Rapid propagation method for seedlings of pipewort in south China

Abstract

The invention belongs to the technical field of agricultural biology, and relates to a rapid propagation method of pipewort seedlings, which comprises the following steps: disinfecting robust pipewort plants with 84 disinfectant, stripping stem tips, carrying out induced differentiation to form adventitious bud clusters, cutting the adventitious bud clusters into small blocks, carrying out subculture multiplication, carrying out strong seedling culture, cutting single plants, and carrying out rooting culture to obtain complete plants. Transplanting to a culture medium after seedling hardening, spraying sufficient root fixing water, and carrying out water and fertilizer management and pest control measures, so as to obtain the tissue culture seedlings of the eriocaulon chinense. According to the method, the pipewort seedlings are propagated in a tissue culture mode, the defects of disease and pest propagation caused by division propagation, low sowing propagation germination rate, low efficiency and the like are overcome, stable inheritance of excellent characters of stock plants is kept, the method is not limited by seasons and weather conditions, production can be conducted all the year round, and the method is easy to implement, easy to operate, low in input cost and suitable for large-scale popularization and application. The seedling forming speed is high, and a technical foundation is laid for effective utilization of the Eriocaulum arvense resources.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to a plant tissue culture method, specifically, a method for rapid propagation of *Eriocaulon buergerianum* seedlings from South China. Background Technology

[0002] *Eriocaulon buergerianum* Koern. is an annual herb belonging to the genus *Eriocaulon* in the family Eriocaulaceae. Also known as star-bearing grass, meteor grass, and literary star grass, it is mainly distributed in Jiangsu, Anhui, and Zhejiang provinces. According to the *Compendium of Materia Medica*, "Eriocaulon buergerianum is light and buoyant, able to ascend to the Yangming meridian. It is very effective in treating various eye diseases. Its effect in brightening the eyes and clearing corneal opacity seems to surpass that of chrysanthemum." It also treats headaches, blindness, corneal opacity, post-smallpox corneal opacity, and stops bleeding. Its taste is pungent and sweet, and its nature is neutral. It enters the liver and lung meridians.

[0003] The propagation methods for Eriocaulon buergerianum are generally seed propagation and division propagation. Because Eriocaulon buergerianum seeds are very small and have poor resistance to adverse environments, their germination rate is low. Outdoor sowing requires covering with a thin film to prevent strong winds from blowing the seeds away. Furthermore, seed propagation is seasonally limited, affecting large-scale production and industrial development. Division propagation, on the other hand, easily leads to the spread of pests and diseases, affecting growth and yield to varying degrees, and its propagation efficiency is low.

[0004] Plant tissue culture has advantages such as low raw material requirements, no time or location restrictions, high propagation coefficient, and preservation of superior traits of the parent plant, making it widely used in plant seedling propagation. Currently, there are no reports on research into tissue culture techniques for *Eriocaulon buergerianum*, particularly *Eriocaulon buergerianum* from South China. Summary of the Invention

[0005] The purpose of this invention is to provide a method for rapid propagation of *Eriocaulon buergerianum* seedlings. Using robust *Eriocaulon buergerianum* plants as material, the stem tips are removed after disinfection and used as explants for tissue culture. Through adventitious bud induction culture, cluster bud proliferation culture, robust seedling culture, rooting and transplanting, in vitro regeneration of *Eriocaulon buergerianum* is achieved.

[0006] The technical solution adopted in this invention is:

[0007] A method for rapid propagation of *Eriocaulon buergerianum* seedlings from South China includes the following processes: obtaining explants, inducing adventitious buds, propagation and seedling strengthening culture, rooting, and transplanting. The specific steps are as follows:

[0008] (1) Obtaining explants

[0009] Stem tips were taken from healthy plants of *Eriocaulon buergerianum* after disinfection and used as explants.

[0010] (2) Adventitious bud induction

[0011] Shoot tips were inoculated onto an induction medium to induce differentiation and form adventitious shoot clusters; the induction medium was a modified MS medium containing 10.0-15.0 mg / L 6-BA, 0.3-0.8 mg / L IAA, 10-20 mg / L Vc, 30 g / L sucrose, and 3.0 g / L Gelrite, with a pH of 5.8.

[0012] (3) Proliferation and seedling cultivation

[0013] The adventitious bud clusters obtained by induced differentiation were cut into small pieces and transferred to a seedling propagation and strengthening medium to induce the proliferation and growth of the clustered buds. The seedling propagation and strengthening medium consisted of WPM + 6-BA 5.0-10.0 mg / L + NAA 0.1-0.3 mg / L + sucrose 30 g / L + carrageenan 6.5 g / L + activated carbon 0.5 g / L, with a pH of 5.8.

[0014] (4) Rooting culture

[0015] When the clump of shoots grows to 3-4 cm in height, the clump of shoots is divided into individual plants and inoculated into a rooting medium to induce rooting, thus obtaining rooted seedlings. The rooting medium consists of WPM + 6-BA 1.0-3.0 mg / L + NAA 0.1-1.0 mg / L + IBA 0.4-0.7 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.8.

[0016] In one optional embodiment, the improved MS uses MS medium as the basic medium, with the concentration of ammonium nitrate adjusted from 1650 mg / L to 825 mg / L, the concentration of potassium nitrate adjusted from 1900 mg / L to 950 mg / L, and the content of trace elements adjusted to 1 / 2 of the original content.

[0017] In an optional embodiment, in step (2), the induction medium is a modified MS medium containing 12 mg / L 6-BA, 0.5 mg / L IAA, 15 mg / L Vc, 30 g / L sucrose, and 3 g / L Gelrite; the culture conditions are: temperature 24–26°C, light duration 10–12 h / d, and light intensity 20–30 μmol·m⁻¹. -2 ·s -1 .

[0018] In an optional embodiment, in step (3), the proliferating and strengthening culture medium is WPM + 6-BA 8 mg / L + NAA 0.1 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.8; the culture conditions are: temperature 24–26 °C, light duration 10–12 h / d, and light intensity 20–30 μmol·m⁻¹. -2 ·s-1 .

[0019] In an optional embodiment, in step (4), the rooting medium is WPM + 6-BA 3.0 mg / L + NAA 0.5 mg / L + IBA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.8; the culture conditions are: temperature 24–26 °C, light duration 10–12 h / d, and light intensity 20–30 μmol·m⁻¹. -2 ·s -1 .

[0020] In one optional embodiment, the disinfection process in step (1) involves rinsing the plant clean of mud and sand under running water, removing rotten leaves, cutting the plant 3.0 cm above the base, removing surface moisture, disinfecting with 84 disinfectant for 8-12 minutes, rinsing with sterile water 4-5 times, and absorbing surface moisture with sterile absorbent paper before use.

[0021] In one alternative implementation, the method further includes step (6), namely, hardening off and transplanting: selecting the rooted seedlings that have taken root and are growing well in step (5) for hardening off and then transplanting them.

[0022] In one optional embodiment, the seedling hardening method in step (6) is as follows: the rooted Eriocaulon buergerianum seedlings are moved from the culture room to the outdoors and placed in a cool place for hardening for 3 to 5 days. After that, the seedlings are taken out of the container, the root culture medium is washed with clean water, and then soaked in a fungicide solution for 0.5 to 1 minute before being taken out and prepared for transplanting.

[0023] In one alternative implementation, the transplanting substrate in step (6) is soil:coconut coir:river sand = 2:1:1, and the substrate moisture is maintained above 80% after transplanting.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention uses the stem tips of *Eriocaulon buergerianum* as explants and employs tissue culture technology to induce the formation of adventitious bud clusters. Through processes such as proliferation, seedling strengthening, rooting, and transplanting, regenerated *Eriocaulon buergerianum* plants are obtained, enabling mass production of *Eriocaulon buergerianum* tissue culture seedlings. Its beneficial effects include:

[0026] 1. This invention has the advantages of being easy to implement, simple to operate, not limited by season, fast seedling growth, and strong and uniform seedling growth, laying a technical foundation for the effective utilization of Eriocaulon buergerianum in South China.

[0027] 2. Since diseases and pests such as nematode disease in Eriocaulon fragilis are quite serious in some areas, using tissue culture to propagate Eriocaulon fragilis seedlings can avoid seedlings carrying diseases and pests, improve cultivation yield and quality, reduce production costs, and provide technical support for the industrialization of Eriocaulon fragilis and the promotion and application of superior varieties. Attached Figure Description

[0028] Figure 1 This is an image showing the effect of the invention of inducing the differentiation of adventitious bud clusters from the shoot tips of *Eriocaulon buergerianum*.

[0029] Figure 2 This is an illustration of the effect of the invention on the proliferation of shoots and strong seedlings of *Eriocaulon buergerianum* in South China.

[0030] Figure 3 This is a diagram showing the effect of rooting seedlings of *Eriocaulon buergerianum* in South China according to the present invention.

[0031] Figure 4 This is a diagram illustrating the effect of transplanting rooted seedlings of *Eriocaulon buergerianum* (a type of grass) according to the present invention. Detailed Implementation

[0032] The specific embodiments of the present invention will be described in further detail below with reference to the examples. The following examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0033] In this invention:

[0034] 1. The improved MS medium uses MS medium as the basal medium, with the concentration of ammonium nitrate adjusted from 1650 mg / L to 825 mg / L, the concentration of potassium nitrate adjusted from 1900 mg / L to 950 mg / L, and the content of trace elements adjusted to half of the original content. (MS medium is an internationally recognized basal medium; its composition and preparation method can be found in the book (Zheng Chengmu and Liu Jinping, eds., Micropropagation of Tropical and Subtropical Plants, Hunan Science and Technology Press, 2001).

[0035] 2. Culture conditions for adventitious bud induction, proliferation, seedling strengthening, and rooting: Culture temperature 24–26℃, light duration 10–12 h / d, light intensity 20–30 μmol·m⁻². -2 ·s -1 .

[0036] I. Propagation of *Eriocaulon buergerianum* seedlings in South China

[0037] Example 1

[0038] 1. Adventitious bud induction process

[0039] Take healthy *Eriocaulon buergerianum* plants with a diameter of approximately 0.5 cm, rinse them under running water to remove mud and sand, remove rotten leaves, and cut them about 3.0 cm above the base of the plant. Absorb (blow) the surface moisture of the material, disinfect it with 84 disinfectant for 8 minutes on a clean bench, rinse it 5 times with sterile water, and dry the surface with absorbent paper. Using a microscope, peel off stem tips of about 1.0 mm in size as explants, and inoculate them on induction medium (modified MS + 6-BA 10.0 mg / L + IAA 0.5 mg / L + Vc 15 mg / L + sucrose 30 g / L + Gelrite 3.0 g / L, pH 5.8) to induce shoot differentiation. After about 7 days of culture, the surviving stem tips begin to turn green, and after 30 days of culture, they begin to differentiate into adventitious bud clusters. Figure 1 ).

[0040] 2. Proliferation and Seedling Cultivation Process

[0041] The adventitious shoot clusters obtained from induced differentiation were divided into small pieces and transferred to a proliferation and seedling strengthening medium (WPM + 6-BA 5.0 mg / L + NAA 0.1 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, pH 5.8) for proliferation and seedling strengthening culture to obtain clustered shoots ( Figure 2 ).

[0042] 3. Rooting Culture Process

[0043] When the clustered shoots grow to 3-4 cm in height, they are divided into individual plants and inoculated into rooting medium (WPM medium + 6-BA 1.0 mg / L + NAA 0.1 mg / L + IBA 0.5 mg / L + sucrose 30 g / L + carrageenan 7.0 g / L + activated charcoal 1.0 g / L) to induce rooting and obtain rooted seedlings. Figure 3 ).

[0044] 4. Seedling hardening and transplanting process

[0045] After transplanting the rooted Eriocaulon seedlings from the culture room to the outdoors and placing them in a cool, shady place for 3-5 days to harden off, remove the seedlings from the container, wash the roots of the culture medium with clean water, soak them in a fungicide solution for 0.5-1 minutes, remove them, and transplant them into a substrate made of peat moss, coconut coir, and river sand. Figure 4 The substrate ratio is calculated by volume: soil: coconut coir: river sand = 2:1:1. After that, water thoroughly to settle the roots and keep the substrate moisture above 80%, or place the substrate half-submerged in a tray with water and keep the tray moist.

[0046] The substrate preparation involves mixing soil, coconut coir, and river sand in a volume ratio of 2:1:1, filling the mixture into non-woven fabric seedling bags, soaking the substrate with a fungicide solution, and covering it with a film for 3-5 days. After the transplanted seedlings develop new leaves, foliar spraying with a 0.1%–0.2% urea solution is applied on sunny mornings, and fertilization is carried out every 7–15 days.

[0047] Example 2

[0048] 1. Adventitious bud induction process

[0049] Take healthy plants of *Eriocaulon buergerianum* with a diameter of about 0.5 cm, rinse them under running water to remove mud and sand, peel off rotten leaves, cut them about 3 cm above the base of the plant, absorb (blow) the surface moisture, disinfect them with 84 disinfectant for 10 minutes on a clean bench, rinse them 5 times with sterile water, and dry the surface moisture with absorbent paper. Then, using a microscope, peel off stem tips of about 1.0 mm in size as explants and inoculate them on induction medium (modified MS + 6-BA 12.0 mg / L + IAA 0.7 mg / L + Vc 12 mg / L + sucrose 30 g / L + Gelrite 3.0 g / L + activated carbon 0.5 g / L, pH 5.8) to induce shoot differentiation. After about 7 days of culture, the surviving stem tips begin to turn green, and after 30 days of culture, they begin to differentiate into adventitious bud clusters.

[0050] 2. Proliferation and Seedling Cultivation Process

[0051] The adventitious bud clusters obtained by induced differentiation were cut into small pieces and transferred to a proliferation and seedling strengthening medium (WPM + 6-BA 8.0 mg / L + NAA 0.3 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, pH 5.8) for proliferation and seedling strengthening culture to obtain clustered buds.

[0052] 3. Rooting Culture Process

[0053] When the clump of shoots grows to 3-4 cm in height, the clump of shoots is divided into individual plants and inoculated into rooting medium (WPM medium + 6-BA 3.0 mg / L + NAA 0.5 mg / L + IBA 0.4 mg / L + sucrose 30 g / L + carrageenan 7.0 g / L + activated carbon 1.0 g / L) to induce rooting and obtain rooted seedlings.

[0054] 4. Seedling hardening and transplanting process

[0055] After transferring the rooted tissue culture seedlings of Eriocaulon buergerianum from the culture room to the outdoors and placing them in a cool, shady place for 3-5 days to harden off, remove the rooted seedlings from the container, wash the roots of the culture medium with clean water, soak them in a fungicide solution for 0.5-1 minute, remove them, and transplant them into a substrate made of soil, coconut coir, and river sand. Figure 4The substrate ratio is calculated by volume: soil: coconut coir: river sand = 2:1:1. After that, water thoroughly to settle the roots and keep the substrate moisture above 80%, or place the substrate half-submerged in a tray with water and keep the tray moist.

[0056] Example 3

[0057] 1. Adventitious bud induction process

[0058] Take healthy plants of *Eriocaulon buergerianum* with a diameter of about 0.5 cm, rinse them under running water to remove mud and sand, peel off rotten leaves, cut them about 3 cm above the base of the plant, disinfect them with 84 disinfectant for 8 minutes on a clean bench, rinse them 5 times with sterile water, and dry the surface with absorbent paper. Then, using a microscope, peel off stem tips of about 1.0 mm in size as explants and inoculate them on induction medium (modified MS + 6-BA 15.0 mg / L + IAA 0.3 mg / L + Vc 18 mg / L + sucrose 30 g / L + Gelrite 3 g / L, pH 5.8) to induce shoot differentiation. After about 7 days of culture, the surviving stem tips begin to turn green, and after 30 days of culture, they begin to differentiate into adventitious bud clusters.

[0059] 2. Proliferation and Seedling Cultivation Process

[0060] The adventitious bud clusters obtained by induced differentiation were cut into small pieces and transferred to a proliferation and seedling strengthening medium (WPM + 6-BA 10.0 mg / L + NAA 0.2 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, pH 5.8) for proliferation and seedling strengthening culture to obtain clustered buds.

[0061] 3. Rooting Culture Process

[0062] When the clustered shoots grow to 3-4 cm in height, they are divided into individual plants and inoculated into rooting medium (WPM medium + 6-BA 2.0 mg / L + NAA 0.8 mg / L + IBA 0.7 mg / L + sucrose 30 g / L + carrageenan 7.0 g / L + activated charcoal 1.0 g / L) to induce rooting and obtain rooted seedlings. Figure 3 ).

[0063] 4. Seedling hardening and transplanting process

[0064] After transplanting the rooted Eriocaulon seedlings from the culture room to the outdoors and placing them in a cool, shady place for 3-5 days to harden off, remove the seedlings from the container, wash the roots of the culture medium with clean water, soak them in a fungicide solution for 0.5-1 minutes, remove them, and transplant them into a substrate made of soil, coconut coir, and river sand. Figure 4 The substrate ratio is calculated by volume: soil: coconut coir: river sand = 2:1:1. After that, water thoroughly to settle the roots and keep the substrate moisture above 80%, or place the substrate half-submerged in a tray with water and keep the tray moist.

[0065] II. Evaluation of the Propagation Effect of South China Valley Grass Seedlings

[0066] 1. Identification of adventitious buds induced by *Eriocaulon buergerianum* in South China

[0067] To evaluate the effect of *Eriocaulon buergerianum* shoot tip induction on adventitious buds, the induction of adventitious buds in the three examples above was observed, the number of induced differentiated buds was counted, and the induction rate was calculated. The results are shown in Table 1.

[0068] Table 1. Shoot tip induction of *Eriocaulon buergerianum* in South China

[0069] project Number of vaccinations Number of explants that induced adventitious buds Induction rate Example 1 30 19 63.0％ Example 2 30 15 50.0％ Example 3 30 17 56.7％

[0070] The table above shows the effect of inducing adventitious shoots in *Eriocaulon buergerianum*, indicating that the modified MS medium of this invention induces shoot differentiation from shoot tips. The shoot tips begin to turn green after about 7 days, and adventitious shoots are formed after 30 days. The induction rate is as high as 63% or more, demonstrating a very good differentiation effect.

[0071] 2. Identification of the Proliferation Effect of *Eriocaulon buergerianum* in South China

[0072] To assess the propagation effect of *Eriocaulon buergerianum*, the propagation of adventitious buds in the three examples described above was observed, the number of propagated buds was counted, and the propagation coefficient was calculated. The results are shown in Table 2.

[0073] Table 2. Proliferation of adventitious buds

[0074] project number of adventitious buds number of proliferations Proliferation coefficient Example 1 50 310 6.2 Example 2 50 265 5.3 Example 3 50 210 4.2

[0075] The table above shows that the adventitious bud proliferation effect of this invention, using WPM as the basal medium, can proliferate adventitious buds after 30 days of culture, with a proliferation coefficient of up to about 6.0, indicating a significant proliferation effect.

[0076] 3. Identification of the rooting effect of adventitious buds (clustered buds)

[0077] To assess the rooting effect of adventitious buds, the rooting of adventitious buds in the three embodiments described above was observed, the number of roots was counted, and the rooting rate was calculated. The results are shown in Table 3.

[0078] Table 3 Rooting status of adventitious buds

[0079] project number of adventitious buds Number of roots Rooting rate Example 1 150 147 98％ Example 2 150 150 100％ Example 3 150 149 98.7％

[0080] The rooting effect of adventitious buds in the table above shows that, with WPM as the basal medium of this invention, the rooting rate of single buds 3-4 cm high can reach up to 100%.

[0081] 4. Evaluation of the transplanting effect of rooted seedlings

[0082] To assess the survival rate of transplanted seedlings after rooting, the growth of the rooted seedlings in the three examples was observed. The number of surviving seedlings was counted 20 days after transplanting, and the survival rate was calculated. The results are shown in Table 4.

[0083] Table 4. Growth of seedlings after transplanting from seed trays.

[0084]

[0085]

[0086] The transplanting results shown in the table above indicate that this invention, using seedlings with well-induced root systems for transplanting (seedling cups) and a mixture of soil, coconut coir, and river sand as the cultivation substrate, achieves a high transplant survival rate, with survival rates all above 95% and reaching as high as 100%. This can meet the needs of mass seedling propagation and lay the foundation for the breeding and industrial production of *Vigna angularis* seedlings in South China.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of *Eriocaulon buergerianum* seedlings in South China, characterized in that, Includes the following steps: (1) Obtaining explants: After disinfection, the stem tips of healthy plants of *Eriocaulon buergerianum* were peeled off as explants; (2) Adventitious bud induction Shoot tips were inoculated onto an induction medium to induce differentiation and form adventitious shoot clusters; the induction medium was a modified MS medium containing 10.0-15.0 mg / L 6-BA, 0.3-0.8 mg / L IAA, 10-20 mg / L Vc, 30 g / L sucrose, and 3.0 g / L Gelrite, with a pH of 5.

8. (3) Proliferation and seedling cultivation The adventitious bud clusters obtained by induced differentiation were cut into small pieces and transferred to a propagation and seedling strengthening medium to induce the proliferation and growth of the clustered buds; the propagation and seedling strengthening medium consisted of WPM + 6-BA 5.0-10.0 mg / L + NAA 0.1-0.3 mg / L + sucrose 30 g / L + carrageenan 6.5 g / L + activated carbon 0.5 g / L, with a pH of 5.

8. (4) Rooting culture When the clump of shoots grows to 3-4 cm in height, the clump of shoots is divided into individual plants and inoculated into a rooting medium to induce rooting, thus obtaining rooted seedlings. The rooting medium consists of WPM + 6-BA 1.0-3.0 mg / L + NAA 0.1-1.0 mg / L + IBA 0.4-0.7 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.

8.

2. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, The disinfection process in step (1) involves rinsing the plant clean of mud and sand under running water, removing rotten leaves, cutting the plant 3.0 cm above the base, removing surface moisture, disinfecting with 84 disinfectant for 8-12 minutes, rinsing with sterile water 4-5 times, and absorbing surface moisture with sterile absorbent paper before use.

3. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, The improved MS medium uses MS medium as the basic medium, with the concentration of ammonium nitrate adjusted from 1650 mg / L to 825 mg / L, the concentration of potassium nitrate adjusted from 1900 mg / L to 950 mg / L, and the content of trace elements adjusted to 1 / 2 of the original content.

4. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, In step (2), the induction medium is a modified MS medium containing 12 mg / L 6-BA, 0.5 mg / L IAA, 15 mg / L Vitamin C, 30 g / L sucrose, and 3 g / L Gelrite. The culture conditions are: temperature 24–26 °C, light duration 10–12 h / d, and light intensity 20–30 μmol·m⁻². -2 ·s -1 .

5. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, In step (3), the propagation and seedling strengthening culture medium is WPM + 6-BA 8 mg / L + NAA 0.1 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.8; the culture conditions are: temperature 24–26℃, light duration 10–12 h / d, and light intensity 20–30 μmol·m -2 ·s -1 .

6. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, In step (4), the rooting medium is WPM + 6-BA 3.0 mg / L + NAA 0.5 mg / L + IBA 0.5 mg / L + activated carbon 1.0 g / L + sucrose 30 g / L + carrageenan 6.5 g / L, with a pH of 5.8; the culture conditions are: temperature 24–26℃, light duration 10–12 h / d, and light intensity 20–30 μmol·m -2 ·s -1 .

7. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 1, characterized in that, It also includes step (6), namely hardening and transplanting: select the rooted seedlings that have taken root and are growing well in step (5), harden them, and then transplant them.

8. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 7, characterized in that, The method for hardening off seedlings in step (6) is as follows: Move the rooted tissue culture seedlings of Eriocaulon buergerianum from the culture room to the outdoors and place them in a cool place for hardening off for 3 to 5 days. After that, take the seedlings out of the container, wash the culture medium on the roots with clean water, soak them in a fungicide solution for 0.5 to 1 minute, and then take them out to prepare for transplanting.

9. The method for rapid propagation of *Eriocaulon buergerianum* seedlings according to claim 7, characterized in that, In step (6), the transplanting substrate is soil:coconut coir:river sand = 2:1:

1. After transplanting, the substrate moisture should be kept above 80%.

Images