Simple, convenient and efficient seedling raising method for black boea mellettii and application thereof

Abstract

The invention belongs to the technical field of artificial cultivation of plants, and particularly relates to a simple, convenient and efficient seedling raising method for boea cavaleriei and application thereof. The seedling culture method comprises the following steps: adding disinfected boea cavaleriei seeds and strigolactones analogue solution into a 1 / 2 MS culture medium for seed culture to obtain boea cavaleriei seedlings; the concentration of the striga lactone analogue solution is 0.1 to 10 [mu] mol / L; the volume ratio of the strigolactone analogue solution to the 1 / 2 MS culture medium is (1-3): 15; placing the black boea cavaleriei seedlings in a new 1 / 2MS culture medium, and performing seedling culture to obtain black boea cavaleriei seedlings; in the 1 / 2MS culture medium, the mass volume concentration of the cane sugar is 0.5%-2%. The seedling raising method is easy to operate and low in cost, the germination rate and the seedling rate of the black boea mellea seeds can be greatly increased, the seedling raising period is shortened, and a solid foundation is laid for breaking through the technical bottleneck of artificial cultivation and production of black boea mellea and achieving industrial development of the black boea mellea.

Description

Technical Field

[0001] This invention belongs to the field of plant artificial cultivation technology, specifically relating to a simple and efficient method for raising seedlings of black capsule and its application. Background Technology

[0002] Black capsule ( Alectra avensis Also known as Huaxuedan, Honggencao, and Xiaohuaxuecao, it is an annual herb belonging to the genus *Sargentodoxa* of the family Orobanchaceae (formerly Scrophulariaceae). It is a rare medicinal herb primarily used to treat early-stage leukemia, dysfunctional uterine bleeding, excessive postpartum bleeding, and myocardial ischemia, with immense potential for medicinal development. However, wild resources of *Sargentodoxa dorsalis* are scarce and endangered. As a rare and endangered medicinal plant commonly used by ethnic minorities such as the Yi, Miao, and Lahu, the fresh whole herb has reached a price of approximately 4,000 yuan per kilogram. Artificial cultivation has become an important way to address its resource shortage.

[0003] Black capsule is an obligate root-parasitic plant with extremely small seeds (about 0.25 mm long and 0.15 mm wide). It lacks endogenous nutrients, making it difficult to support the initial growth of seedlings after germination. As a result, the germination rate of black capsule seeds during artificial cultivation is extremely low (less than 0.3%). The difficulty of artificial seedling cultivation has become the core bottleneck restricting its resource development and utilization.

[0004] Existing technologies for improving the germination rate of black capsule seeds are often limited to using the traditional plant hormone gibberellin as a seed germination inducer (CN103004326A) or using tissue culture for rapid propagation (CN103004601A). However, these methods do not take into account their parasitic characteristics, resulting in cumbersome operation, low seed germination rate (about 65%), and long cultivation time (the bud height of seedlings is only 1.3 mm after 45-70 days of growth), which cannot meet the actual needs of large-scale black capsule seedling production.

[0005] The growth and development processes of different types of parasitic plants vary significantly. Holoparasitic plants require strigolactones in the root exudates of the host plant for seed germination induction, and their growth requires nutrients entirely from the host plant. Facultative root hemiparasitic plants do not require strigolactones for seed germination induction; although they can achieve partial nutrient self-sufficiency through photosynthesis, they still need to obtain some nutrients from the host. Obligate root hemiparasitic plants, on the other hand, possess both seed germination induction requirements and a certain degree of autotrophic capacity. Currently, there is a lack of understanding of the parasitic characteristics of *Capsella bursa-pastoris* within the industry, and a lack of systematic research on its key seed germination conditions and seedling growth patterns has prevented current technologies from overcoming the challenge of large-scale artificial seedling cultivation. Therefore, clarifying the parasitic characteristics of *Capsella bursa-pastoris* and overcoming the bottlenecks in artificial seedling cultivation technology has become a core breakthrough for promoting its large-scale application and a key technical problem urgently needing to be solved in this field. Summary of the Invention

[0006] In order to solve the problems of complicated operation, low germination rate and poor seedling effect of artificial culture of black seedlings in the prior art, the application provides a simple and efficient black seedling culture method and application thereof, and specifically comprises the following technical scheme.

[0007] The application provides a simple and efficient black seedling culture method, which comprises the following steps. (1) Seed culture: The sterilized black seed and a strigolactone analogue solution are added into 1 / 2MS culture medium for seed culture, so as to obtain black seedlings. The concentration of the strigolactone analogue in the strigolactone analogue solution is 0.1-10 μmol / L. The volume ratio of the strigolactone analogue solution to the 1 / 2MS culture medium is 1-3:15. (2) Seedling culture: The black seedlings are transferred into new 1 / 2MS culture medium for seedling culture, so as to obtain black seedlings. In the 1 / 2MS culture medium in steps (1) and (2), the mass-volume concentration of sucrose is 0.5%-2%.

[0008] Preferably, the sterilization in step (1) comprises the following steps: the black seed is soaked in 1% sodium hypochlorite solution, oscillation sterilization is performed for 2 min, and then the seed is washed with sterile water for 2-4 times.

[0009] Preferably, after the sterilization, the following step is further included: sterile water is added into the sterilized black seed to prepare a seed suspension, and then the seed suspension is added to the surface of the 1 / 2MS culture medium.

[0010] Preferably, the sterile water comprises reverse osmosis water and / or deionized water.

[0011] Preferably, in step (1), the strigolactone analogue solution is added into the 1 / 2MS culture medium by dropwise adding the strigolactone analogue solution to the surface of the 1 / 2MS culture medium. The dropwise adding position is the seed suspension on the surface of the 1 / 2MS culture medium.

[0012] Preferably, the seed culture in step (1) is performed under the following conditions: the culture time is 14-28 d; the culture temperature is 23-27℃; the light cycle is 12 h light / 12 h darkness; and the light intensity is 20-25 μmol·m -2 ·s -1 .

[0013] Preferably, the seedling culture condition in step (2) is as follows: culture temperature 18-28℃; light cycle 16 h light / 8 h dark; light intensity 115-120 μmol·m -2 ·s -1 .

[0014] Preferably, the black pod seedling comprises a black pod seedling at the stage of 2-4 true leaves.

[0015] The application also provides the use of the black pod seedling method as described above, which comprises one or more of the following aspects: 1) improving the germination rate of black pod seeds; 2) promoting the seedling formation of black pod seeds; 3) promoting the growth of black pod seedlings and / or black pod seedlings.

[0016] Preferably, the promotion of the growth of black pod seedlings and / or black pod seedlings comprises one or more of the following aspects: 1) improving the survival rate of black pod seedlings and / or black pod seedlings; 2) improving the plant height of black pod seedlings and / or black pod seedlings; 3) increasing the leaf number of black pod seedlings and / or black pod seedlings; 4) increasing the branch number of black pod seedlings and / or black pod seedlings; 5) promoting the root length of black pod seedlings and / or black pod seedlings; 6) increasing the root number of black pod seedlings and / or black pod seedlings.

[0017] The application has the following beneficial effects: (1) The germination rate is significantly improved: for the first time, the application applies GR24 to the sterile seedling of black pod, and the germination rate is increased from 65% in the prior art to more than 90%, and the highest can reach 97.40%.

[0018] (2) Good seedling formation and growth effect: by combining the seed germination inducer and the operation of adapting the culture condition, the application successfully realizes the transformation of black pod from "host-dependent" to "independent autotrophy", and the seedling is healthy, and the plant height can reach about 16 cm after 50 d.

[0019] (3) Simple operation: the seedling method of the application has clear process and is easy to standardize operation, which lays a solid foundation for the large-scale and factory seedling of black pod. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows.

[0021] Figure 1Growth of black peper plantlets cultivated by the method of Example 2 (scale = 2 mm); Figure 2 Growth of black peper plantlets cultivated by the method of Example 3 (scale = 2 mm); Figure 3 Growth of black peper plantlets cultivated by the method of Example 4 (scale = 2 mm); Figure 4 Growth of black peper plantlets cultivated by the method of Comparative Example 3 (scale = 2 mm); Figure 5 Growth of black peper plantlets cultivated by the method of Comparative Example 4 (scale = 2 mm); Figure 6 Growth of black peper plantlets cultivated by the method of Comparative Example 5 (scale = 2 mm); Figure 7 Growth of black peper plantlets cultivated by the method of Comparative Example 5 (scale = 2 mm); Figure 8 Growth of black peper plantlets cultivated by the method of Comparative Example 5 (scale = 2 mm). DETAILED DESCRIPTION

[0022] The present application provides a simple and efficient black peper plantlet cultivation method, comprising the following steps: adding the sterilized black peper seeds and a GR24 solution into 1 / 2MS medium to culture the seeds, to obtain black peper plantlets; the concentration of the GR24 solution is 0.1-10 μmol / L; the volume ratio of the GR24 solution to the 1 / 2MS medium is 1-3:15; placing the black peper plantlets into new 1 / 2MS medium to culture the plantlets, to obtain black peper seedlings; the mass concentration of sucrose in the 1 / 2MS medium is 0.5%-2%.

[0023] The present application places the sterilized black seed and GR24 solution in 1 / 2MS medium for seed culture to obtain black seedlings. As an embodiment, the sterilization step includes: soaking the black seed in 1% sodium hypochlorite solution, shaking for 2 minutes, and then rinsing the seed with sterile water for 2-4 times. As an embodiment, after the sterilization, it further includes adding sterile water to the sterilized black seed to form a seed suspension, and then adding the seed suspension to the surface of the 1 / 2MS medium. As an embodiment, the sterile water includes reverse osmosis water and / or deionized water. As an embodiment, the GR24 solution is an aqueous solution of strigolactone analog. As an embodiment, the addition of the GR24 solution to the 1 / 2MS medium is: dropping the GR24 solution on the surface of the 1 / 2MS medium; the dropping position is: the seed suspension on the surface of the 1 / 2MS medium. As an embodiment, the concentration of the GR24 solution added to the 1 / 2MS medium is 0.1-10 μmol / L, and the concentration of the GR24 solution refers to the concentration of GR24 in the GR24 solution. As another embodiment, the concentration of the GR24 solution can be any one of 0.1 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L and 10 μmol / L or the intermediate value of any two point values. As an embodiment, the mass concentration of sucrose in the 1 / 2MS medium is 0.5%-2%. As an embodiment, the mass concentration of sucrose in the 1 / 2MS medium can be any one of 0.5%, 1.0%, 1.5% and 2% or the intermediate value of any two point values. As an embodiment, the volume ratio of the GR24 solution to the 1 / 2MS medium is: 1-3:15. As another embodiment, the volume ratio of the GR24 solution to the 1 / 2MS medium can be any one of 1:15, 2:15 and 3:15 or the intermediate value of any two point values. As an embodiment, the seed culture time is 14-28 d. As another embodiment, the seed culture time can be any one of 14 d, 15 d, 16 d, 17 d, 18 d, 19 d, 20 d, 21 d, 22 d, 23 d, 24 d, 25 d, 26 d, 27 d and 28 d or the intermediate value of any two point values. As an embodiment, the seed culture temperature is 23-27 ℃. As another embodiment, the seed culture temperature can be any one of 23 ℃, 24 ℃, 25 ℃, 26 ℃ and 27 ℃ or the intermediate value of any two point values.As an implementation form, the light period of the seed culture is 12 h; as an implementation form, the light intensity of the seed culture is 20-25 μmol·m. -2 ·s -1 As another implementation form, the light intensity of the seed culture can be any one or intermediate value of any two point values in 20 μmol·m -2 ·s -1 , 21 μmol·m -2 ·s -1 , 22 μmol·m -2 ·s -1 , 23 μmol·m -2 ·s -1 , 24 μmol·m -2 ·s -1 and 25 μmol·m -2 ·s -1 . The adding mode of the GR24 solution in the application can be adding on the surface of the 1 / 2MS culture medium or mixing with the 1 / 2MS culture medium. There is no significant difference between the effects of the two adding modes on the germination of the seeds and the formation of seedlings of the black husk, but in order to save costs, the preferred adding mode of the GR24 solution is to add the GR24 solution on the surface of the 1 / 2MS culture medium. In this way, not only a good effect can be achieved, but also the use amount of the GR24 solution added on the surface is less than that of the GR24 solution mixed in the culture medium, which can reduce the cost by about 93.33%. Moreover, the present application first adds the seed suspension of the black husk into the 1 / 2MS culture medium, and then drops the GR24 solution on the seed suspension, which can make the GR24 solution drive the seed suspension to be uniformly dispersed on the surface of the culture medium, so that the dispersion effect of the seeds is better. The present application optimizes the adding mode of the GR24 solution by dropping the GR24 solution on the surface of the culture medium, which can save 93.33% of the use amount of the GR24 solution compared with the mode of mixing the GR24 solution into the culture medium, greatly reduces the production cost on the premise of improving the cultivation effect of the black husk.

[0024] After obtaining the black capsule seedling, the black capsule seedling is placed in a new 1 / 2MS medium for seedling culture to obtain a black capsule seedling. As an embodiment, the time for seedling culture is not particularly limited. After obtaining the black capsule seedling, the black capsule seedling is transferred to a culture tube containing the 1 / 2MS medium for culture. As an embodiment, the temperature for seedling culture is 18-28 ℃. As another embodiment, the temperature for seedling culture can be any one or an intermediate value of any two point values in 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃ and 28 ℃. As an embodiment, the light cycle for seedling culture is 12 h. As an embodiment, the light intensity for seedling culture is 115-120 μmol·m -2 ·s -1 . As another embodiment, the light intensity for seedling culture can be any one or an intermediate value of any two point values in 115 μmol·m -2 ·s -1 , 116 μmol·m -2 ·s -1 , 117 μmol·m -2 ·s -1 , 118 μmol·m -2 ·s -1 , 119 μmol·m -2 ·s -1 and 120 μmol·m -2 ·s -1 . As an embodiment, the black capsule seedling comprises a black capsule seedling at the stage of 2-4 true leaves. As an embodiment, the mass-volume concentration of sucrose in the 1 / 2MS medium is 0.5%-2%. As another embodiment, the mass-volume concentration of sucrose in the 1 / 2MS medium is 1%.

[0025] The application also provides the use of the black capsule seedling culture method as described above, which comprises one or more of the following aspects: 1) improving the germination rate of black capsule seeds; 2) promoting the seedling formation of black capsule seeds; 3) promoting the growth of black capsule seedlings and / or black capsule seedlings.

[0026] As an embodiment, in the use as described above, the promotion of the growth of black capsule seedlings and / or black capsule seedlings comprises one or more of the following aspects: 1) improving the survival rate of black capsule seedlings and / or black capsule seedlings; 2) improving the plant height of black capsule seedlings and / or black capsule seedlings; 3) increasing the number of leaves of black capsule seedlings and / or black capsule seedlings; 4) increasing the number of branches of black capsule seedlings and / or black capsule seedlings; 5) promoting the root length of black capsule seedlings and / or black capsule seedlings; 6) increasing the number of roots of black capsule seedlings and / or black capsule seedlings.

[0027] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a simple and efficient method for cultivating black capsule seedlings and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] The technical means used in this invention are conventional methods well known to those skilled in the art, and the materials and reagents used are all commercially available. Unless otherwise specified, all conventional operations described in this invention are performed in a clean bench. The black capsule seeds used in the embodiments and comparative examples of this invention were all obtained from the Southwest Wild Germplasm Bank of the Kunming Institute of Botany, Chinese Academy of Sciences. The strigolactone analogue (rac-GR24) described in this invention was purchased from Beijing Coolplay Technology Co., Ltd., product number: CR9420.

[0029] Example 1: A simple and efficient method for raising seedlings of black capsule. The steps are as follows: A. Preparation of culture medium: 1 / 2MS medium: Add the following to 1 L of deionized water: 950 mg potassium nitrate, 825 mg ammonium nitrate, 220 mg calcium chloride, 185 mg magnesium sulfate, 85 mg potassium dihydrogen phosphate, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.3 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.025 mg cobalt chloride, 27.8 mg ferrous sulfate, 100 mg inositol, 2 mg glycine, 0.1 mg thiamine hydrochloride (vitamin B1), 0.5 mg pyridoxine hydrochloride (vitamin B6), 37.3 mg disodium EDTA, 10 g agar, and 10 g sucrose.

[0030] Adjust the pH of the 1 / 2MS medium to 5.8, autoclave at 121 °C for 20 min, and then dispense 30 mL of the medium into disposable sterile petri dishes with a diameter of 9 cm and a height of 1.2 cm. After cooling and solidification, the petri dishes containing the 1 / 2MS medium are obtained.

[0031] B. Seed disinfection and seed suspension preparation: Take an appropriate amount of black capsule seeds and put them into a 5 mL centrifuge tube. Add 3 mL of 1% sodium hypochlorite solution, shake thoroughly, and disinfect for 2 min. Then, use a 10 mL sterile syringe to draw out the sodium hypochlorite solution, add 3 mL of sterile water to rinse the seeds 3 times to obtain disinfected black capsule seeds. Suspend the disinfected black capsule seeds in 1 mL of sterile water to prepare a seed suspension.

[0032] The seed suspension was prepared as follows: Because the seeds of *Capsella bursa-pastoris* are extremely small (thousand-seed weight <0.001 g), 0.0003 g (approximately 300 seeds) was weighed for the seed suspension. The *Capsella bursa-pastoris* seeds were sterilized and then suspended in 1 ml of sterile water. Some seed loss may occur during sterilization, but the final seed concentration should be approximately 200-300 seeds per ml of sterile water.

[0033] C. Inoculation: Use a pipette to transfer 100 μL of seed suspension (containing about 20-30 seeds) into a culture dish containing 1 / 2 MS medium.

[0034] D. Seed germination: Using a pipette, add 2 mL of 0.1 μmol / L GR24 solution to the seed suspension in the 1 / 2 MS medium. Gently rotate the petri dish to spread the seeds to most of the dish using the liquid. After sealing the petri dish with sealing film, place it in a 25 ℃ light and temperature incubator and provide 12 h of light per day at a light intensity of 22.22 μmol·m⁻²·s⁻¹. The sucrose concentration in the 1 / 2 MS medium is 1% (w / v).

[0035] Comparative Example 1: A method for raising seedlings of black capsule The procedure is the same as in Example 1, except that in step B, after disinfecting with sodium hypochlorite and rinsing the seeds three times with 3 mL of sterile water to obtain disinfected black capsule seeds, the seeds are soaked in 0.1 μmol / L GR24 solution for 12 h, rinsed again with sterile water, and then a seed suspension is prepared. In step D, during seed germination, sterile water is used instead of GR24 solution.

[0036] Comparative Example 2: A method for raising seedlings of black capsule The procedure was the same as in Comparative Example 1, except that in step D, a 300 mg / L GA3 solution was used instead of a 0.1 μmol / L GR24 solution.

[0037] Experiment Example 1: Effects of Aseptic Culture Methods on Seed Germination Rate and Seedling Rate of Black Capsule Black capsule seeds were cultured using the aseptic culture methods described in Example 1 and Comparative Examples 1-2, respectively, and designated as Group 1 (Example 1), Group 1 (Comparative Example 1), and Group 2 (Comparative Example 2). The germination rate of black capsule seeds in the culture dishes of different groups was statistically analyzed on days 7 and 14 of culture, and the seedling rate was statistically analyzed on days 14 and 28. The seedling determination criterion was that the black capsule seed shed its seed coat and developed 2-4 true leaves. The statistical results are summarized in Table 1.

[0038] The formula for calculating the seedling survival rate is: Seedling survival rate = Number of seedlings / Total number of seeds × 100%.

[0039] Table 1. Germination rate and seedling survival rate of black capsule seeds in different groups (%)

[0040] Note: In Table 1, due to occasional contamination of individual petri dishes, the number of culture medium samples in different groups is not exactly the same, but the number of petri dish samples (n) is between 5 and 6; different letters in the same column of the table represent the treatments. P Significant differences at the <0.05 level (based on one-way ANOVA and LSD multiple comparisons).

[0041] As shown in Table 1, the germination rate of *Capsella bursa-pastoris* seeds in Comparative Example 2 was 0% at 14 days. The germination rate in Comparative Example 1 was 22.90%, both significantly lower than the 85.91% germination rate in Example 1. Furthermore, in Example 1, the seedling survival rate reached 77.33% on day 14, and all germinating seeds developed into seedlings by day 28. In contrast, the seedling survival rate in Comparative Example 2 was only 4.44% on day 14 and only 22.90% on day 28.

[0042] In conclusion, GA3 soaking treatment did not promote the germination and seedling formation of *Capsella bursa-pastoris* seeds, while GR24 soaking could promote seed germination to some extent. However, in Comparative Example 1, the seedling formation rate and survival rate of *Capsella bursa-pastoris* on days 14 and 28 were significantly lower than those treated in Example 1. This indicates that adding GR24 to the surface of the culture medium is more conducive to the formation of *Capsella bursa-pastoris* seedlings.

[0043] Example 2: A simple and efficient method for raising seedlings of black capsule. The procedure is the same as in Example 1, except that the concentration of the GR24 solution used in step D is 2.5 μmol / L.

[0044] Example 3: A simple and efficient method for raising seedlings of black capsule. The steps are the same as in Example 2, except that the concentration of the GR24 solution used in step D is 5 μmol / L.

[0045] Example 4: A simple and efficient method for raising seedlings of black capsule. The steps are the same as in Example 2, except that the concentration of the GR24 solution used in step D is 10 μmol / L.

[0046] Comparative Example 3: A method for raising seedlings of black capsule The steps are the same as in Example 2, except that the culture medium used is water agar medium.

[0047] Comparative Example 4: A method for raising seedlings of black capsule The steps are the same as in Example 3, except that the culture medium used is water agar medium.

[0048] Comparative Example 5: A method for raising seedlings of black capsule The steps are the same as in Example 4, except that the culture medium used is water agar medium.

[0049] Comparative Example 6: A method for raising seedlings of black capsule The steps are the same as in Example 1, except that in step D, GR24 was not used to treat the black capsule seeds, but sterile water was used to spread the seed suspension in the dish.

[0050] Comparative Example 7: A method for raising seedlings of black capsule The steps are the same as in Example 1, except that in step D, GR24 was not used to treat the seeds of *Capsella bursa-pastoris*, but sterile water was used to disperse the seeds in the seed suspension into the dish; and the culture medium used was water agar medium.

[0051] Experiment Example 2: Validation of the effects of different culture media and GR24 concentrations Black capsule seeds were cultured using the aseptic culture methods described in Examples 2-4 and Comparative Examples 3-7. The germination rate of black capsule seeds was counted on day 7 of culture, and the seedling rate was counted on day 28 of culture. The results are summarized in Table 2.

[0052] Table 2. Germination rate and seedling rate of *Capsella bursa-pastoris* seeds under different culture media and GR24 treatment (%)

[0053] Note: Sample size n=5; the same letter in the same column of the table represents the treatment interval. P There was no significant difference at the <0.05 level.

[0054] Analysis of variance was performed on the data in Table 2. The results showed that the types of culture media (F=2.654) were significantly different. P =0.116) and GR24 concentration (F=1.041, P =0.368) and the interaction between the two (F=2.859, P =0.077) had no significant effect on the germination rate of *Capsella bursa-pastoris* seeds. In Comparative Examples 6 and 7, *Capsella bursa-pastoris* seeds did not germinate without the addition of GR24; however, in Examples 2-4 and Comparative Examples 3-5, the addition of GR24 resulted in high germination rates (>90%, Table 2). In Example 3, the germination rate reached as high as 97.40%. Comparison of the results from Examples 2-4 and Comparative Examples 3-5 revealed that although the germination rates of *Capsella bursa-pastoris* seeds were relatively high in both 1 / 2 MS sucrose medium and water agar medium, there were significant differences in their seedling survival rates.

[0055] The seeds of *Capsella burmannii* in the culture media of Examples 2-4 and Comparative Examples 3-5 were photographed on day 28 after germination. The results are as follows: Figures 1-6As shown, in the water agar medium of Comparative Examples 3-5, the seeds of *Capsella bursa-pastoris* only germinated but could not shed their seed coat to form seedlings; while in the 1 / 2 MS sucrose medium of Examples 2-4, the seeds of *Capsella bursa-pastoris* germinated and could normally shed their seed coat and grow into seedlings.

[0056] Example 5: A simple and efficient method for raising seedlings of black capsule. The procedure is the same as in Example 1, except that in step D, the concentration of the GR24 solution is 5 μmol / L, and the sucrose concentration in the 1 / 2 MS medium is 0.5% (w / v).

[0057] Example 6: A simple and efficient method for raising seedlings of black capsule. The procedure is the same as in Example 5, except that the sucrose concentration is 1% (w / v).

[0058] Example 7: A simple and efficient method for raising seedlings of black capsule. The procedure is the same as in Example 5, except that the sucrose concentration is 2% (w / v).

[0059] Comparative Example 8: A method for raising seedlings of black capsule The procedure is the same as in Example 5, except that no sucrose is added to the 1 / 2MS medium, i.e., the sucrose concentration used is 0%.

[0060] Experiment Example 3: Verification of the effects of different sucrose concentrations in the culture medium Black capsule seedlings were cultured using the methods shown in Examples 5-7 and Comparative Example 8. The germination rate of seedlings was recorded on day 7 and day 14. The seedling survival rate and the above-ground height of the seedlings were recorded on day 28. The results are summarized in Table 3.

[0061] Table 3. Germination rate and seedling survival rate of *Capsella bursa-pastoris* seeds under different sucrose concentrations (%)

[0062] Note: In Table 3, due to occasional contamination of individual petri dishes, the number of culture medium samples in different groups is not exactly the same, but the sample number n of petri dishes is between 9 and 11; different letters in the same column of the table represent the treatments. P Significant differences at the <0.05 level (based on one-way ANOVA and LSD multiple comparisons).

[0063] As shown in Table 3, the addition of sucrose had no significant effect on the germination of *Capsella bursa-pastoris* seeds, but it significantly affected seedling formation. In Comparative Example 8, the *Capsella bursa-pastoris* seeds only germinated on day 14, but the seed coat did not shed, and no seedlings formed. However, at other sucrose concentrations, the seeds germinated successfully, shed their seed coats, and grew 2-4 true leaves, forming seedlings. But the seedling formation rate and seedling growth varied under different sucrose concentrations. Specifically, the seedling formation rate and above-ground height of *Capsella bursa-pastoris* seedlings were lower under the 0.5% sucrose concentration treatment than under the 1% and 2% sucrose treatments; the above-ground height of seedlings was highest under the 2% sucrose treatment. This indicates that the addition of sucrose is crucial for the formation of *Capsella bursa-pastoris* seedlings. Sucrose, as an important energy source, plays a vital role in promoting plant growth. *Capsella bursa-pastoris* seeds are extremely small and lack endogenous nutrients; the addition of sucrose can provide sufficient energy for the growth of *Capsella bursa-pastoris* seedlings.

[0064] Example 8: A simple and efficient method for raising seedlings of black capsule. The procedure is the same as in Example 1, except that the concentration of GR24 is 1 μmol / L.

[0065] Comparative Example 9: A method for raising seedlings of black capsule The procedure is the same as in Example 8, except that in step A, the GR24 solution is directly added to 1 / 2 MS medium to prepare a medium containing 1 μmol / L GR24; there is no restriction on the order of adding the GR24 solution, it can be added when mixing the medium raw materials. In step D, GR24 is not used to treat the black capsule seeds, but sterile water is used to spread the seed suspension in the dish.

[0066] Experiment Example 4: Verification of the Effect of GR24 Usage Black capsule seedlings were cultured using the methods shown in Example 8 and Comparative Example 9. The germination rate of seedlings was counted on day 7 and day 14, and the seedling survival rate was counted on day 28. The results are summarized in Table 4.

[0067] Table 4. Germination rate (%) of black capsule seeds under different GR24 application methods

[0068] Note: n=10; the same letter in the same column of the table represents the processing interval. P No significant difference at the <0.05 level (based on two independent samples t-test).

[0069] Table 4 shows that adding GR24 to the surface of the culture medium in the dish and adding GR24 to the culture medium had no significant effect on the germination of *Capsella bursa-pastoris* seeds (t=0.739, t=0.739). P=0.469), the germination rate of *Capsella bursa-pastoris* seeds can reach over 90% under both GR24 addition methods. However, the amount of GR24 used in the two treatments is completely different. For example, if a stock solution of 10 μmol / L GR24 is used to prepare a culture medium containing 1 μmol / L GR24, then 100 ml of GR24 stock solution needs to be added to each liter of culture medium. At a dosage of 30 ml per dish, approximately 33.33 dishes are needed per liter of culture medium. If GR24 is added to the surface of the culture medium in the dish, at a dosage of 2 ml per dish, 33.33 dishes would require 66.67 ml of 1 μmol / L GR24 (6.67 ml of stock solution diluted 10 times is sufficient). Therefore, the cost saving rate = (100 - 6.67) / 100 × 100 = 93.33%. Given that the price of 1 mg GR24 ranges from 500 to 1200 yuan, which is relatively expensive, adding GR24 solution to the surface of the culture medium can not only achieve better results, but also greatly save costs.

[0070] Experiment Example 5: Verification of Seedling Growth Effect After cultivation in Examples 2-4, the seedlings at the 2-4 true leaf stage were transferred to new square petri dishes containing 1 / 2 MS medium (1% sucrose concentration) for further cultivation. Four seedlings were placed per dish, with 10 dishes per treatment. A 16-hour light period was provided daily at a light intensity of 117.85 μmol·m⁻¹. -2 · s -1 The cultivation temperature was 18-25 ℃. After 40 days, black capsule seedlings were obtained. The growth of the seedlings was observed, and the survival rate was recorded. Ten seedlings were randomly selected from each group to measure their growth indicators, and the results are summarized in Table 5.

[0071] The survival rate is calculated as: (Number of surviving black capsule seedlings / Number of transplanted black capsule seedlings) × 100%.

[0072] Table 5. Growth indicators of *Capsella bursa-pastoris* seedlings treated with different GR24 in 1 / 2 MS medium

[0073] Note: Survival rate n=12; other growth indicators n=10; different letters in the same column of the table represent the differences between treatments. P Significant differences at the <0.05 level (based on one-way ANOVA and LSD multiple comparisons).

[0074] As shown in Table 5, seedlings treated with different GR24 concentrations all grew normally after being transferred to new 1 / 2 MS medium, with a survival rate of over 90%. After 40 days of culture, the seedlings of *Capsella bursa-pastoris* were generally over 1.5 cm tall, with more than 14 leaves, 2-3 branches, and the longest root reaching over 4 cm in length, with more than 7 roots. Among them, most growth indicators of seedlings after germination induced by different GR24 concentrations showed no significant differences, except that the seedlings treated with 5 μmol / L GR24 were significantly taller than those treated with other GR24 concentrations.

[0075] Experiment Example 6: Verification of Seedling Growth Effect The 2-4 true leaf stage seedlings obtained in Examples 5-7 were transferred to new glass tubes containing 1 / 2 MS medium of the corresponding sucrose concentration for culture, with one seedling per tube and 10 replicates per treatment. A 16-hour light exposure was provided daily at a light intensity of 117.85 μmol·m⁻². -2 ·s -1 The cultivation temperature was 18-28℃. The growth of the black capsule seedlings was observed during the cultivation process, and plant growth was recorded and photographed on day 50. The results are as follows: Figure 7 As shown in Table 6.

[0076] Table 6. Growth of *Capsella burmannii* seedlings under different sucrose concentrations

[0077] During the experiment, some black capsule seedlings exhibited clump growth (clumped seedlings), slow growth, and no obvious dominant branching. Figure 7 Some black capsule seedlings have only one above-ground branch or one dominant above-ground branch (single-stem seedlings), and their growth is normal. Figure 8 The proportion of clump-forming seedlings varied under different sucrose concentrations. The lowest proportions were 20% and 10% under 0.5% and 1% sucrose treatments, respectively. The proportion of clump-forming seedlings was higher in the 2% sucrose treatment, reaching 40%, and the clump-forming phenomenon was more pronounced.

[0078] Five normally growing *Capsella burmannii* seedlings were randomly selected under different sucrose concentration treatments to statistically analyze growth indicators such as plant height, number of leaves, and fresh weight of above-ground parts. The results are as follows: Figure 8 As shown in Table 7.

[0079] Table 7. Growth indicators of *Capsella bursa-pastoris* seedlings treated with different sucrose concentrations after 50 days of culture in 1 / 2 MS medium.

[0080] Note: n=5; different letters in the same column of the table represent the processing intervals. P Significant differences at the <0.05 level (based on one-way ANOVA and LSD multiple comparisons).

[0081] The results showed that the growth of *Capsella bursa-pastoris* seedlings treated with 1% and 2% sucrose was better than that treated with 0.5% sucrose. The seedlings treated with 1% sucrose exhibited the best growth, reaching a height of 16.40 cm and 15.80 leaves. The seedlings treated with 2% sucrose showed slightly less vigor than those treated with 1% sucrose, and a higher proportion of clump-forming seedlings were observed. This indicates that, unlike Experiment 3 where a high sucrose concentration (2%) was beneficial for the growth of newly sprouted seedlings, the sucrose requirement decreased as the seedlings grew larger.

[0082] In summary, this invention provides a simple and efficient method for cultivating black capsule seedlings and its application. By combining the novel plant hormone strigolactone analog GR24 with a 1 / 2 MS medium containing mineral nutrients and sucrose, a high seed germination rate (up to 97%), seedling establishment rate (>90%), and seedling survival rate (nearly 100%) were achieved. Black capsule seedlings at the 2-4 true leaf stage grew well when transferred to glass tubes containing 1% sucrose in 1 / 2 MS medium, reaching a height of approximately 16 cm after 50 days, exhibiting robust growth. This method is simple to operate, low in cost, and can obtain a large number of black capsule seedlings in a short period, laying the foundation for the large-scale propagation of this rare and precious medicinal herb from Yunnan, and has high potential for widespread application.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A simple and efficient method for raising seedlings of black capsule, characterized in that, Includes the following steps: (1) Seed culture: Sterilized black capsule seeds and strigolactone analogue solution were added to 1 / 2 MS medium for seed culture to obtain black capsule seedlings; The concentration of strigolactone analog in the strigolactone analog solution is 0.1~10 μmol / L; The volume ratio of the strigolactone analog solution to 1 / 2 MS medium is 1~3:15; (2) Seedling cultivation: The black capsule seedlings were transferred to a new 1 / 2 MS medium for seedling culture to obtain black capsule seedlings; In the 1 / 2MS culture medium described in steps (1) and (2), the sucrose concentration is 0.5% to 2% by volume.

2. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, The disinfection described in step (1) includes: soaking the seeds of black capsule in a 1% sodium hypochlorite solution, shaking for 2-3 minutes, and then rinsing the seeds with sterile water 2-4 times.

3. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, After disinfection, the process also includes adding sterile water to the disinfected black capsule seeds to prepare a seed suspension, and then adding the seed suspension to the surface of 1 / 2MS culture medium.

4. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 3, characterized in that, The sterile water includes reverse osmosis water and / or deionized water.

5. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, The addition of the strigolactone analog solution to the 1 / 2 MS medium in step (1) is as follows: the strigolactone analog solution is dropped onto the surface of the 1 / 2 MS medium; The strigolactone analog solution was added at the seed site of *Capsella bursa-pastoris* on the surface of 1 / 2 MS medium.

6. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, The seed culture conditions described in step (1) are as follows: culture time 14-28 days; culture temperature 23-27℃; photoperiod 12 h light / 12 h dark; light intensity 20-25 μmol·m -2 ·s -1 .

7. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, The conditions for seedling cultivation in step (2) are as follows: cultivation temperature 18~28℃; photoperiod of 16 h light / 8 h dark; light intensity of 115~120 μmol·m -2 ·s -1 .

8. The method for raising seedlings of *Capsella bursa-pastoris* as described in claim 1, characterized in that, The black capsule seedlings include black capsule seedlings at the 2-4 true leaf stage.

9. The application of the method for raising seedlings of *Capsella bursa-pastoris* according to any one of claims 1 to 8, characterized in that, The application includes one or more of the following aspects: 1) Improve the germination rate of black capsule seeds; 2) Promotes seedling emergence from black capsule seeds; 3) Promote the growth of black capsule seedlings and / or black capsule seedlings.

10. The application as described in claim 9, characterized in that, The promotion of black capsule seedlings and / or black capsule seedling growth includes one or more of the following aspects: 1) Improve the survival rate of black capsule seedlings and / or black capsule seedlings; 2) Increase the plant height of black capsule seedlings and / or black capsule seedlings; 3) Increase the number of leaves in black capsule seedlings and / or black capsule seedlings; 4) Increase the number of branches in black capsule seedlings and / or black capsule seedlings; 5) Promotes root growth in black capsule seedlings and / or black capsule seedlings; 6) Increase the number of roots of black capsule seedlings and / or black capsule seedlings.

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