present application relates to the technical field of plant induced regeneration, and particularly relates to a high-efficiency artificial propagation method of jixiang grass, comprising the following steps: cutting the year-old stem with bud roots after sterilization treatment into about 0.5*0.5cm, and inoculating the stem with bud roots into culture medium A; cutting the stem with bud roots in the culture medium A to 1.5-2.0cm high with petiole, and transferring the stem with 3-4 leaf blades into culture medium B for culture for 60 days; cutting the cluster culture in the culture medium B into 1.0*1.0cm with 2-4 leaf blades reserved and cut to 1.5-2.0cm high, and transferring a single plant into culture medium C; when the test tube seedling is as high as 8-10cm, and the seedling is strong and thick, the test tube seedling is taken out and transferred into a substrate for culture, and the survival rate is 100% after 60 days. The present application realizes high-efficiency occurrence of the jixiang grass stem cluster buds, and the whole culture process can be completed only by two culture media except for the ex-plant starting culture, so that the artificial in-vitro propagation efficiency is greatly improved, the cost is low, the cycle is short, the seedling quality and survival rate are high, and technical support can be provided for factory scale growth.

Description

Highly efficient artificial propagation methods for *Lysimachia christinae* Technical Field

[0001] This invention belongs to the field of plant induced regeneration technology, and particularly relates to an efficient artificial propagation method for *Heliotropium indicum*. Background Technology

[0002] *Reineckea carnea* (Andrews) Kunth., also known as Chinese evergreen, is a monotypic perennial evergreen herb belonging to the genus *Reineckea* in the family Liliaceae. It prefers warm, humid environments, is relatively cold-hardy and shade-tolerant, and is highly adaptable. It typically grows on shady, damp mountain slopes, in valleys, or under dense forests at altitudes of 170-3200m. *Reineckea carnea* is a traditional herbal medicine of the Miao ethnic group in southwestern China, and is also widely distributed in Japan and India. In my country, it is mainly distributed in Jiangsu, Hunan, Hubei, Henan, Shaanxi, Guizhou, Sichuan, and Yunnan provinces. *Asparagus setaceus* has a bitter taste and neutral properties. The whole plant is used medicinally. It contains steroids, flavonoids, terpenes, and other chemical components, with saponins being the main active ingredient. It possesses hemolytic, anti-inflammatory, lung-moistening, and cough-relieving effects. In Miao areas, it is commonly used to treat coughs, bronchitis, and tuberculosis coughs. It can also be used to tonify the kidneys and strengthen the lower back, treat hemoptysis, and rheumatoid arthritis. Externally, it can treat bruises and sprains. The name *Asparagus setaceus* first appeared in *Compendium of Materia Medica*. *Illustrated Compendium of Plants* states: "*Asparagus setaceus* has slightly broad leaves, six relatively large flowers, blooms in winter, and is planted in pots. In autumn, it bears fruit similar to *Asparagus officinalis*, with a reddish-purple color and pointed tips." *Asparagus setaceus* has a beautiful plant shape and emerald green leaves, making it an excellent houseplant. It is evergreen year-round, provides good cover, and can be planted extensively along garden paths to beautify the environment. Its well-developed root system also makes it one of the best herbaceous plants for ecological restoration in karst desertification areas. In recent years, the commercial and medicinal value of *Asparagus setaceus* has been increasingly recognized, leading to a significant increase in demand. Traditional propagation methods for *Gynostemma pentaphyllum* mainly include seed propagation and division. Both methods suffer from problems such as long propagation cycles, high susceptibility to variation, low survival rates, weak plants, and susceptibility to viruses, falling far short of the requirements for large-scale production. Therefore, a fast and efficient method is needed to obtain a large number of genetically stable seedlings, and plant tissue culture technology is one of the best solutions for large-scale production of high-quality *Gynostemma pentaphyllum*.

[0003] Tissue culture technology not only boasts advantages such as rapid growth, short cycle, and high reproducibility, but also offers a single source of material, preserving the original superior traits of the plant and enabling the rapid production of large quantities of genetically highly consistent seedlings. The development of plant in vitro culture technology provides an effective guarantee for the supply of scarce medicinal plant resources. Faced with the gradual depletion of wild resources of *Gynostemma pentaphyllum* and the extreme shortage of seedlings needed for artificial cultivation, there is an urgent need to find a new, low-cost, short-time, high-quality, and high-survival-rate asexual reproduction method that can fix the superior traits of the parent plant to expand the propagation of *Gynostemma pentaphyllum* seedlings and facilitate the industrialized production of high-quality seedlings to meet planting demands. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of seedling shortage of *Gynostemma pentaphyllum* and avoid the problems of long growth cycle, weak and small size, variation, and low survival rate of seedlings obtained from seed. This method can lay a technical foundation for fixing the superior traits of the parent plant and developing artificial cultivation. Through this invention, high-quality *Gynostemma pentaphyllum* seedlings with consistent genotypes can be provided to meet the needs of artificial cultivation.

[0005] To solve the above technical problems, the present invention adopts the following technical solution:

[0006] The efficient artificial propagation method of *Gynostemma pentaphyllum* includes the following steps: cutting the sterilized rhizome buds into appropriate sizes, inoculating them into a culture medium to induce basal stem cluster buds, rooting, hardening off, and transplanting.

[0007] Furthermore, the highly efficient artificial propagation method for the aforementioned auspicious grass includes the following steps:

[0008] (1) Obtaining explants: Select healthy plants with good growth and no pests or diseases, and take their one-year-old root buds;

[0009] (2) Disinfect the root and stem buds from step (1);

[0010] (3) Cut the sterilized root and stem buds from step (2) into appropriate sizes and inoculate them into the following culture medium A, wherein culture medium A includes the following raw materials:

[0011] MS basic culture medium

[0012] 6-Benzylaminopurine (6-BA)

[0013] sucrose

[0014] Agar powder

[0015] By controlling the light intensity, temperature, and light duration during cultivation, sterile test-tube seedlings with a slight proliferation effect can be obtained.

[0016] (4) Development and proliferation of basal shoot clusters: The proliferating seedlings cultured in step (3) are transferred to the following culture medium B, which includes the following raw materials:

[0017] MS basic culture medium

[0018] 6-Benzylaminopurine (6-BA)

[0019] α-Naphthaleneacetic acid (NAA)

[0020] 2,4-Dichlorophenoxyacetic acid (2,4-D)

[0021] Uniconazole (S-3307)

[0022] sucrose

[0023] Agar powder

[0024] By controlling the light intensity, temperature, and light duration during cultivation, a large number of clustered shoots can be obtained at the base of the culture.

[0025] (5) Take the strong cluster of buds from step (4), cut them into appropriate sizes, and inoculate each seedling individually in the following C medium, wherein the C medium includes the following raw materials:

[0026] 1 / 2MS basic culture medium

[0027] Indolebutyric acid (IBA)

[0028] sucrose

[0029] Agar powder

[0030] By cultivating the seedlings under controlled light intensity, temperature, and light duration, robust seedlings with thick roots can be obtained.

[0031] (6) Hardening and transplanting: Take the rooted plants from step (5) and harden them at room temperature. Take the seedlings out of the culture medium, clean the residual culture medium, disinfect them in carbendazim solution, and then transplant them into the disinfected humus soil for warm and moist cultivation to obtain transplanted seedlings.

[0032] Further, the method for disinfecting the root and stem buds in step (2) is as follows: after washing the surface with tap water, soak it in a 10% (w / w) laundry detergent solution for 10 minutes and gently shake and stir it, then rinse it with running water for 30 minutes, place it on a clean bench and treat it with a 75% (w / w) ethanol solution for 10-15 seconds, then sterilize it with a 0.1% (w / w) mercuric chloride aqueous solution for 10-12 minutes, and finally rinse it with sterile water 3 times, each time for no less than 3 minutes, and shake the container thoroughly throughout the disinfection process.

[0033] Further, the culture medium A mentioned in step (3) includes the following raw materials:

[0034] MS basic culture medium:

[0035] 6-Benzylaminopurine (6-BA) 1.5-2.0 mg / L

[0036] 30000mg / L of sucrose

[0037] Agar powder 4700mg / L.

[0038] Furthermore, the pH value of the culture medium A is 5.4-5.8.

[0039] Further, the material from step (3) is divided into pieces of approximately 0.5 × 0.5 cm with a bud height of about 2.0 cm and transferred to culture medium B, which includes the following raw materials:

[0040] MS basic culture medium

[0041]

[0042]

[0043] Furthermore, the pH value of the culture medium B is 5.4-5.8.

[0044] Furthermore, it also includes the following steps: dividing the clump-forming seedlings in step (4) into single buds and transferring them into culture medium C in step (5), wherein culture medium C includes the following raw materials:

[0045] 1 / 2MS basic culture medium

[0046] Indolebutyric acid (IBA) 1.5-2.0 mg / L

[0047] 30000mg / L of sucrose

[0048] Agar powder 4700mg / L.

[0049] Furthermore, the pH value of the culture medium B is 5.4-5.8.

[0050] Further, in step (6), the substrate disinfection method is to spray a potassium permanganate solution with a mass concentration of 0.05-0.1% onto the humus soil, and after slightly wetting it, seal it with plastic film for one week for later use.

[0051] Further, in step (6), the mass concentration of the carbendazim solution is 0.1-0.2%.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The present invention uses tissue culture technology to achieve year-round production in the culture room, which saves land resources and improves economic efficiency, and overcomes the difficulty of traditional vegetative propagation methods that cannot carry out year-round production.

[0054] (2) This invention utilizes the root and stem buds of the auspicious grass to fix the superior traits of the parent plant to the greatest extent through direct organogenesis, thus avoiding the possible variations that may occur through indirect organogenesis.

[0055] (3) After the aseptic system is established, the present invention can complete the proliferation and rooting process by direct organogenesis in only two culture media. This can meet the needs of large-scale production while maintaining the excellent traits of the parent plant, making it the most effective proliferation method for artificial rapid propagation and improving the quality of seedlings.

[0056] (4) This invention achieves the goal of efficient and rapid propagation. A propagation culture cycle is 60 days, and the propagation coefficient can reach more than 10.0.

[0057] (5) The test-tube seedlings of auspicious grass produced by this invention have a high survival rate after transplanting because each plant has small tubers of different sizes and grows rapidly. Currently, Yunnan Youyang Biotechnology Co., Ltd. has carried out demonstration cultivation in Puge County, Liangshan, Sichuan Province, and the results are very good.

[0058] (6) This invention is of great significance and value for the rapid artificial propagation of *Gynostemma pentaphyllum*, and can also provide technical reference for the rapid propagation of other perennial rhizomatous medicinal plants. Attached Figure Description

[0059] Figure 1 shows the growth stages of the root, stem, and bud materials and the establishment of the aseptic system;

[0060] Among them, 1A and 1B are the source diagrams of the culture materials; 1C and 1D are the growth diagrams after 15 days of culture; 1E is the growth diagram after 30 days of culture; and 1F is the growth diagram after 60 days of culture.

[0061] Figure 2 shows the growth stages of basal stem cluster bud induction and proliferation culture;

[0062] Among them, 2A and 2B are growth figures after 15 days of culture; 2C and 2D are growth figures after 30 days of culture; 2E and 2F are growth figures after 45 days of culture; and 2G and 2H are growth figures after 60 days of culture.

[0063] Figure 3 shows the various growth stages of a single basal stem cluster bud;

[0064] Among them, 3A shows the growth after 15 days of culture; 3B shows the growth after 30 days of culture; 3C, 3D, and 3E show the growth after 45 days of culture; and 3F shows the growth after 60 days of culture.

[0065] Figure 4 shows the growth stages of rooting culture;

[0066] Among them, 4A and 4B are growth figures after 15 days of culture; 4C and 4D are growth figures after 30 days of culture; 4E and 4F are growth figures after 45 days of culture; and 4G and 4H are growth figures after 60 days of culture.

[0067] Figure 5 shows the growth stages of the acclimatized transplanted plants;

[0068] Among them, 5A shows the growth after 15 days of acclimatization; 5B shows the growth after 30 days of acclimatization; and 5C and 5D show the growth after 60 days of transplanting into flower pots.

[0069] Figure 6 shows the effect of a single exogenous plant hormone on the growth of rhizome and shoot of *Heliotropium indicum*.

[0070] Among them, 6A was MS + 3.0 mg / L LM6-BA; 6B was MS + 4.0 mg / L LM6-BA; 6C was MS + 0.5 mg / L KT; 6D was MS + 1.0 mg / L NAA; 6E was MS + 1.5 mg / L IBA; and 6F was MS + 0.01 mg / L 2,4-D.

[0071] Figure 7 shows the effects of a combination of exogenous plant hormones on the growth of rhizome and shoots of *Heliotropium indicum*.

[0072] Among them, 7A is blank MS medium; 7B is MS + 3.0 mg / L LM6-BA + 0.5 mg / L NAA; 7C is MS + 3.0 mg / L LM6-BA + 0.01 mg / L 2,4-D; 7D is MS + 3.0 mg / L LM6-BA. Detailed Implementation

[0073] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0074] Based on this invention, the efficient artificial propagation method for *Heliotropium indicum* includes the following steps:

[0075] S1. Take the rhizome buds of the auspicious grass, disinfect them, and then trim them into explants with a height of ≤2.0cm;

[0076] It is readily understood by those skilled in the art that healthy plants with good growth and no pests or diseases should be selected, and one-year-old root buds should be taken, disinfected, and cut into pieces of 0.5×0.5cm with a bud height of ≤2.0cm as explants; in a more preferred embodiment, the size of the pieces is 0.5×0.5cm and the bud height is 1.8-2.0cm.

[0077] S2. The treated explants were inoculated into culture medium A and cultured for 50-60 days at 21-23℃ with a light intensity of 1800-2500 lx and 9-11 hours of light per day to form proliferating seedlings with a proliferation coefficient of 1.5-2.0 (see Figure 1 for each growth stage); wherein, culture medium A is based on MS medium, with the addition of 1.5-2.0 mg / L 6-benzylaminopurine, 30000 mg / L sucrose, 4700 mg / L agar powder, and a pH of 5.4-5.8;

[0078] As shown in Figure 1 (scale bar = 2.0 cm), Figures 1C and 1D show that after 15 days of cultivation, the leaves of the root, stem, and bud gradually elongate; Figure 1E shows that after 30 days of cultivation, new buds appear at the base of the material; and Figure 1D shows that after 60 days of cultivation, the new buds grow rapidly and roots develop.

[0079] S3. After cutting the propagated seedlings into 0.5×0.5cm sections with a height of 1.8-2.0cm, inoculate them into culture medium B and culture them for 50-60 days at 21-23℃ with a light intensity of 1800-2500lx and 9-11 hours of light per day to form robust clusters of shoots with a propagation coefficient >10 (see Figures 2-3 for each growth stage); wherein, the culture medium B is MS medium as the basic medium, supplemented with 2.5-3.5mg / L 6-benzylaminopurine, 1.0-1.5mg / L α-naphthaleneacetic acid, 0.5-1.0mg / L 2,4-dichlorophenoxyacetic acid, 3.0-5.0mg / L paclobutrazol, 30000mg / L sucrose, 4700mg / L agar powder, and a pH of 5.4-5.8;

[0080] As shown in Figure 2 (scale bar = 2.0 cm), Figures 2A and 2B show that after 15 days of cultivation, the base of the material begins to swell slightly and new buds appear as the main bud grows; Figures 2C and 2D show that after 30 days of cultivation, the new shoots grow rapidly, and new buds appear at their base; Figures 2E and 2F show that after 45 days of cultivation, new shoots continuously appear at the base of the material as the new shoots grow; Figures 2G and 2H show that after 60 days of cultivation, the entire surface of the culture medium is covered by clustered buds from the basal stem.

[0081] As shown in Figure 3 (scale bar = 1.0 cm), Figure 3A shows that after 15 days of culture, the base of the material (as indicated by the arrow) is white and swollen; Figure 3B shows that after 30 days of culture, a large number of buds appear in the part of the rhizome submerged in the culture medium (as indicated by the arrow); Figures 3C-3E show that after 45 days of culture, the basal buds differentiate into clustered buds; and Figure 3F shows that after 60 days of culture, a clustered culture is formed.

[0082] S4. Cut the clustered buds from the rhizome into individual buds measuring 1.0 × 1.0 cm and 1.5-2.0 cm in height. Each individual bud retains 2-4 leaves before inoculating it into culture medium C. Culture the buds at 21-23℃ under a light intensity of 1800-2500 lx for 9-11 hours daily for 50-60 days to form rooted seedlings (see Figure 4 for each growth stage). Culture medium C uses 1 / 2 MS medium as the basal medium, supplemented with 1.5-2.0 mg / L indolebutyric acid, 30000 mg / L sucrose, 4700 mg / L agar powder, and a pH of 5.4-5.8.

[0083] As shown in Figure 4 (scale bar = 1.0 cm), Figures 4A and 4B show that after 15 days of culture, pale yellow adventitious roots appear at the base of the buds (as indicated by the arrows); Figures 4C and 4D show that after 30 days of culture, the number of adventitious roots (as indicated by the arrows) gradually increases; Figures 4E and 4F show that after 45 days of culture, the adventitious root system becomes well-developed and robust as the plant grows; Figures 4G and 4H show that after 60 days of culture, the induction of adventitious roots is completed. Throughout the process, there is no significant swelling at the base of the test-tube seedlings, and only occasionally 1-2 lateral buds are seen, which is very suitable for subsequent hardening and acclimatization.

[0084] S5. Once the rooted seedlings from S4 have grown to 8-10cm in height, after disinfection, they are transferred into the substrate for warm and moist cultivation to obtain transplanted seedlings (see Figure 5 for each growth stage).

[0085] As shown in Figure 5 (scale bar = 3.0cm), Figure 5A shows that the test-tube seedlings showed obvious growth after 15 days of acclimatization; Figure 5B shows that the test-tube seedlings grew vigorously after 30 days of acclimatization, with a survival rate of 100%; Figures 5C and 5D show that the test-tube seedlings grew strong after 60 days of transplanting into flower pots.

[0086] As one possible implementation method, the root and stem shoots of the present invention are disinfected by the following method: after washing the surface with tap water, soak in a 10% (w / w) laundry detergent solution for 10 minutes, gently shake and agitate, rinse with running water for 30 minutes, treat with a 75% (w / w) ethanol solution for 10-15 seconds, sterilize with a 0.1% (w / w) mercuric chloride aqueous solution for 10-12 minutes, and finally rinse with sterile water 3 times. The container is thoroughly shaken throughout the disinfection process.

[0087] As one possible implementation method, the seedling hardening and transplanting method of the present invention is as follows: when the rooted seedlings in the culture bottle grow to 8-10cm in height, place them under natural light for 7 days, then open the bottle cap and harden the seedlings for 3 days. Take out the rooted seedlings, carefully wash off the residual culture medium, and soak their roots in 0.1-0.2% carbendazim solution for 5 minutes. Transplant them into a foam box containing humus soil that has been disinfected with 0.05-0.1% potassium permanganate by mass concentration. The substrate thickness is 6.0cm. Under the conditions of temperature 20-25℃ and humidity of about 70%, the transplanted seedlings are obtained after 60 days.

[0088] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0089] Example 1

[0090] The efficient artificial propagation method for *Heliotropium indicum* includes the following steps:

[0091] 1. Obtaining explants: Select vigorous plants with good growth and no pests or diseases, and take their current year's rhizome buds.

[0092] 2. After washing the surface of the root and stem shoots from step 1 with tap water, soak them in a 10% (w / w) laundry detergent solution for 10 minutes and gently shake and agitate them. Then rinse them with running water for 30 minutes. Place them on a clean bench and treat them with a 75% (w / w) ethanol solution for 10 seconds. Then sterilize them with a 0.1% (w / w) mercuric chloride solution for 10 minutes. Finally, rinse them three times with sterile water for at least 3 minutes each time. Shake the container thoroughly throughout the entire disinfection process.

[0093] 3. Establishment of a sterile system: Trim the sterilized rootstock buds from step 2 to approximately 0.5 × 0.5 cm in length and 2.0 cm in height, and inoculate them into the following culture medium A:

[0094] MS basic culture medium:

[0095]

[0096]

[0097] Culture conditions: The plants were cultured under the following conditions: light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control of 22±1℃. After 15 days, the leaves gradually elongated; after 30 days, new buds sprouted at the base; after 60 days, the new buds grew rapidly and roots formed, and the aseptic system was established; at this time, the proliferation coefficient was 1.58.

[0098] 4. Induction and proliferation of basal shoot clusters: The aseptic seedlings from step 3 were cut into sections approximately 0.5 × 0.5 cm in size and 2.0 cm in height, and inoculated into culture medium B below:

[0099]

[0100] Culture conditions: Cultured under the conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature control. After 15 days, as the main bud grows, the base of the material begins to swell slightly and new buds appear. After 30 days, the new shoots grow rapidly, and new buds appear at their base. After 45 days, as the new shoots grow, new shoots continue to appear at the base of the material. After 60 days, the entire surface of the culture medium is covered by the material, and at this time, the proliferation coefficient of regenerated shoots generated through the clustered buds of the basal stem can reach 10.85.

[0101] 5. Rooting Culture: Cut the clustered culture from step 4 into 1.0×1.0cm sections from the rhizome, retaining 2 leaves and cutting them to 1.5cm in height. Transfer each plant individually to the following culture medium C:

[0102] 1 / 2MS basic culture medium

[0103]

[0104] Culture conditions: Cultured under conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature. After 15 days of culture, new leaves begin to elongate, and pale yellow roots emerge from the base of the stem. After 30 days, the root system gradually increases. After 45 days, the basal root system is well-developed and robust, and the plant has basically completed its growth. After 60 days, the rooting process is complete, with a rooting rate of 100%. Throughout the rooting process, there is no significant swelling at the base of the test-tube seedlings, with only occasionally 1-2 lateral buds appearing, making it very suitable for subsequent hardening-off and acclimatization.

[0105] 6. Hardening off and Transplanting: After 60 days of rooting culture, when the test-tube seedlings reach a height of 8cm, place the culture bottles under natural light for 7 days, then open the caps and harden off for 3 days. Remove the rooted seedlings, carefully wash away any remaining culture medium, and soak the roots in a 0.1% carbendazim solution for 5 minutes. Transplant them into a foam box (36cm × 26cm × 15cm inner dimensions, sterilized with 0.05% potassium permanganate), with a substrate thickness of 4.5cm. Maintain moisture and provide shade (temperature 20-25℃, humidity around 70%). Transplanted seedlings will be obtained after 60 days.

[0106] Example 2

[0107] The efficient artificial propagation method for *Heliotropium indicum* includes the following steps:

[0108] 1. Obtaining explants: Select vigorous plants with good growth and no pests or diseases, and take their current year's rhizome buds.

[0109] 2. After washing the surface of the root and stem shoots from step 1 with tap water, soak them in a 10% (w / w) laundry detergent solution for 10 minutes and gently shake and agitate them. Then rinse them with running water for 30 minutes. Place them on a clean bench and treat them with a 75% (w / w) ethanol solution for 12 seconds. Then sterilize them with a 0.1% (w / w) mercuric chloride solution for 11 minutes. Finally, rinse them with sterile water 3 times, each time for no less than 3 minutes. Shake the container thoroughly throughout the entire disinfection process.

[0110] 3. Establishment of a sterile system: Trim the sterilized rootstock buds from step 2 to approximately 0.5 × 0.5 cm in length and 2.0 cm in height, and inoculate them into the following culture medium A:

[0111] MS basic culture medium:

[0112]

[0113] Culture conditions: The plants were cultured under the following conditions: light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control at 22±1℃. After 15 days, the leaves gradually elongated; after 30 days, new buds sprouted at the base; after 60 days, the new buds grew rapidly and roots formed, and the aseptic system was established; at this time, the proliferation coefficient was 1.87.

[0114] 4. Induction and proliferation of basal shoot clusters: Cut the aseptic seedlings from step 3 into sections of approximately 0.5 × 0.5 cm with shoot height of about 2.0 cm, and inoculate them into the following culture medium B:

[0115]

[0116] Culture conditions: Cultured under the conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature. After 15 days, as the main bud grows, the base of the material begins to swell slightly and new buds appear. After 30 days, the new shoots grow rapidly, and new buds appear at their base. After 45 days, as the new shoots grow, new shoots continue to appear at the base of the material. After 60 days, the entire surface of the culture medium is covered by the material, and at this time, the proliferation coefficient of the regenerated shoots generated through the clustered buds of the basal stem can reach 11.12.

[0117] 5. Rooting Culture: Cut the clustered culture from step 4 into 1.0×1.0cm sections from the rhizome, retaining 3 leaves and cutting them to a height of 1.8cm. Transfer each plant individually to the following culture medium C:

[0118] 1 / 2MS basic culture medium

[0119]

[0120] Culture conditions: Cultured under conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature. After 15 days of culture, new leaves begin to elongate, and pale yellow roots emerge from the base of the stem. After 30 days, the root system gradually increases. After 45 days, the basal root system is well-developed and robust, and the plant has basically completed its growth. After 60 days, the rooting process is complete, with a rooting rate of 100%. Throughout the rooting process, there is no significant swelling at the base of the test-tube seedlings, with only occasionally 1-2 lateral buds appearing, making it very suitable for subsequent hardening-off and acclimatization.

[0121] 6. Hardening off and Transplanting: After 60 days of rooting culture, when the test-tube seedlings reach a height of 9cm, place the culture bottles under natural light for 7 days, then open the caps and harden off for 3 days. Remove the rooted seedlings, carefully wash away any remaining culture medium, and soak the roots in a 0.2% carbendazim solution for 5 minutes. Transplant them into a foam box (internal dimensions 36cm × 26cm × 15cm) filled with humus soil sterilized with 0.08% potassium permanganate, with a substrate thickness of 5.0cm. Maintain moisture and provide shade (temperature 20-25℃, humidity around 70%). Transplanted seedlings will be obtained after 60 days.

[0122] Example 3

[0123] The efficient artificial propagation method for *Heliotropium indicum* includes the following steps:

[0124] 1. Obtaining explants: Select vigorous plants with good growth and no pests or diseases, and take their current year's rhizome buds.

[0125] 2. After washing the surface of the root and stem shoots from step 1 with tap water, soak them in a 10% (w / w) laundry detergent solution for 10 minutes and gently shake and agitate them. Then rinse them with running water for 30 minutes. Place them on a clean bench and treat them with a 75% (w / w) ethanol solution for 15 seconds. Then sterilize them with a 0.1% (w / w) mercuric chloride solution for 12 minutes. Finally, rinse them three times with sterile water for at least 3 minutes each time. Shake the container thoroughly throughout the entire disinfection process.

[0126] 3. Establishment of a sterile system: Trim the sterilized rootstock buds from step 2 to approximately 0.5 × 0.5 cm in length and 2.0 cm in height, and inoculate them into the following culture medium A:

[0127] MS basic culture medium:

[0128]

[0129] Culture conditions: The plants were cultured under the following conditions: light intensity of 1800-2500 lx, light duration of 10 h / d, and temperature control of 22±1℃. After 15 days, the leaves gradually elongated; after 30 days, new buds sprouted at the base; after 60 days, the new buds grew rapidly and roots formed, and the aseptic system was established; at this time, the proliferation coefficient was 2.05.

[0130] 4. Induction and proliferation of basal shoot clusters: The aseptic seedlings from step 3 were cut into sections approximately 0.5 × 0.5 cm in size and 2.0 cm in height, and inoculated into culture medium B below:

[0131]

[0132]

[0133] Culture conditions: Cultured under the conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature. After 15 days, as the main bud grows, the base of the material begins to swell slightly and new buds appear. After 30 days, the new shoots grow rapidly, and new buds appear at their base. After 45 days, as the new shoots grow, new shoots continue to appear at the base of the material. After 60 days, the entire surface of the culture medium is covered by the material, and at this time, the proliferation coefficient of the regenerated shoots generated through the clustered buds of the basal stem can reach 10.50.

[0134] 5. Rooting Culture: Cut the clustered culture from step 4 into 1.0×1.0cm sections from the rhizome, retaining 3 leaves and cutting them to a height of 1.8cm. Transfer each plant individually to the following culture medium C:

[0135] 1 / 2MS basic culture medium

[0136]

[0137] Culture conditions: Cultured under conditions of 1800-2500 lx light intensity, 10 h / d light duration, and 22±1℃ temperature. After 15 days of culture, new leaves begin to elongate, and pale yellow roots emerge from the base of the stem. After 30 days, the root system gradually increases. After 45 days, the basal root system is well-developed and robust, and the plant has basically completed its growth. After 60 days, the rooting process is complete, with a rooting rate of 100%. Throughout the rooting process, there is no significant swelling at the base of the test-tube seedlings, with only occasionally 1-2 lateral buds appearing, making it very suitable for subsequent hardening-off and acclimatization.

[0138] 6. Hardening off and Transplanting: After 60 days of rooting culture, when the test-tube seedlings reach a height of 10cm, place the culture bottles under natural light for 7 days, then open the caps and harden off for 3 days. Remove the rooted seedlings, carefully wash away any remaining culture medium, and soak the roots in a 0.1% carbendazim solution for 5 minutes. Transplant them into a foam box (inner dimensions: 36cm × 26cm × 15cm) filled with humus soil sterilized with 0.1% potassium permanganate, with a substrate thickness of 6.0cm. Maintain moisture and provide shade (temperature 20-25℃, humidity approximately 70%). Transplanted seedlings will be obtained after 60 days.

[0139] Example 4 investigated the effects of exogenous plant hormones on the growth of rhizome buds of *Gynostemma pentaphyllum*.

[0140] The same culture conditions as in Example 2 were used, except that 6-benzylaminopurine (6-BA) in culture medium B was replaced with KT, NAA and 2,4-D.

[0141] As shown in Figure 6 (scale bar = 2.0 cm), Figures 6A and 6B show that high concentrations of 6-BA (4.0 mg / L and below) can induce a large number of basal shoots. Figures 6C, 6D, and 6F show that KT is better than NAA and 2,4-D in inducing adventitious shoots, with a suitable concentration range of 0.05-0.5 mg / L. Figure 6E shows that 1.5 mg / L IBA is more conducive to rooting. It can be seen that the addition of exogenous plant hormones alone can have a certain impact on the growth of rhizome shoots of *Gynostemma pentaphyllum*, but compared with 6-BA, other exogenous plant hormones such as KT, NAA, and 2,4-D have a poorer effect on inducing the occurrence of adventitious shoots in basal stems.

[0142] As shown in Figure 7 (scale bar = 2.0 cm), Figure 7A shows that the proliferation was low in the blank group without added hormones after 60 days of culture. Figures 7B and 7C show that in MS + exogenous plant hormone combination culture for 60 days, the 6-BA / NAA combination had a higher effect on basal stem bud formation than the 6-BA / 2,4-D combination. It can be seen that combining 6-BA with other exogenous plant hormones can promote the growth of rhizome buds of *Gynostemma pentaphyllum*. However, compared with 6-BA alone (see Figure 7D), the proliferation coefficient of adventitious buds of basal stems with added hormone combination is lower. It can be seen that the culture medium of the present invention is more conducive to the rooting of plants.

[0143] Technical principle of the invention:

[0144] This invention completes the proliferation and rooting process with only two culture media, making the process simple and easy to implement, which is conducive to the planning of subsequent production. At the same time, the emergence of rhizomes in test-tube seedlings solves the problem of low survival rate after acclimatization.

[0145] This invention utilizes a direct organogenesis method, which allows all seedlings to maintain the same genotype background, making it easy to standardize and industrialize operations, effectively improving seedling quality, and providing high-quality seedlings with unified standards for large-scale planting.

[0146] The core of this invention is the use of clopidogrel (S-3307), which promotes the swelling and lateral growth of the rhizomes of *Gnaphalium affine*, while inducing a large number of internode bud clusters. This provides a very valuable reference for the rapid in vitro propagation of rhizomatous plants.

[0147] The regeneration method of *Gynostemma pentaphyllum* in this invention is very similar to that in the wild state, both involving the swelling of the rhizome. In the test tube, the extremely shortened nodes produce both fixed and adventitious buds simultaneously, eventually forming a cluster structure. The swelling of the rhizome often occurs along with the growth of the residual growth point, resulting from the continuous synthesis of auxin by leaf buds, its downward transport, and its interaction with a high concentration of exogenous mitogens. Due to the abundant tissue at the nodes, numerous white-green bud primordia protrude, subsequently forming adventitious bud clusters, i.e., basal stem cluster buds. The proliferation efficiency of this invention is far higher than that in the natural state, achieving a satisfactory proliferation effect.

[0148] The present invention provides an efficient artificial propagation method for auspicious grass, which is low in cost, short in time, high in quality and high in survival rate. It is also an asexual propagation method that can fix the excellent traits to expand the propagation of seedlings and carry out the industrial production of high-quality auspicious grass seedlings to meet planting needs.

[0149] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A highly efficient artificial propagation method for *Heliotropium indicum*, characterized in that, Includes the following steps: (1) Obtaining explants: Select robust plants with good growth and no pests or diseases, and take the rhizomes that sprout in the current year; (2) Disinfect the rhizomes from step (1), cut them into appropriate sizes, and inoculate them into the culture medium for culture; (3) Cut the disinfected rhizomes from step (2) into appropriate sizes and inoculate them into culture medium A for culture to obtain proliferating seedlings with a slight proliferation effect. Culture medium A uses MS medium as the basic culture medium and adds 1.5-2.0 mg / L 6-benzylaminopurine, 30000 mg / L sucrose, and 4700 mg / L agar powder; (4) Occurrence and proliferation culture of basal shoot clusters: Transfer the proliferating seedlings cultured in step (3) into culture medium B for culture to obtain clustered shoots. Culture medium B uses MS medium as the basic culture medium and adds 2.5-3.5 mg / L 6-benzylaminopurine, 1.0-1.5 mg / L α-naphthaleneacetic acid, and 0.5-1.0 mg / L 2,4-Dichlorophenoxyacetic acid, 3.0-5.0 mg / L uniconazole, 30000 mg / L sucrose, 4700 mg / L agar powder, pH value 5.4-5.8; (5) Take the strong cluster buds from step (4), cut them into appropriate sizes, and inoculate them individually into culture medium C for cultivation. Strong seedlings with thick roots can be obtained. Culture medium C uses 1 / 2MS medium as the basic culture medium, with 1.5-2.0 mg / L indolebutyric acid, 30000 mg / L sucrose, 4700 mg / L agar powder, pH value 5.4-5.8; (6) Hardening and transplanting: Take the rooted plants from step (5) and place them at room temperature for hardening. Take the seedlings out of culture medium C, clean the residual culture medium, disinfect them in carbendazim solution, and then transplant them into disinfected humus soil for warm and moist cultivation. Transplanted seedlings can be obtained.

2. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 1, characterized in that, The method for disinfecting the root and stem buds described in step (2) is as follows: after washing the surface with tap water, soak it in a 10% (w / w) laundry detergent solution for 10 minutes and gently shake and stir it. Then rinse it with running water for 30 minutes, place it on a clean bench and treat it with a 75% (w / w) ethanol solution for 10-15 seconds. Then sterilize it with a 0.1% (w / w) mercuric chloride aqueous solution for 10-12 minutes. Finally, rinse it with sterile water 3 times, each time for no less than 3 minutes. Shake the container thoroughly throughout the disinfection process.

3. The efficient artificial propagation method for *Auspicious Grass* according to claim 1 or 2, characterized in that, The environmental conditions in step (3) are 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day.

4. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 3, characterized in that, In step (3), the pH value of culture medium A is 5.4-5.

8.

5. The efficient artificial propagation method for *Auspicious Grass* according to claim 1 or 2, characterized in that, The environmental conditions in step (4) are 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day.

6. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 1 or 2, characterized in that, In step (4), the pH value of culture medium B is 5.4-5.

8.

7. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 6, characterized in that, The environmental conditions in step (5) are 21-23℃, light intensity of 1800-2500 lx, and 9-11 hours of light per day.

8. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 1 or 2, characterized in that, In step (5), the pH value of culture medium C is 5.4-5.

8.

9. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 8, characterized in that, The size of the rootstock bud cut in step (3) is 0.5×0.5 cm and the bud height is about 2.0 cm.

10. The efficient artificial propagation method for *Gynostemma pentaphyllum* according to claim 1, characterized in that, (6) The mass concentration of the carbendazim solution mentioned in the step is 0.1-0.2%; the substrate disinfection method is to spray a potassium permanganate solution with a mass concentration of 0.05-0.1% onto the humus soil, and after slightly wetting it, seal it with plastic film for one week for later use.

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