Tissue culture method of nine-grade xiangshu lotus and application thereof

Abstract

This invention relates to a tissue culture method for *Ligustrum lucidum* and its application, belonging to the field of plant tissue culture technology. The tissue culture medium combination for *Ligustrum lucidum* of this invention includes a sterile seedling solid-phase medium, a differentiation-inducing medium, a shoot-inducing medium, and a rooting medium. The *Ligustrum lucidum* plants obtained by this invention have a longer culture period, exhibit full growth and more mature development, robust root systems, and significantly improved stress resistance and environmental adaptability. After hardening-off treatment, the *Ligustrum lucidum* plants can gradually adapt to changes in external light, humidity, and temperature, and the final hardening-off survival rate after transplanting is high, providing important technical support for the rapid propagation and further optimization of genetic transformation technology for *Ligustrum lucidum*.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a tissue culture method for the nine-grade fragrant lotus and its application. Background Technology

[0002] Water lilies Nymphaea L.) belongs to the genus *Nymphaeus* of the family Nymphaeaceae. Nymphaea Perfume lily (also known as water lily) is a perennial aquatic herbaceous plant and a world-renowned aquatic ornamental flower. Nymphaea The hybrid *Liriope muscari*, commonly known as the Nine-Grade Fragrant Water Lily, possesses ornamental, ecological, and commercial value. It is widely cultivated in southern China (Guangdong, Guangxi, Fujian, Yunnan) and Southeast Asia (Thailand, Malaysia, Indonesia), primarily in aquatic environments with an average annual temperature above 15°C, abundant water, and ample sunlight. Botanically, the Nine-Grade Fragrant Water Lily is a perennial aquatic herb with a well-developed underground rhizome system. Its leaves mostly float on or slightly above the water surface. The leaves are round or nearly round, with smooth or slightly wavy edges, and a dark green color. The flowers are solitary at the top of the peduncle, usually emerging above the water surface to open. The flowers are relatively large and have a distinct fragrance. Flower colors are rich, including white, pink, purple, blue, red, gold, and yellow. The flowering period is long, and it can continue to bloom under suitable temperature conditions. The flowers generally open in the morning and close in the evening, exhibiting a distinct diurnal rhythm.

[0003] At present, although some progress has been made in the cultivation research of water lilies, the research on tissue culture is still in its initial stage. The existing traditional propagation methods mainly rely on division, sowing and tuber propagation, which are slow and have low coefficients. Summary of the Invention

[0004] The purpose of this invention is to provide a tissue culture method for the nine-grade fragrant lotus and its application, so as to solve the problems of slow seed propagation speed and low survival rate of the nine-grade fragrant lotus in the prior art, and to lay a stable and reliable tissue regeneration technology system for subsequent research on the genetic transformation of fragrant lotus.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a tissue culture medium combination for nine-grade fragrant lotus, including sterile seedling solid culture medium, differentiation induction culture medium, shoot cluster induction culture medium and rooting culture medium; The sterile seedling solid culture medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, activated carbon 0.5–1 g / L, Flower Treasure No. 1 0.5–1 g / L, 6-benzylaminopurine 1–1.5 mg / L, naphthaleneacetic acid 1–1.5 mg / L, and agar 5–10 g / L. The differentiation-inducing medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, zeatin 1–1.5 mg / L, naphthaleneacetic acid 1–1.5 mg / L, and agar 5–10 g / L. The shoot induction medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, 6-benzylaminopurine 0.5–1.5 mg / L, naphthaleneacetic acid 0.5–1.5 mg / L, zeatin 0.2–1 mg / L, and agar 5–10 g / L. The rooting medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, naphthaleneacetic acid 0.5–1.5 mg / L, activated carbon 0.5–1 g / L, and agar 5–10 g / L.

[0006] The present invention also provides the application of the aforementioned nine-grade fragrant lotus tissue culture medium combination in the tissue and / or plant culture of nine-grade fragrant lotus.

[0007] This invention also provides a method for tissue and / or plant culture of the nine-grade fragrant lotus, comprising the following steps: (1) Take the seeds of the nine-grade fragrant lotus, soak them in carbendazim, disinfect them with 75% alcohol, NaClO, Oxytech T-010, and soak them in sterile water to obtain detoxified seeds; (2) Place the virus-free seeds in sterile water and culture for 10-15 days to obtain germinating seedlings; (3) The germinated seedlings were cultured in a sterile seedling solid culture medium for 30-65 days to obtain sterile seedlings; (4) Different tissue parts of sterile seedlings were placed in induction differentiation medium and cultured in the dark for 5-10 days to screen for tissue culture explants; (5) Place the tissue culture explants in a shoot induction medium and culture them in the dark for 20-30 days to obtain shoots; (6) Place the seedlings in a rooting medium and culture for 10-15 days to obtain tissue culture seedlings; (7) Harden the tissue culture seedlings for 3-6 days to obtain nine-grade fragrant lotus plants.

[0008] Preferably, the rotation speed of the carbendazim soaking in step (1) is 150-250 rpm, and the soaking time of the carbendazim is 20-40 min; The alcohol disinfection is performed 1 to 2 times, and the alcohol disinfection time is 0.5 to 2 minutes per time; The concentration of NaClO is 10%–25%; The NaClO sterilization time is 6–8 minutes; The concentration of Oxytech T-010 is 50% to 100%; The Oxytech T-010 disinfection time is 20–60 minutes; The immersion time in sterile water is 10 to 20 minutes.

[0009] Preferably, the culture temperature in steps (2) to (3) is 24 to 27°C, the light exposure time is 10 to 14 h / d, and the light intensity is 4000 to 6500 lx.

[0010] Preferably, the temperature for dark culture in steps (4) to (5) is 24 to 27°C.

[0011] Preferably, the culture temperature in step (6) is 24-27°C, the light exposure time is 10-14 h / d, and the light intensity is 4000-6500 lx.

[0012] Preferably, the temperature for hardening seedlings in step (7) is 24-27°C, the light exposure time for hardening seedlings is 10-14 h / d, and the light intensity for hardening seedlings is 4000-6500 lx.

[0013] The present invention also provides the application of the method in the cultivation of tissues and / or plants of the nine-grade fragrant lotus.

[0014] The present invention has the following technical effects and advantages: This invention uses *Ligustrum lucidum* (also known as the nine-grade fragrant water lily) as the research object and constructs a technical system for the efficient regeneration of tissue culture explants from seed disinfection and germination to sterile seedling acquisition. Specifically, the surface disinfection method of this invention demonstrates excellent effectiveness, stability, and reproducibility in detoxifying *Ligustrum lucidum* seeds. In the sterile seedling solid-phase culture medium, the adsorption properties of AC improve the culture environment, 6-BA and NAA regulate hormones within *Ligustrum lucidum*, and Flower Treasure No. 1 improves the nutrients in the culture medium, thus exerting a synergistic effect, resulting in a large number of buds, robust seedling growth, and well-developed leaves in the sterile seedlings. The hypocotyl explants can normally initiate the differentiation process, successfully inducing the differentiation of new stem and leaf tissues, exhibiting stable organogenesis and plant reconstruction capabilities. In the bud induction medium, 6-BA, NAA, and ZT significantly enhance the stem length and bud proliferation of *Ligustrum lucidum*. In the rooting medium, NAA and AC have a synergistic effect on rooting. The nine-grade fragrant water lily plants obtained by this invention have a longer cultivation period, grow fully and develop more maturely, have robust root systems, and significantly improve their own resistance to adverse conditions and environmental adaptability. After hardening treatment, the nine-grade fragrant water lily plants can gradually adapt to changes in external light, humidity and temperature. The final hardening survival rate after transplanting can reach more than 80%, which provides important technical support for the rapid propagation and further optimization of genetic transformation technology of nine-grade fragrant water lilies. Attached Figure Description

[0015] Figure 1 Nine-grade fragrant lotus with different flower colors and varieties; Figure 2 The mature berries and seeds of the nine-grade fragrant lotus; Figure 3 Germination results of virus-free seeds of the nine-grade fragrant lotus after 7 days of culture; Figure 4 The results show the germination rate and uncontaminated rate of virus-free seeds of the nine-grade fragrant water lily. In the figure, Cl represents NaClO and T represents Oxytech T-010. Figure 5 The images show the growth status of sterile seedlings of the nine-grade fragrant water lily. A represents the growth status after 15 days of cultivation in the first stage, B represents the growth status after 20 days of cultivation in the second stage, and C represents the growth status after 30 days of cultivation in the third stage. Figure 6 These are sterile seedlings of the ninth-grade fragrant water lily. The red circle in the picture represents the explant of the hypocotyl. Figure 7 The growth status of each tissue culture explant of the nine-grade fragrant lotus after 7 days of culture, where A is the root explant, B is the stem segment explant, C is the leaf explant, and D is the hypocotyl explant. Figure 8 The results of the cultivation of each bud of the nine-grade fragrant water lily are shown, where A to I correspond to groups C1 to C9 respectively; Figure 9 The results show the rooting of tissue culture seedlings of the nine-grade fragrant water lily. A shows the front view of the tissue culture seedling, and B shows the rooting on the back side of the tissue culture seedling. Figure 10 The images show the hardening-off process for nine-grade fragrant water lilies. A represents a nine-grade fragrant water lily tissue culture seedling after rooting culture has been completed, B represents a nine-grade fragrant water lily plant after hardening-off, and C represents a nine-grade fragrant water lily plant transplanted into a plastic pot. Figure 11 This document outlines the tissue culture process for *Ligustrum lucidum*, where A represents mature seeds, B represents the germination of virus-free seeds, C represents the first stage of aseptic seedling culture, D represents the second stage of aseptic seedling culture, E represents the culture of *Ligustrum lucidum* buds, F represents the division culture of *Ligustrum lucidum* buds, G represents the rooting of tissue-cultured *Ligustrum lucidum* seedlings, H represents the subculture of tissue-cultured *Ligustrum lucidum* seedlings, and I represents the hardening-off process of *Ligustrum lucidum* seedlings. Detailed Implementation

[0016] This invention provides a tissue culture medium combination for nine-grade fragrant lotus, including sterile seedling solid culture medium, differentiation induction culture medium, shoot cluster induction culture medium and rooting culture medium; The sterile seedling solid culture medium is MS-based and further comprises the following components at the following concentrations: glucose 25–35 g / L, preferably 30 g / L; activated carbon (AC) 0.5–1 g / L, preferably 1 g / L; Flower Treasure No. 1 0.5–1 g / L, preferably 1 g / L; 6-benzylaminopurine (6-BA) 1–1.5 mg / L, preferably 1 mg / L; naphthaleneacetic acid (NAA) 1–1.5 mg / L, preferably 1 mg / L; agar 5–10 g / L, preferably 10 g / L; pH 5.8. The differentiation-inducing medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, preferably 30 g / L; zeatin (ZT) 1–1.5 mg / L, preferably 1 mg / L; NAA 1–1.5 mg / L, preferably 1 mg / L; agar 5–10 g / L, preferably 8 g / L; pH 5.8. The shoot induction medium is MS-based and further comprises the following components at the following concentrations: glucose 25–35 g / L, preferably 30 g / L; 6-BA 0.5–1.5 mg / L, preferably 1 mg / L; NAA 0.5–1.5 mg / L, preferably 1 mg / L; ZT 0.2–1 mg / L, preferably 1 mg / L; agar 5–10 g / L, preferably 8 g / L; pH 5.8. The rooting medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, preferably 30 g / L; NAA 0.5–1.5 mg / L, preferably 1 mg / L; AC 0.5–1 g / L, preferably 1 mg / L; agar 5–10 g / L, preferably 8 g / L; pH 5.8.

[0017] The present invention also provides the application of the aforementioned nine-grade fragrant lotus tissue culture medium combination in the tissue and / or plant culture of nine-grade fragrant lotus.

[0018] This invention also provides a method for tissue and / or plant culture of the nine-grade fragrant lotus, comprising the following steps: (1) Take the seeds of the nine-grade fragrant lotus, soak them in carbendazim, disinfect them with 75% alcohol, disinfect them with NaClO, disinfect them with Oxytech T-010, and soak them in sterile water to obtain virus-free seeds; (2) Place the virus-free seeds in sterile water and culture for 10-15 days, preferably 15 days, to obtain germinating seedlings; (3) The germinated seedlings are placed in a sterile seedling solid culture medium and cultured for 30 to 65 days, preferably 65 days, to obtain sterile seedlings; (4) Different tissue parts of sterile seedlings were placed in induction differentiation medium and cultured in the dark for 5-10 days, preferably 7 days, and tissue culture explants were obtained by screening. (5) Place the tissue culture explants in a shoot induction medium and culture them in the dark for 20-30 days, preferably 21 days, to obtain shoots; (6) Place the seedlings in a rooting medium and culture them for 10-15 days, preferably 14 days, to obtain tissue culture seedlings; (7) Harden the tissue culture seedlings for 3 to 6 days, preferably 6 days, to obtain the nine-grade fragrant lotus plants.

[0019] In this invention, the rotation speed of the carbendazim soaking in step (1) is 150-250 rpm, preferably 200 rpm; the soaking time of the carbendazim is 20-40 min, preferably 30 min; The carbendazim is a 500-1000 times diluted solution, preferably a 500 times diluted solution; The alcohol disinfection is performed 1 to 2 times, preferably 2 times; the alcohol disinfection time is 0.5 to 2 minutes per time, preferably 1 minute per time. The concentration of NaClO is 10% to 25%, preferably 25%; The effective chlorine concentration of the NaClO is ≥8%; The NaClO sterilization time is 6 to 8 minutes, preferably 8 minutes; The concentration of Oxytech T-010 is 50% to 100%, preferably 100%; The disinfection time of the Oxytech T-010 is 20 to 60 minutes, preferably 40 minutes; Each step of the disinfection process is followed by rinsing with sterile water 3 to 5 times. The soaking time in sterile water is 10 to 20 minutes, preferably 15 minutes.

[0020] In this invention, the culture temperature in steps (2) to (3) is 24 to 27°C, preferably 26°C; the light exposure time for culture is 10 to 14 h / d, preferably 12 h / d; and the light intensity for culture is 4000 to 6500 lx, preferably 6500 lx.

[0021] In this invention, the temperature of the dark culture in steps (4) to (5) is 24 to 27°C, preferably 26°C.

[0022] In this invention, the culture temperature in step (6) is 24-27°C, preferably 26°C; the light exposure time is 10-14 h / d, preferably 12 h / d; and the light intensity is 4000-6500 lx, preferably 6500 lx.

[0023] In this invention, the temperature for hardening seedlings in step (7) is 24-27°C, preferably 26°C; the light exposure time for hardening seedlings is 10-14 h / d, preferably 12 h / d; and the light intensity for hardening seedlings is 4000-6500 lx, preferably 6500 lx.

[0024] In this invention, the seedling hardening step (7) is as follows: (a) Open the cover of the tissue culture seedlings by 1 / 4 and harden them off for 2 days; (b) Open the cover of the tissue culture seedlings by 1 / 2 and harden them off for 2 days; (c) Fully open the cover of the tissue culture seedlings and harden them off for 2 days.

[0025] The present invention also provides the application of the method in the cultivation of tissues and / or plants of the nine-grade fragrant lotus.

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] The test material for this invention is seeds of nine-colored fragrant water lilies of different flower colors in a tropical subgenus, sourced from the Haikou base of Hainan Fodu Lianyuan Ecological Agriculture Co., Ltd. (110°19′48″E, 19°57′00″N). Figures 1-2 As shown.

[0028] Example 1: Surface disinfection and germination of seeds of the nine-grade fragrant lotus Mature berries of the nine-grade fragrant lotus were collected and soaked in water for 7 days until the berries burst open and released mature seeds. The mature seeds were washed with detergent and then rinsed with sterile water for 5 minutes. They were then soaked in a 500-fold diluted carbendazim solution at 200 rpm for 30 minutes, followed by rinsing with sterile water 3–5 times. They were then disinfected twice with 75% alcohol for 1 minute each time, followed by rinsing with sterile water 3 times. Finally, they were disinfected by soaking in NaClO (effective chlorine concentration ≥8%) and rinsing with sterile water 5 times. Oxytech T-010 was used for disinfection, followed by rinsing with sterile water 5 times. Finally, the seeds were soaked in sterile water for 15 minutes and then rinsed once with sterile water to obtain virus-free seeds. Based on the disinfection concentration and time of NaClO and Oxytech T-010, the mature seeds were divided into groups A1–A7, as shown in Table 1. Virus-free seeds were placed in sterile water and cultured for 15 days at 26℃, with a light duration of 12 h / d and a light intensity of 6500 lx to obtain germinating seedlings, and the germination rate was calculated. Based on the contamination status of the germinating seedlings after transplanting, the uncontaminated rate after surface disinfection was calculated. The results are shown in Table 1 and... Figures 3-4 As shown.

[0029] Table 1 Results of surface disinfection of nine-grade fragrant water lily seeds The results showed that group A6 had the highest uncontamination rate and germination rate of virus-free seeds, reaching 95% and 65.33%, respectively. After 7 days of culture in sterile water, the virus-free seeds germinated into seedlings approximately 1 cm long. Repeated experiments verified that the A6 group's disinfection effect of soaking in 25% NaClO for 8 min followed by soaking in 100% Oxytech T-010 for 40 min was highly effective, stable, and reproducible in detoxifying *Liriope muscari* seeds. Compared to group A6, the germination rate did not change significantly under low-concentration disinfectant combinations, but the uncontamination rate decreased significantly. Appropriately increasing the concentration of one disinfectant could improve the uncontamination rate to some extent, but had varying degrees of impact on the germination rate. High-concentration disinfectants combined with longer treatment times significantly inhibited the germination of virus-free seeds.

[0030] Example 2: Obtaining sterile seedlings of the nine-grade fragrant water lily Seedlings with uniform growth from Example 1 were selected and placed in a sterile seedling solid-phase medium (pH=5.8) based on MS, including 30 g / L glucose, 10 g / L agar, AC, Flower Treasure No. 1, 6-BA, and NAA, with sterile water added for the first stage of culture. The seedlings were cultured for 15 days at 26°C, 12 h / d photoperiod, and 6500 lx light intensity to obtain sterile seedlings. Then, the seedlings were transplanted to a new sterile seedling solid-phase medium and sterile water was added for the second stage of culture. After 21 days of culture at 26°C, 12 h / d photoperiod, and 6500 lx light intensity, the plant height, leaf length, and average number of bud clusters were recorded, as shown in Table 2. Finally, the seedlings were transplanted to a new sterile seedling solid-phase medium and sterile water was added for the third stage of culture. After 30 days of culture at 26°C, 12 h / d photoperiod, and 6500 lx light intensity, the seedlings were cultured. Based on the concentrations of NAA, 6-BA, AC, and Flower Treasure No. 1 in the sterile seedling solid culture medium, germinating seedlings were divided into groups B1 to B6, as shown in Table 2. Figure 5 As shown.

[0031] Table 2. Growth Indicators of Aseptic Seedlings of Nine-Grade Fragrant Water Lily After Stage 2 Culture The results showed significant differences in the mean number of shoot clusters, plant height, and leaf length among the groups. The B3 group of sterile seedlings exhibited high shoot proliferation, robust growth, and well-developed leaves, demonstrating superior overall growth compared to other groups. Repeated experiments confirmed that the optimal culture medium for B3 sterile seedlings was 1 mg / L NAA + 1 mg / L 6-BA + 1 g / L AC + 1 g / L Flower Treasure No. 1. Compared to the B3 group, the B5 group showed a slightly higher mean number of shoot clusters after increasing the 6-BA concentration, but significantly lower plant height and leaf length, indicating weaker seedling growth. The B1 and B2 groups, without the addition of Flower Treasure No. 1, showed low shoot proliferation and relatively low plant height and leaf length. In the B4 and B6 groups, increasing the concentrations of 6-BA and NAA resulted in a decrease in the mean number of shoot clusters, with no improvement in any growth indicators.

[0032] Example 3: Screening of tissue culture explants of the nine-grade fragrant lotus Sterile seedlings of the nine-grade fragrant lotus with uniform growth, selected from Example 2, were used. Roots, stem segments, leaves, and hypocotyls were cut and placed in induction differentiation medium (pH=5.8) based on MS, including 30 g / L glucose, 1 mg / L ZT, 1 mg / L NAA, and 8 g / L agar. The explants were cultured in the dark at 26℃ for 7 days. The growth status of each explant was observed, and the explant materials for subsequent tissue culture and genetic transformation were selected based on their regeneration effect. The results are as follows: Figures 6-7 As shown.

[0033] The results showed that all stem explants died, with no surviving individuals; only some root and leaf explants maintained basic survival, without browning or mold, but they never initiated the dedifferentiation and redifferentiation process, and no callus, adventitious buds, or new organs were formed; hypocotyl explants could normally initiate the differentiation process, successfully inducing the differentiation of new stem and leaf tissues, and possessing stable organogenesis and plant reconstruction capabilities.

[0034] Example 4: Obtaining the sprouts of the nine-grade fragrant lotus The hypocotyl explants from Example 3 were placed in a shoot induction medium (pH=5.8) based on MS, containing 30 g / L glucose, 8 g / L agar, 6-BA, NAA, and ZT. The explants were cultured in the dark at 26°C for 21 days to obtain shoots, and the stem length and average number of shoot clusters were measured. Based on the concentrations of 6-BA, NAA, and ZT, the hypocotyl explants were divided into groups C1–C9, as shown in Table 3. Figure 8 As shown.

[0035] Table 3. Growth Indicators of Nine-Grade Fragrant Lotus Seedlings The results showed significant differences in stem length and the mean number of bud clusters among the groups. Group C5 exhibited the best results in both stem length and the mean number of bud clusters, and both were significantly higher than those of the other groups. This indicates that the 1 mg / L 6-BA + 1 mg / L NAA + 1 mg / L ZT medium in the bud cluster induction medium of the present invention has a prominent effect on stem length and bud proliferation.

[0036] Example 5: Obtaining tissue culture seedlings of the nine-grade fragrant water lily Vigorous, uniformly growing, and uncontaminated seedlings from Example 4 were selected and placed in rooting medium (pH=5.8) based on MS, including 30 g / L glucose, 8 g / L agar, NAA, and AC. The culture was carried out at 26℃, with a light duration of 12 h / d and a light intensity of 6500 lx for 14 days to obtain tissue culture seedlings. The rooting rate and rooting coefficient of the tissue culture seedlings were then measured. Based on the concentrations of NAA and AC, the seedlings were divided into groups D1–D6, as shown in Table 4. Figure 9 As shown.

[0037] Table 4. Rooting Indicators of Nine-Grade Fragrant Water Lily Tissue Culture Seedlings The results showed that NAA significantly improved rooting. Among the D1-D3 groups without added AC, the D2 group (1 mg / L NAA) showed the best rooting effect. Both excessively low and high NAA concentrations inhibited root induction and growth. Adding 1 g / L AC significantly increased the rooting rate and rooting coefficient of all groups, with the D5 group showing significantly better rooting than the other groups. This indicates that the 1 mg / L NAA + 1 g / L AC in the D5 group rooting medium of this invention has a synergistic effect on rooting.

[0038] Example 6: Hardening off tissue culture seedlings of the nine-grade fragrant water lily Tissue culture seedlings from Example 5 were placed in the sterile solid-phase culture medium of Group B3 from Example 2. Subculture was performed according to the method in Example 2 for seedling establishment and strengthening. Then, seedlings that had undergone three subcultures (30 days each), exhibited vigorous growth, no browning, and intact root systems, were selected for hardening and transplanting under conditions of 26℃, 12h / d light, and 6500lx light intensity. A gradient hardening method was used: the seedlings were hardened gradually by opening the cover to 1 / 4, 1 / 2, and then fully opening, with each stage lasting 2 days. After hardening, the seedlings were obtained as *Hydrocotyle sibthorpioides* plants. The plants were gently removed, and the roots were washed with sterile water to remove the agar, avoiding damage to the roots and leaves. A loose, well-drained, and moderately fertile aquatic soil substrate with a top layer of sand was used for planting. After spreading the roots, the substrate was backfilled and compacted. After transplanting, place the seedlings in plastic pots, cover them with water, and allow them to recover in a diffused light environment with a temperature of 26℃ and an air humidity of 92%. Initially, avoid direct sunlight. Once the *Ligustrum lucidum* plants have resumed growth and new leaves have sprouted, gradually restore normal light and water / fertilizer management. The results are as follows... Figure 10 As shown, the tissue culture procedure for the nine-grade fragrant lotus is as follows: Figure 11 As shown.

[0039] The results showed that the nine-grade fragrant water lily plants underwent a longer cultivation period, resulting in fuller growth, more mature development, stronger root systems, and significantly improved resistance and environmental adaptability. After hardening-off treatment, the nine-grade fragrant water lily plants could gradually adapt to changes in external light, humidity, and temperature, and the final survival rate after transplanting could reach over 80%.

[0040] As can be seen from the above embodiments, the present invention provides a tissue culture method for the nine-grade fragrant water lily and its application. The nine-grade fragrant water lily plants obtained according to the method of the present invention have a longer culture period, are more fully grown and mature, have robust root systems, and significantly improved resistance and environmental adaptability. After hardening-off treatment, the nine-grade fragrant water lily plants can gradually adapt to changes in external light, humidity, and temperature, and the final hardening-off survival rate after transplanting is high, providing important technical support for further optimization of rapid propagation and genetic transformation technology research of the nine-grade fragrant water lily.

[0041] 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 principle 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 tissue culture medium combination for Agapetes formosana, characterized by, This includes sterile seedling solid culture medium, differentiation induction medium, shoot cluster induction medium, and rooting medium; The sterile seedling solid culture medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, activated carbon 0.5–1 g / L, Flower Treasure No. 1 0.5–1 g / L, 6-benzylaminopurine 1–1.5 mg / L, naphthaleneacetic acid 1–1.5 mg / L, and agar 5–10 g / L. The differentiation-inducing medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, zeatin 1–1.5 mg / L, naphthaleneacetic acid 1–1.5 mg / L, and agar 5–10 g / L. The shoot induction medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, 6-benzylaminopurine 0.5–1.5 mg / L, naphthaleneacetic acid 0.5–1.5 mg / L, zeatin 0.2–1 mg / L, and agar 5–10 g / L; The rooting medium is MS-based and also includes the following components at the following concentrations: glucose 25–35 g / L, naphthaleneacetic acid 0.5–1.5 mg / L, activated carbon 0.5–1 g / L, and agar 5–10 g / L.

2. The application of the tissue culture medium combination for the nine-grade fragrant lotus as described in claim 1 in the tissue and / or plant culture of the nine-grade fragrant lotus.

3. A method for tissue and / or plant culture of the nine-grade fragrant lotus, characterized in that, Includes the following steps: (1) Take the seeds of the nine-grade fragrant lotus, soak them in carbendazim, disinfect them with 75% alcohol, NaClO, Oxytech T-010, and soak them in sterile water to obtain detoxified seeds; (2) Place the virus-free seeds in sterile water and culture for 10-15 days to obtain germinating seedlings; (3) Place the germinating seedlings in a sterile seedling solid culture medium and culture for 30-65 days to obtain sterile seedlings; (4) Different tissue parts of sterile seedlings were placed in induction differentiation medium and cultured in the dark for 5-10 days to screen out tissue culture explants; (5) Place the tissue culture explants in a shoot induction medium and culture them in the dark for 20-30 days to obtain shoots; (6) Place the seedlings in a rooting medium and culture for 10-15 days to obtain tissue culture seedlings; (7) Harden the tissue culture seedlings for 3-6 days to obtain nine-grade fragrant lotus plants; The sterile seedling solid culture medium, differentiation induction culture medium, shoot cluster induction culture medium, and rooting culture medium mentioned in steps (3) to (6) are the sterile seedling solid culture medium, differentiation induction culture medium, shoot cluster induction culture medium, and rooting culture medium described in claim 1.

4. The method according to claim 3, characterized in that, In step (1), the rotation speed of the carbendazim soaking is 150-250 rpm, and the soaking time is 20-40 min. The alcohol disinfection is performed 1 to 2 times, and the alcohol disinfection time is 0.5 to 2 minutes per time; The concentration of NaClO is 10%–25%; The NaClO sterilization time is 6–8 minutes; The concentration of Oxytech T-010 is 50% to 100%; The Oxytech T-010 disinfection time is 20–60 minutes; The immersion time in sterile water is 10 to 20 minutes.

5. The method according to claim 4, characterized in that, The culture temperature in steps (2) to (3) is 24 to 27°C, the light exposure time is 10 to 14 h / d, and the light intensity is 4000 to 6500 lx.

6. The method according to claim 5, characterized in that, The temperature for dark culture in steps (4) to (5) is 24 to 27°C.

7. The method according to claim 6, characterized in that, The culture temperature in step (6) is 24-27℃, the light exposure time is 10-14 h / d, and the light intensity is 4000-6500 lx.

8. The method according to claim 7, characterized in that, The temperature for hardening seedlings in step (7) is 24-27℃, the light exposure time for hardening seedlings is 10-14 h / d, and the light intensity for hardening seedlings is 4000-6500 lx.

9. The application of the method according to any one of claims 3 to 8 in the cultivation of tissues and / or plants of the nine-grade fragrant lotus.

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