A tissue culture rapid propagation method and application of a superior clone of Sharp-pointed Mountain Fragrant Round

Abstract

This invention relates to the field of plant tissue culture technology, specifically to a method and application for the rapid propagation of superior clonal lines of *Rhizophora stylosa*. The method for rapid propagation of superior clonal lines of *Rhizophora stylosa* includes the following steps: sterilizing superior single-bud stem segments of *Rhizophora stylosa* as explants, and then inducing differentiation of adventitious buds in a primary culture medium; placing the adventitious buds in a secondary culture medium for proliferation culture to obtain secondary seedlings; placing the secondary seedlings in a rooting culture medium for rooting culture to obtain rooted seedlings; and transplanting the rooted seedlings into a substrate, controlling humidity and light for hardening off. This invention's method for tissue culture propagation of superior clonal lines of *Rhizophora stylosa* has the advantages of high induction rate, high proliferation rate, and high rooting rate, enabling the production of more high-quality *Rhizophora stylosa* seedlings in a short time, meeting the needs of large-scale commercial production.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically disclosing a method and application for rapid propagation of the superior clonal line of *Rhizophora stylosa* through tissue culture. Background Technology

[0002] Ruijianshan Xiangyuan, belonging to the genus *Cinnamomum* of the family Caryophyllaceae, is an evergreen shrub or small tree that grows in valleys, forest edges, or thickets. It is distributed in Jiangxi, Hunan, and Fujian provinces. A national protected traditional Chinese medicine, it is used to treat pharyngitis, tonsillitis, tonsillar abscess, upper respiratory tract infections, bronchitis, and other diseases, exhibiting rapid and high efficacy. Traditional seed propagation is commonly used, but the offspring's traits segregate after seed propagation, failing to maintain the superior characteristics of the parent plant. Tissue culture propagation of Ruijianshan Xiangyuan, however, preserves the superior traits of the parent plant, providing a pathway for the rapid propagation of superior individual plants.

[0003] The prior art discloses a rapid propagation method for *Syzygium serratum* regenerated plants, including disinfection of *Syzygium serratum* leaves, callus induction, callus differentiation, root induction, hardening-off, and transplanting. The main steps are as follows: Young leaves of *Syzygium serratum* are taken and disinfected; young leaves are selected, rinsed thoroughly with running water, soaked in 1% sodium hypochlorite for 8 minutes, immersed in 70% alcohol for 1 minute, and rinsed 5 times with sterile water until no residue remains. The disinfected leaves are inoculated into a culture medium MSB + 1.0 mg / L 6-BA + 0.02 mg / L NAA for callus induction, with 30 g / L sucrose and 7 g / L agar added. The temperature is 24℃, in the dark, and the pH is 5.8. The induced callus is then placed in a culture medium MSB + 1.0-1.2 g / L. Callus differentiation was initiated using 6-BA + 0.8-1.0 mg / L KT + adenine sulfate 10-20 mg / L, supplemented with 30 g / L sucrose and 7 g / L agar. The temperature was 24℃, the light intensity was 2500 lx, the daily light duration was 10 h, and the pH was 5.8. The adventitious shoots induced by differentiation were placed in MS medium for rooting induction. After rooting, the test-tube seedlings were hardened off and transplanted. The hardening method was to wash off the agar from the rooted seedlings, transplant them into a seedling tray containing stone: peat = 1:1, cover them with plastic film, remove the film after one week, and grow them in a glass greenhouse.

[0004] The aforementioned technique utilizes the indirect organogenesis pathway in tissue culture, where explants undergo dedifferentiation to form callus, which then differentiates into organs, resulting in regenerated plants. However, this pathway still has certain drawbacks. The cells in the callus are genetically unstable, making it impossible to maintain the superior traits of the parent plant. Furthermore, as the subculture time of the callus increases, the initial plant regeneration ability gradually declines or even disappears completely. Therefore, developing a rapid tissue culture propagation method for superior clones of *Rhizophora stylosa* that can maintain the superior traits of the parent plant, achieve high induction rates, high proliferation rates, high rooting rates, and short cultivation times is of significant practical importance for promoting the development and utilization of *Rhizophora stylosa*. Summary of the Invention

[0005] In view of this, the present invention provides a tissue culture method for rapid propagation of the superior clone of Ruijianshan Xiangyuan, which can maintain the excellent traits of the parent plant, with high induction rate, high multiplication rate, high rooting rate and short cultivation time.

[0006] In a first aspect, the present invention provides a method for rapid propagation of a superior clonal line of *Ruijianshan Xiangyuan* through tissue culture, comprising the following steps:

[0007] (1) Selection and disinfection of explants: Select single-bud stem segments of *Rhizophora stylosa* as explants. First, disinfect with 70-75% alcohol, rinse once with sterile water, then disinfect with 0.1-0.2% mercuric chloride solution containing surfactant, rinse twice with sterile water, and set aside.

[0008] (2) Primary culture: The sterilized explants were placed in the primary culture medium for induction culture to induce the differentiation of adventitious shoots;

[0009] The primary culture medium consists of: MS medium, 20-30 g / L sucrose and 5-7 g / L agar;

[0010] (3) Subculture: The adventitious buds are placed in a subculture medium for proliferation culture to obtain subculture seedlings;

[0011] The subculture medium comprises: MS medium, 0.5-1 mg / L of 6-benzylaminopurine, 0.02-0.06 mg / L of naphthaleneacetic acid, 0.5-1 mg / L of gibberellin, 20-30 g / L of sucrose and 5-7 g / L of agar;

[0012] (4) Rooting culture: The subcultured seedlings are placed in a rooting culture medium for rooting culture to obtain rooted seedlings;

[0013] The rooting medium comprises: modified MS medium, 0.5-1 mg / L indolebutyric acid and 0.5-1 mg / L naphthaleneacetic acid, 20-30 g / L sucrose and 5-7 g / L agar; the modified MS medium is prepared by halving the content of macroelements in MS medium, while keeping the content of iron salts, trace elements, organic matter and inositol unchanged.

[0014] (5) Transplanting and acclimatization: Transplant the rooted seedlings into the substrate and control the humidity and light to harden them off;

[0015] The cultivation conditions in steps (2) and (3) include: temperature 24±2℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks.

[0016] The cultivation conditions in step (4) include: temperature 22±3℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks.

[0017] In one optional embodiment, the pH of the primary culture medium, the subculture medium, and the rooting medium are all 5.8-6.0.

[0018] In one optional embodiment, the primary culture medium, the subculture medium, and the rooting medium are all sterilized before use, and the sterilization conditions include: temperature 121-126℃ and sterilization time 18-20min.

[0019] In one optional embodiment, the MS culture medium comprises macroelements, iron salts, trace elements, and organic matter. The macroelements include ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, magnesium sulfate heptahydrate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, and calcium chloride dihydrate 440 mg / L. The iron salts include ferrous sulfate heptahydrate 27.8 mg / L and disodium ethylenediaminetetraacetate 37.3 mg / L. The trace elements... The elements include manganese sulfate monohydrate 16.9 mg / L, zinc sulfate heptahydrate 8.6 mg / L, boric acid 6.2 mg / L, potassium iodide 0.83 mg / L, copper sulfate pentahydrate 0.025 mg / L, cobalt chloride hexahydrate 0.025 mg / L, and sodium molybdate dihydrate 0.25 mg / L. The organic compounds include glycine 2 mg / L, pyridoxine 0.5 mg / L, nicotinic acid 0.5 mg / L, thiamine 0.1 mg / L, and inositol 100 mg / L.

[0020] In one alternative implementation, the proliferation coefficient of the subculture is 2-3.

[0021] In one alternative embodiment, the length of the adventitious bud is 1.5-2 cm.

[0022] In one optional implementation, the height of the subculture seedling is 2-2.5 cm.

[0023] In one optional implementation, the hardening-off step includes: when the new roots of the rooted seedlings grow to 1 cm, they are moved to a place with natural light of 3000-4000 Lux and humidity of 50-90% for hardening-off for 1 week.

[0024] In one alternative embodiment, the matrix comprises a mixture of vermiculite and peat moss in a mass ratio of 3-7:1.

[0025] In one optional embodiment, the mass ratio of the surfactant to the aqueous mercuric chloride solution is 1:490-510.

[0026] In one alternative embodiment, the surfactant is at least one of Tween 20 and Tween 80.

[0027] In one optional implementation, the number of cleaning cycles is 2-4.

[0028] In one optional implementation, the secondary cleaning is performed 5-7 times.

[0029] Secondly, the present invention provides an application of the tissue culture rapid propagation method of the above-mentioned excellent clonal line of Ruijianshan Xiangyuan in the industrial production of Ruijianshan Xiangyuan.

[0030] In tissue culture experiments, cytokinins and auxins are generally used together. Different addition ratios often lead to different culture effects. For example, a high auxin / cytokinin ratio is beneficial for root differentiation, while a low auxin / cytokinin ratio is beneficial for shoot differentiation.

[0031] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0032] 1. This invention provides a method for rapid propagation of superior clones of *Rhizophora stylosa* through tissue culture, comprising the following steps: (1) Selection and disinfection of explants: Select single-bud stem segments of *Rhizophora stylosa* as explants, disinfect them with 70-75% alcohol, rinse them once with sterile water, then disinfect them again with 0.1-0.2% mercuric chloride aqueous solution containing surfactant, rinse them twice with sterile water, and set aside; (2) Primary culture: Place the disinfected explants in a primary culture medium for induction culture to induce differentiation of adventitious buds; (3) Subculture: Place the adventitious buds in a subculture medium for proliferation culture to obtain subculture seedlings; (4) Rooting culture (5) Transplanting and acclimatization: The subcultured seedlings are placed in a rooting medium for rooting culture to obtain rooted seedlings; (6) Transplanting and acclimatization: The rooted seedlings are transplanted into a substrate, and humidity and light are controlled for hardening; The culture conditions in steps (2) and (3) include: temperature 24±2℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks; The culture conditions in step (4) include: temperature 22±3℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks. This invention directly differentiates explants with primary differentiation ability into organs to obtain regenerated plants, that is, adventitious buds are induced from single bud stem segments without forming callus tissue. In the process of direct organ formation, since the ploidy of cell chromosomes remains basically stable, the frequency of mutation is low, which can ensure that the Ruijianshan Xiangyuan seedlings maintain the good seed characteristics of the mother plant.

[0033] After disinfection, the explants can be thoroughly cleaned to establish a sterile system. In the primary culture, no plant hormones are added to the MS medium, which better simulates the growth environment of cells in the natural environment, helping to maintain the original characteristics and functions of the tissue. At the same time, it reduces experimental variables, simplifies the culture process, and improves the reproducibility of experiments. In the subculture, the addition of high concentrations of cytokinins (6-BA, GA3) and low concentrations of auxin (NAA) is beneficial to bud differentiation, increases the proliferation coefficient, produces a large number of plants, and enables the plants to rapidly reproduce into new individuals. In rooting culture, a modified MS medium (with macroelements halved and others remaining unchanged) is used to reduce the concentration of inorganic salts. Lower inorganic salts require more developed root systems for absorption, thus inducing root development. The modified MS medium is more suitable for the root growth environment. Increasing the addition of high concentrations of auxins (NAA, IBA) is beneficial for root differentiation and improves the rooting rate. During the rooting culture process, naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) are added simultaneously. NAA mainly promotes cell division and tissue expansion, while IBA helps promote cell differentiation and root hair formation. The combined effect of the two can better promote the rooting process of plants.

[0034] Among them, 6-BA promotes cell division and bud formation, promotes the differentiation of undifferentiated tissues, and induces callus formation; GA3 accelerates cell growth and also promotes cell division; IBA promotes stem and branch growth and can promote rooting, especially the growth of adventitious roots; NAA can promote stem and branch growth and also promote rooting. The tissue culture seedling cultivation method of the superior clonal line of *Rhizophora stylosa* of this invention has the advantages of high induction rate, high proliferation rate, and high rooting rate, and can obtain more high-quality *Rhizophora stylosa* seedlings in a short time to meet the needs of large-scale commercial production.

[0035] In tissue culture, except for the need for darkness to induce callus, a certain amount of light is generally required. Light intensity and duration significantly affect cell proliferation and organ differentiation. Current research indicates that light intensity has a clear impact on the initial division of plant cells. Generally, stronger light results in robust seedling growth, while weaker light leads to etiolation. In this invention, the light intensity for both primary and subculture cultures is 2000-2500 Lux, with a photoperiod of 14h / 10h day / dark.

[0036] When using mercuric chloride disinfectant to sterilize inoculation materials, adding surfactants can moisten the entire explant tissue, promoting full contact of the sterilizing solution with the surface tissue and achieving better disinfection results. Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 This describes the growth of adventitious buds of *Rhizoma Scutellariae rubra* in Example 1 of the present invention;

[0039] Figure 2 This describes the cultivation of *Rhizophora stylosa* subculture seedlings in Example 1 of this invention;

[0040] Figure 3 This describes the cultivation of *Rhizoma Sinensis* rooted seedlings in Example 1 of this invention.

[0041] Figure 4 This is the growth status of the transplanted seedlings of Ruijianshan Xiangyuan in Embodiment 1 of the present invention. Detailed Implementation

[0042] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0043] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0044] To address the problems existing in the aforementioned related technologies, according to a first aspect of the present invention, the present invention provides a method for rapid tissue culture propagation of a superior clonal line of *Rhizophora stylosa*, comprising the following steps: (1) Selection and disinfection of explants: Selecting single-bud stem segments of *Rhizophora stylosa* as explants, first disinfecting with 70-75% alcohol, rinsing once with sterile water, then disinfecting with a 0.1-0.2% mercuric chloride aqueous solution containing surfactant, rinsing twice with sterile water, and setting aside; (2) Primary culture: Placing the disinfected explants in a primary culture medium for induction culture to induce differentiation of adventitious buds; (3) Subculture: Placing the adventitious buds in a subculture medium for proliferation culture. (3) Cultivation to obtain subculture seedlings; (4) Rooting culture: The subculture seedlings are placed in a rooting culture medium for rooting culture to obtain rooted seedlings; (5) Transplanting and acclimatization: The rooted seedlings are transplanted into a substrate, and humidity and light are controlled for hardening; The culture conditions in steps (2) and (3) include: temperature 24±2℃, light intensity 2000-2500Lux, photoperiod is day / dark = 14h / 10h, and growth cycle is 4-5 weeks; The culture conditions in step (4) include: temperature 22±3℃, light intensity 2000-2500Lux, photoperiod is day / dark = 14h / 10h, and growth cycle is 4-5 weeks. This invention is a process of directly differentiating explants with primary differentiation ability into organs to obtain regenerated plants, that is, inducing adventitious buds from single bud stem segments without forming callus tissue. During the direct organogenesis process, the ploidy of cell chromosomes remains relatively stable, resulting in a low frequency of variation, which ensures that the Ruijianshan Xiangyuan seedlings maintain the good genetic characteristics of the mother plant.

[0045] After disinfection, the explants can be thoroughly cleaned to establish a sterile system. In primary culture, no plant hormones are added to the MS medium. In subculture, high concentrations of cytokinins (6-BA, GA3) and low concentrations of auxin (NAA) are added, which promotes shoot differentiation, increases the proliferation coefficient, and produces a large number of plants, enabling rapid propagation into new individuals. In rooting culture, a modified MS medium (with macroelements halved and others unchanged) is used to reduce the inorganic salt concentration. Lower inorganic salts require more developed root systems for absorption, thus inducing root development. The modified MS medium is more suitable for root growth. Adding higher concentrations of auxin (NAA, IBA) promotes root differentiation and increases the rooting rate. During rooting culture, naphthaleneacetic acid (NAA) and indolebutyric acid (IBA) are added simultaneously. NAA primarily promotes cell division and tissue expansion, while IBA helps promote cell differentiation and root hair formation. The combined effect of both enhances the rooting process.

[0046] Among them, 6-BA promotes cell division and bud formation, promotes the differentiation of undifferentiated tissues, and induces callus formation; GA3 accelerates cell growth and also promotes cell division; IBA promotes stem and branch growth and can promote rooting, especially the growth of adventitious roots; NAA can promote stem and branch growth and also promote rooting. The tissue culture seedling cultivation method of the superior clonal line of *Rhizophora stylosa* of this invention has the advantages of high induction rate, high proliferation rate, and high rooting rate, and can obtain more high-quality *Rhizophora stylosa* seedlings in a short time to meet the needs of large-scale commercial production.

[0047] In tissue culture, except for the need for darkness to induce callus, a certain amount of light is generally required. Light intensity and duration significantly affect cell proliferation and organ differentiation. Current research indicates that light intensity has a clear impact on the initial division of plant cells. Generally, stronger light results in robust seedling growth, while weaker light leads to etiolation. In this invention, the light intensity for both primary and subculture cultures is 2000-2500 Lux, with a photoperiod of 14h / 10h day / dark.

[0048] When using mercuric chloride disinfectant to sterilize inoculation materials, adding surfactants can moisten the entire explant tissue, promoting full contact of the sterilizing solution with the surface tissue and achieving better disinfection results.

[0049] The initial culture is conducted via axillary bud germination, which is the direct developmental pathway. Most forest plants suitable for rapid tissue culture propagation can be cultured via this pathway, resulting in less genetic variation in regenerated plants compared to callus induction. From the physiological perspective of the explant, the developmental stage and degree of tissue differentiation also influence the differentiation pathway. Earlier explants, such as young leaves, may more easily form callus, while more mature explants, such as stem segments, may more easily develop organs directly. The concentration of hormones and nutrients in the culture medium can also affect the differentiation pathway of the explant. Adjusting the concentration and type of hormones in the culture medium can promote callus formation or direct organogenesis. This invention uses axillary bud germination in the initial culture process, which can rapidly obtain a large number of sterile tissue culture seedlings with a fast propagation speed. In contrast, the indirect developmental pathway, i.e., the formation of callus, is more difficult for woody plants to re-differentiate from callus, making it difficult to obtain a large number of high-quality *Ilex chinensis* seedlings in a short time, and thus unable to meet the needs of large-scale commercial production.

[0050] This invention provides an MS culture medium comprising macroelements, iron salts, trace elements, and organic matter. The macroelements include ammonium nitrate (1650 mg / L), potassium nitrate (1900 mg / L), magnesium sulfate heptahydrate (370 mg / L), potassium dihydrogen phosphate (170 mg / L), and calcium chloride dihydrate (440 mg / L). The iron salts include ferrous sulfate heptahydrate (27.8 mg / L) and disodium EDTA (37.3 mg / L). The trace elements include manganese sulfate monohydrate (16.9 mg / L), zinc sulfate heptahydrate (8.6 mg / L), boric acid (6.2 mg / L), potassium iodide (0.83 mg / L), copper sulfate pentahydrate (0.025 mg / L), cobalt chloride hexahydrate (0.025 mg / L), and sodium molybdate dihydrate (0.25 mg / L). The organic matter includes glycine (2 mg / L), pyridoxine (0.5 mg / L), nicotinic acid (0.5 mg / L), thiamine (0.1 mg / L), and inositol (100 mg / L). The preparation process of culture media at each stage of tissue culture includes the following steps:

[0051] (1) Method for preparing macro-element solutions, iron salt solutions, micro-element solutions, and organic matter solutions in MS culture medium: Macro-element solutions are prepared into a 20-fold 1L stock solution (stock solution 1); iron salt solutions are prepared into a 100-fold 1L stock solution (stock solution 2); micro-element solutions are prepared into a 100-fold 1L stock solution (stock solution 3); and organic matter solutions are prepared into a 100-fold 1L stock solution (stock solution 4). Specifically:

[0052] Preparation of Mother Solution 1: Weigh out 33g of ammonium nitrate, 38g of potassium nitrate, 7.4g of magnesium sulfate heptahydrate, 3.4g of potassium dihydrogen phosphate, and 8.8g of calcium chloride dihydrate. Dissolve them thoroughly in sequence, mix them evenly, and transfer them to a volumetric flask to make up to 1L.

[0053] Preparation of Mother Solution 2: Weigh 2.78g of ferrous sulfate heptahydrate and 3.73g of disodium ethylenediaminetetraacetate, dissolve and mix them thoroughly, and transfer them to a volumetric flask to make up to 1L.

[0054] Preparation of Mother Solution 3: Weigh 1.69g of manganese sulfate monohydrate, 0.86g of zinc sulfate heptahydrate, 0.62g of boric acid, 0.083g of potassium iodide, 0.0025g of copper sulfate pentahydrate, 0.0025g of cobalt chloride hexahydrate and 0.025g of sodium molybdate dihydrate, dissolve them thoroughly in sequence, mix them evenly, and transfer them to a volumetric flask to make up to 1L.

[0055] Preparation of Mother Solution 4: Weigh 0.2g of glycine, 0.05g of pyridoxine, 0.05g of nicotinic acid, 0.01g of thiamine and 10g of inositol, dissolve and mix them thoroughly in sequence, and transfer them to a volumetric flask to make up to 1L.

[0056] (2) Prepare 100 mL of each of the plant growth regulators 6-benzylaminopurine, gibberellin, indolebutyric acid and naphthaleneacetic acid at a concentration of 1 mg / mL.

[0057] Preparation of 6-benzylaminopurine (6-BA) solution: Weigh 0.1 g of 6-benzylaminopurine powder, dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, add pure water and transfer to a 100 mL volumetric flask, make up to volume, mix well, and store in a 4℃ refrigerator for later use.

[0058] Preparation of gibberellin (GA3) solution: Weigh 100 mg of gibberellin powder, dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, add pure water and transfer to a 100 mL volumetric flask, make up to volume, mix well, and store in a 4℃ refrigerator for later use.

[0059] Preparation of indolebutyric acid (IBA) solution: Weigh 100 mg of indolebutyric acid powder, dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, add pure water and transfer to a 100 mL volumetric flask, make up to volume, mix well, and store in a 4℃ refrigerator for later use.

[0060] Preparation of naphthaleneacetic acid (NAA) solution: Weigh 100 mg of naphthaleneacetic acid powder, dissolve it in 1 mL of 1 mol / L sodium hydroxide solution, add pure water and transfer to a 100 mL volumetric flask, make up to volume, mix well, and store in a 4℃ refrigerator for later use.

[0061] (3) Prepare primary culture medium, proliferation culture medium and rooting culture medium.

[0062] Preparation of primary culture medium: Take 50 mL of mother liquor 1, 10 mL of mother liquor 2, 10 mL of mother liquor 3 and 10 mL of mother liquor 4 from step (1), weigh 20-30 g of sucrose and 5-7 g of agar powder, add pure water to mix and dissolve, make up to 1 L, adjust the pH to 5.8-6.0, heat to boiling, dispense into 100 mL test tube bottles, put them in a high temperature and high pressure sterilizer at 121℃ for 20 min, and then put them in a condenser to cool for later use.

[0063] Preparation of subculture medium: Measure 50 mL of mother liquor 1, 10 mL of mother liquor 2, 10 mL of mother liquor 3 and 10 mL of mother liquor 4 from step (1), weigh 20-30 g of sucrose and 5-7 g of agar powder, add pure water to mix and dissolve, then add 0.5-1 mL of 1 mg / mL 6-benzylaminopurine solution, 0.02-0.06 mL of 1 mg / mL naphthaleneacetic acid solution and 0.5-1 mL of 1 mg / mL gibberellin solution from step (2), make up to 1 L, adjust pH to 5.8-6.0, heat to boiling, dispense into 350 mL glass bottles, sterilize in a high temperature and high pressure autoclave at 121℃ for 20 min, and then cool in a condenser for later use.

[0064] Preparation of rooting medium: Measure 25 mL of mother liquor 1, 10 mL of mother liquor 2, 10 mL of mother liquor 3 and 10 mL of mother liquor 4 from step (1), weigh 20-30 g of sucrose and 5-7 g of agar powder, add pure water to mix and dissolve, then add 500-1000 μL of indolebutyric acid solution and 500-1000 μL of naphthaleneacetic acid solution from step (2), make up to 1 L, adjust the pH to 5.8-6.0, heat to boiling, dispense into 350 mL glass bottles, put them in a high temperature and high pressure sterilizer at 121℃ for 20 min, and then cool in the condenser for later use.

[0065] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0066] Example 1

[0067] This embodiment provides a method for rapid propagation of the superior clonal line of Ruijianshan Xiangyuan through tissue culture, including the following steps:

[0068] (1) Selection of explants: Select healthy, disease-free, and current-year-old sharp-tipped succulents with axillary buds, remove the leaves, and cut them into 2cm single-bud stem segments. Rinse with running water for 2 hours to remove dust and any adhering impurities from the surface of the explants. Then transfer them to a clean bench and sterilize the utensils and tools under high temperature and pressure. Use tweezers to transfer the single-bud stem segments to a sterile bottle, pour in 75% alcohol for 50 seconds, and then quickly pour it out. Rinse with sterile water 3 times, and then sterilize with 0.1% mercuric chloride for 6 minutes. At the same time, add 1-2 drops of Tween 80. The mass ratio of surfactant to mercuric chloride solution is 1:500. After pouring out the mercuric chloride, rinse with sterile water 6 times until there is no residue. Shake continuously during each rinse to ensure that the explants are thoroughly cleaned.

[0069] (2) Primary culture: The explants described in step (1) were vertically inoculated into the primary culture medium, one explant per bottle, and then placed in a culture room for induction culture. The culture temperature was 25℃, the light intensity was 2000 Lux, the photoperiod was day / dark = 14h / 10h, and the growth cycle was 4 weeks. Adventitious shoots were induced to differentiate. The growth of adventitious shoots is shown in the figure. Figure 1 As shown, the induction rate was 55%; the primary culture medium consisted of MS medium supplemented with 30 g / L sucrose and 7 g / L agar, with a pH of 5.8.

[0070] (3) Subculture: Cut 1.5-2cm adventitious buds from step (2) and transfer them to subculture for proliferation culture to increase the number of tissue culture seedlings. The culture temperature is 25℃, the light intensity is 2000Lux, the photoperiod is day / dark = 14h / 10h, the growth cycle is 4 weeks, and the proliferation coefficient is 2.3 to obtain subculture seedlings. The culture details of the subculture seedlings are shown in [link to relevant documentation]. Figure 2 As shown; the subculture medium includes MS medium supplemented with 0.5 mg / L 6-BA, 0.03 mg / L NAA, 0.75 mg / L GA3, 30 g / L sucrose and 7 g / L agar, with a pH of 5.8.

[0071] (4) Rooting Culture: Select robust plants with a height of 2-2.5cm from the subculture seedlings, remove the basal leaves, and vertically inoculate them into the rooting medium for rooting culture. The culture temperature is 25℃, the light intensity is 2000Lux, the photoperiod is day / dark = 14h / 10h, and the growth cycle is 4 weeks to obtain rooted seedlings. The culture process of the rooted seedlings is shown in [link to relevant documentation]. Figure 3 As shown, the rooting medium is a modified MS medium with the addition of 1 mg / L IBA, 1 mg / L NAA, 30 g / L sucrose and 7 g / L agar, pH 5.8, and a rooting rate of 93%, with an average of 2.4 roots per plant.

[0072] (5) Transplanting: When the roots of the seedlings reach 1cm, place them under natural light at 3500Lux and humidity at 70% for one week to harden them off. Fill 50-cell trays with the prepared substrate (vermiculite and peat moss in a 5:1 mass ratio). Remove the seedlings from the trays, wash off the substrate with clean water, and vertically plant the entire plant into the prepared substrate. Place the trays in a small greenhouse to begin acclimatization. See below for the growth status of the transplanted seedlings. Figure 4 As shown, by providing appropriate shading to the seedlings, covering them with plastic film, and maintaining a humidity of 80%, the survival rate of the seedlings can reach 95%.

[0073] Example 2

[0074] This embodiment provides a method for rapid propagation of the superior clonal line of Ruijianshan Xiangyuan through tissue culture, including the following steps:

[0075] (1) Selection of explants: Select healthy, disease-free, and current-year-old sharp-tipped succulents with axillary buds, remove the leaves, and cut into 2cm single-bud stem segments. Rinse with running water for 2 hours to remove dust and any adhering impurities from the surface of the explants. Then transfer them to a clean bench and sterilize the utensils and tools under high temperature and pressure. Use tweezers to transfer the single-bud stem segments to a sterile bottle, pour in 70% alcohol for 50 seconds, and then quickly pour it out. Rinse twice with sterile water, and then sterilize with 0.15% mercuric chloride for 6 minutes. At the same time, add 1-2 drops of Tween 20. The mass ratio of surfactant to mercuric chloride solution is 1:490. After pouring out the mercuric chloride, rinse with sterile water 7 times until there is no residue. Shake continuously during each rinse to ensure that the explants are thoroughly cleaned.

[0076] (2) Primary culture: The explants described in step (1) were vertically inoculated into the primary culture medium, one explant per bottle, and then placed in the culture room for induction culture. The culture temperature was 24℃, the light intensity was 2250 Lux, the photoperiod was day / dark = 14h / 10h, the growth cycle was 4 weeks, and adventitious shoots were induced to differentiate, with an induction rate of 49%. The primary culture medium included MS medium, with 25 g / L sucrose and 5 g / L agar added, and the pH was 5.8.

[0077] (3) Subculture: Cut 1.5-2cm of the adventitious buds mentioned in step (2) and transfer them to subculture for proliferation culture to increase the number of tissue culture seedlings. The culture temperature is 26℃, the light intensity is 2500Lux, the photoperiod is day / dark = 14h / 10h, the growth cycle is 5 weeks, and the proliferation coefficient is 2.1 to obtain subculture seedlings. The subculture medium includes MS medium and is supplemented with 0.75mg / L 6-BA, 0.02mg / L NAA, 0.5mg / L GA3, 25g / L sucrose and 6g / L agar, with a pH of 5.9.

[0078] (4) Rooting culture: Select robust plants with a height of 2-2.5cm from the subculture seedlings, cut off the basal leaves, and vertically inoculate them into the rooting medium for rooting culture; the culture temperature is 22℃, the light intensity is 2250Lux, the photoperiod is day / dark = 14h / 10h, the growth cycle is 5 weeks, and rooted seedlings with roots are obtained. The rooting medium is a modified MS medium, with 0.75mg / L IBA and 0.5mg / L NAA, 20g / L sucrose and 6g / L agar added, the pH is 6.0, the rooting rate is 92%, and the average number of roots per plant is 2.1.

[0079] (5) Transplanting: Once the root system of the seedlings reaches 1cm, place them under natural light at 3000Lux and humidity at 90% for one week to harden them off. Fill 50-cell trays with the prepared substrate (vermiculite and peat moss in a 3:1 mass ratio), remove the seedlings from the bottles, wash off the culture medium with clean water, and vertically plant the entire plant into the prepared substrate. Place the trays in a small greenhouse to begin acclimatization. Provide appropriate shading for the seedlings, cover them with plastic film, and maintain humidity at 80%. The survival rate of the seedlings can reach 94%.

[0080] Example 3

[0081] This embodiment provides a method for rapid propagation of the superior clonal line of Ruijianshan Xiangyuan through tissue culture, including the following steps:

[0082] (1) Selection of explants: Select healthy, disease-free, and current-year-old sharp-tipped succulents with axillary buds, remove the leaves, and cut them into 2cm single-bud stem segments. Rinse with running water for 2 hours to remove dust and any impurities that may be attached to the surface of the explants. Then transfer them to a clean bench and sterilize the utensils and tools under high temperature and pressure. Use tweezers to transfer the single-bud stem segments to a sterile bottle, pour in 73% alcohol for 50 seconds and then quickly pour it out. Rinse with sterile water 4 times, then sterilize with 0.2% mercuric chloride for 6 minutes. At the same time, add 1-2 drops of Tween 80. The mass ratio of surfactant to mercuric chloride solution is 1:510. After pouring out the mercuric chloride, rinse with sterile water 5 times until there is no residue. Shake continuously during each rinse to ensure that the explants are thoroughly cleaned.

[0083] (2) Primary culture: The explants described in step (1) were vertically inoculated into the primary culture medium, one explant per bottle, and then placed in the culture room for induction culture. The culture temperature was 26℃, the light intensity was 2500 Lux, the photoperiod was day / dark = 14h / 10h, the growth cycle was 5 weeks, and adventitious shoots were induced to differentiate, with an induction rate of 60%. The primary culture medium included MS medium, with 20 g / L sucrose and 6 g / L agar added, and the pH was 6.0.

[0084] (3) Subculture: Cut 1.5-2cm of the adventitious buds mentioned in step (2) and transfer them to subculture for proliferation culture to increase the number of tissue culture seedlings. The culture temperature is 24℃, the light intensity is 2250Lux, the photoperiod is day / dark = 14h / 10h, the growth cycle is 4 weeks, and the proliferation coefficient is 3 to obtain subculture seedlings. The subculture medium includes MS medium and adds 1mg / L 6-BA, 0.06mg / L NAA, 1mg / L GA3, 20g / L sucrose and 5g / L agar, with a pH of 5.8.

[0085] (4) Rooting culture: Select robust plants with a height of 2-2.5cm from the subculture seedlings, cut off the basal leaves, and vertically inoculate them into the rooting medium for rooting culture; the culture temperature is 19℃, the light intensity is 2500Lux, the photoperiod is day / dark = 14h / 10h, the growth cycle is 4 weeks, and rooted seedlings with roots are obtained. The rooting medium is a modified MS medium, with 0.5mg / L IBA and 0.75mg / L NAA, 25g / L sucrose and 5g / L agar added, the pH is 5.9, the rooting rate is 96%, and the average number of roots per plant is 3.

[0086] (5) Transplanting: Once the root system of the seedlings reaches 1 cm, place them under natural light (4000 Lux) and humidity (50%) for one week to harden them off. Fill 50-cell seedling trays with the prepared substrate (vermiculite and peat moss in a 7:1 mass ratio). Remove the seedlings from the bottles, wash off the substrate with clean water, and vertically plant the entire plant into the prepared substrate. Place the trays in a small greenhouse to begin acclimatization. Provide appropriate shading for the seedlings, cover them with plastic film, and maintain a humidity of 80%. The survival rate of the seedlings can reach 97%.

[0087] Comparative Example 1

[0088] This comparative example provides a rapid tissue culture propagation method for a superior clonal line of *Ligusticum jirunense*, which is basically the same as the steps in Example 1, except that callus induction is performed in step (2), and adventitious shoots are differentiated in step (3). The results show that the survival rate is 88%. This comparative example uses the indirect organogenesis pathway in tissue culture, where explants undergo dedifferentiation to form callus, which then differentiates into organs to obtain regenerated plants. However, due to the genetic instability of callus cells, the regeneration capacity of the plant gradually decreases or even disappears completely as the subculture time of the callus tissue is prolonged.

[0089] Comparative Example 2

[0090] This comparative example provides a rapid tissue culture propagation method for *Rhizophora stylosa*, which is basically the same as the steps in Example 1, except that young leaves of *Rhizophora stylosa* are selected as explants. The results show that the survival rate is 89%. This is because the subculture proliferation of stem segments and leaves as explants differs in tissue culture. Stem segments have more dividing and differentiating cells, and their subculture proliferation capacity is usually higher than that of leaf explants. They are more likely to form new plants through division and differentiation, and have a shorter growth cycle. Leaf explants, on the other hand, need to form plants through callus tissue, which usually takes longer and has higher requirements for plant species, tissue type, and culture conditions. In addition, the quality and quantity of callus tissue are also worse than those of stem segment explants.

[0091] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of a superior clonal line of *Ligusticum striatum* through tissue culture, characterized in that... Includes the following steps: (1) Selection and disinfection of explants: Select single-bud stem segments of *Rhizophora stylosa* as explants. First, disinfect with 70-75% alcohol, rinse once with sterile water, then disinfect with 0.1-0.2% mercuric chloride solution containing surfactant, rinse twice with sterile water, and set aside. (2) Primary culture: The sterilized explants were placed in the primary culture medium for induction culture to induce the differentiation of adventitious shoots; The primary culture medium consisted of MS medium, 20-30 g / L sucrose, and 5-7 g / L agar. (3) Subculture: The adventitious buds are placed in a subculture medium for proliferation culture to obtain subculture seedlings; The subculture medium consisted of: MS medium, 0.5-1 mg / L of 6-benzylaminopurine, 0.02-0.06 mg / L of naphthaleneacetic acid, 0.5-1 mg / L of gibberellin, 20-30 g / L of sucrose, and 5-7 g / L of agar. (4) Rooting culture: The subcultured seedlings are placed in a rooting culture medium for rooting culture to obtain rooted seedlings; The rooting medium consists of: modified MS medium, 0.5-1 mg / L indolebutyric acid and 0.5-1 mg / L naphthaleneacetic acid, 20-30 g / L sucrose and 5-7 g / L agar; the modified MS medium is prepared by halving the content of macroelements in MS medium, while keeping the content of iron salts, trace elements, organic matter and inositol unchanged. (5) Transplanting and acclimatization: Transplant the rooted seedlings into the substrate and control the humidity and light to harden them off; The cultivation conditions in steps (2) and (3) include: temperature 24±2℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks. The cultivation conditions in step (4) include: temperature 22±3℃, light intensity 2000-2500Lux, photoperiod of day / dark = 14h / 10h, and growth cycle of 4-5 weeks.

2. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The pH values ​​of the primary culture medium, the subculture medium, and the rooting medium are all 5.8-6.

0.

3. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The primary culture medium, the subculture medium, and the rooting medium were all sterilized before use. The sterilization conditions included a temperature of 121-126℃ and a sterilization time of 18-20 minutes.

4. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The MS medium comprises macroelements, iron salts, trace elements, and organic matter. The macroelements include ammonium nitrate (1650 mg / L), potassium nitrate (1900 mg / L), magnesium sulfate heptahydrate (370 mg / L), potassium dihydrogen phosphate (170 mg / L), and calcium chloride dihydrate (440 mg / L). The iron salts include ferrous sulfate heptahydrate (27.8 mg / L) and disodium EDTA (37.3 mg / L). The trace elements include manganese sulfate monohydrate (16.9 mg / L), zinc sulfate heptahydrate (8.6 mg / L), boric acid (6.2 mg / L), potassium iodide (0.83 mg / L), copper sulfate pentahydrate (0.025 mg / L), cobalt chloride hexahydrate (0.025 mg / L), and sodium molybdate dihydrate (0.25 mg / L). The organic matter includes glycine (2 mg / L), pyridoxine (0.5 mg / L), nicotinic acid (0.5 mg / L), thiamine (0.1 mg / L), and inositol (100 mg / L).

5. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The proliferation coefficient of the subculture is 2-3.

6. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The length of the adventitious bud is 1.5-2 cm; and / or, The height of the sub-generation seedlings is 2-2.5cm.

7. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to any one of claims 1-6, characterized in that, The hardening-off process includes: when the new roots of the seedlings grow to 1cm, they are moved to a place with natural light of 3000-4000 Lux and humidity of 50-90% for hardening-off for 1 week.

8. The method for rapid propagation of superior clones of *Ligusticum striatum* according to any one of claims 1-6, characterized in that, The matrix comprises a mixture of vermiculite and peat moss in a mass ratio of 3-7:

1.

9. The method for rapid propagation of the superior clonal line of *Ligusticum chuanxiong* according to claim 1, characterized in that, The mass ratio of the surfactant to the mercuric chloride aqueous solution is 1:490-510; And / or, the surfactant is at least one of Tween 20 and Tween 80; And / or, the number of cleaning cycles in a single cycle is 2-4 times; And / or, the number of secondary cleaning cycles is 5-7.

10. The use of the tissue culture rapid propagation method of the superior clonal line of Ruijianshan Xiangyuan according to any one of claims 1-9 in the industrial production of Ruijianshan Xiangyuan.

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