A method for culturing the tooth germs of the mandibular first premolar

Abstract

The application discloses a tissue culture method of Pterospermum acerifolium, which comprises the following steps: (1) preparation of explants, (2) tissue culture induction medium, (4) subculture, (5) rooting culture, and (6) transplanting of tissue culture seedlings. Through a series of processes such as disinfection, basic culture, proliferation culture and rooting culture of the tissue culture seedlings of Pterospermum acerifolium, a large number of Pterospermum acerifolium tissue culture seedlings with stable quality can be quickly obtained.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a method for the propagation and cultivation of *Rhododendron simsii*. Background Technology

[0002] *Agave titanota*, also known as the King's Crown, is native to the arid tropical regions of the Americas, primarily distributed in Oaxaca, Mexico. It is a perennial plant belonging to the genus *Agave* in the family Agavaceae. The plant lacks a distinct main stem, and its fleshy leaves are broad and thick, growing in a rosette pattern, reaching a maximum width of 35-40 cm. The upper half of the leaves is narrowly elliptical, with a pointed tip bearing a sharp, dark brown spine. *Agave titanota* has the ability to absorb harmful gases such as formaldehyde, benzene, and trichloroethylene, making it valuable for landscaping. It can be placed on indoor desks or tables, or used in desert plant displays, urban landscapes, gardens, and public green spaces. With its high pollution resistance and air-purifying capabilities, it is a commonly used ornamental plant in parks, greenbelts, and homes.

[0003] 'Bucktooth' is one of the variegated varieties of 'Renwangguan' agave. Its leaves have prominent yellow markings along both edges, creating a striking and beautiful appearance. The small, delicate plant has high ornamental value, but its care and propagation are relatively difficult. During propagation, it is prone to bleaching, leading to death. Like most agaves, 'Bucktooth' only flowers once in its lifetime, after which the plant gradually withers and dies, making seed collection difficult. 'Bucktooth' grows slowly, typically only 1-2 cm per year. It requires ample sunlight and a dry environment to thrive. Traditional propagation methods such as division and cuttings result in slow seedling growth and extremely low propagation efficiency. Currently, traditional propagation methods cannot meet the market demand for 'Bucktooth' seedlings and the development of its cultivation industry.

[0004] Existing technology, in a method for inducing bulbils in agave (Yang Zhijian et al., Jiangsu Agricultural Sciences, 2019), discloses using agave stem segments as culture material and obtaining different culture media through different concentrations of hormone combinations, thereby establishing a tissue culture system for variegated agave. However, the inventors found in experiments that when using the above method to tissue culture agave bulbils, severe whitening occurred in the tissue culture seedlings, leading to low tissue culture propagation efficiency. Therefore, this invention utilizes a rapid tissue culture propagation technology system to provide a method for the propagation and cultivation of variegated agave bulbils, providing a theoretical basis for the efficient cultivation and widespread application of variegated agave bulbils, and laying the foundation for its large-scale production. Summary of the Invention

[0005] The main objective of this invention is to provide a method for culturing the protruding teeth of the Niohō-no-Tsurugi (a type of crown).

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A method for culturing tissue from protruding teeth of the Emperor Nioh (a mythical creature), comprising the following steps:

[0008] (1) Preparation of explants

[0009] Select healthy, disease-free young plants of *Gynostemma pentaphyllum*, remove the withered leaves, cut off the roots, disinfect, and obtain *Gynostemma pentaphyllum* explants.

[0010] (2) Tissue culture induction

[0011] Under aseptic conditions, sterilized explants were inoculated into YMS medium containing the following macroelements: KNO3 1700 mg·L⁻¹ -1 NH4NO3 1430mg·L -1 KH2PO4 340mg·L -1 MgSO4 370 mg·L -1 CaCl2 220 mg·L -1 Ca(NO3)2 220 mg·L -1 ;

[0012] Trace element: KI 0.83 mg·L -1 H3BO3 6.2 mg·L -1 MnSO4·4H2O 22.3 mg·L -1 ZnSO4·7H2O 8.6 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CuSO4·5H2O 0.025 mg·L -1 CoCl2·6H2O 0.025 mg·L -1 ;

[0013] Iron salt: FeSO4·7H2O 55.6 mg·L -1 Na2-EDTA·2H2O 74.6 mg·L -1 ;

[0014] Organic ingredient: Inositol 100mg / L -1 Niacin 0.5 mg·L -1 pyridoxine hydrochloride 0.5 mg·L -1 Thiamine hydrochloride 0.5 mg·L -1 Glycine 2.0 mg·L -1 ;

[0015] The above induction medium also contains 0.3 mg / L NAA. -130g / L of sucrose -1 Carrageenan 8.0 g·L -1 pH = 5.8;

[0016] (3) Subsidiary training

[0017] The induced buck-toothed material was inoculated into different subculture proliferation media, and adventitious buds grew after subculture.

[0018] The subculture medium in step (3) is YMS + NAA 0.3 mg·L⁻¹. -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 pH = 5.8;

[0019] (4) Rooting culture

[0020] Adventitious buds with uniform variegation, a height of over 2.5 cm, and 4-5 leaves, obtained from propagation culture, were selected and transferred to rooting medium for rooting culture for 30-40 days. The rooting medium consisted of 1 / 2 YMS + 1.0 mg / L IBA. -1 +NAA 0.4 mg·L -1 +20g / L sucrose -1 + Carrageenan 8.0g·L -1 ;

[0021] (5) Transplanting of tissue culture seedlings

[0022] Select robust tissue culture seedlings that are over 4cm tall, have uniform variegation, and possess at least 4 clean white roots. Place them in a greenhouse with diffused light or in a location with ample natural light but no direct sunlight for hardening-off. After hardening-off, transplant the seedlings into seedling trays. The transplanting substrate should have a volume ratio of peat moss:coconut coir:coarse sand = 2:2:1. After transplanting, place them in a seedling shed for further cultivation. Once the transplanted seedlings have developed well in terms of leaves and roots and have reached a height of over 10cm, they can be planted outdoors or in pots.

[0023] (6) Management of transplanted seedlings.

[0024] Further, in step (1), the disinfection method is as follows: after cutting off the roots of the young protruding tooth plant, rinse it under running water for 20-30 minutes, put the cleaned material into an ultrasonic cleaner for 15 minutes; put the cleaned explant into 75% alcohol and shake for 30 seconds, disinfect with 2% NaClO for 8 minutes, and then disinfect with 0.1% mercuric chloride solution for 12 minutes each. Finally, rinse with sterile water 4-6 times, and dry with sterile filter paper to obtain the protruding tooth explant of Renwangguan.

[0025] Furthermore, in step (2), avoid using 6-BA.

[0026] Furthermore, in step (3) of subculture, avoid using 6-BA.

[0027] Furthermore, in step (4), the cycle is 34 to 36 days.

[0028] Further, in step (5), after the seedling hardening is completed, the tissue culture seedlings are taken out of the culture bottle, the culture medium on the tissue culture seedlings is cleaned with water, the roots of the tissue culture seedlings are immersed in a 1000-fold diluted solution of hymexazol for 30 seconds, and then placed in a cool and ventilated place to dry for 5 days. Then the tissue culture seedlings are transplanted into the seedling trays, and after the transplanting is completed, they are placed in the seedling shed for cultivation.

[0029] Furthermore, in step (5), the best planting time is in spring (April-May) or autumn (September-October).

[0030] Furthermore, in step (6), the temperature, humidity, and light management of the transplanted seedlings are as follows: avoid direct sunlight for 15 days after transplanting, control the temperature at 22-27℃, and the relative humidity should not be too high. Adjust the humidity by spraying or ventilation, generally keeping it at 40%-50% to prevent disease. After 15 days, full sunlight can be used for seedling cultivation. If the sunlight is too strong for the next 1-5 days, provide appropriate shading at noon.

[0031] Furthermore, in step (6), water and fertilizer management is as follows:

[0032] Water the roots until the soil or substrate is moist, but do not overwater. Water again 2-3 days after watering or when the soil or substrate is slightly dry. Reduce watering or stop watering in winter depending on the weather. Apply fertilizer in spring and autumn each year, with a light fertilizer applied to the roots once in May and once in September, mainly nitrogen, phosphorus and potassium compound fertilizer. The fertilizer should be applied 10-15cm away from the roots and away from the branches and leaves.

[0033] Further, in step (6), disease management is as follows: If root rot occurs, the substrate should be dried in time, and the seedlings with milder symptoms should be removed and the rotten roots removed; if leaf rot occurs, watering should be increased to make the substrate completely moist, and yellowed and wilted leaves should be removed to reduce water loss and allow the seedlings to recover. If light management is poor, sunburn may occur, causing brown spots that gradually enlarge and become sunken on the surface of new leaves. At this time, shade nets should be added in time to avoid direct sunlight, ventilation in the greenhouse should be strengthened, and water should be sprayed appropriately to prevent the disease from worsening.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) This invention can quickly obtain a large number of high-quality and stable Renwangguan buck-tooth tissue culture seedlings by performing a series of processes such as disinfection, basic culture, proliferation culture and rooting culture on Renwangguan tissue culture seedlings.

[0036] (2) This invention obtains a modified YMS medium by adjusting the macroelements and iron salts in MS. This invention found that using MS and YMS medium has little effect on the induction rate, but a significant impact on the growth of the material. Using YMS medium can effectively alleviate the whitening phenomenon that occurs during the tissue culture of *Rhododendron simsii* with protruding teeth, reducing the whitening rate. Simultaneously, this invention found that 6-BA easily leads to mutations and deformities in tissue culture seedlings; removing 6-BA during tissue culture can help maintain its phenotypic stability.

[0037] (3) This invention found that a peat moss:coconut coir:coarse sand ratio of 2:2:1 in the substrate during transplanting can significantly increase the survival rate of transplanted seedlings. Peat moss is rich in nutrients and has good water retention; coconut coir has good water retention and air permeability, and is also rich in organic matter, making it suitable for tissue culture seedling growth; coarse sand has larger particles, which can increase the air content in the soil, which is beneficial to plant respiration and growth. The combined use of peat moss, coconut coir, and coarse sand is beneficial to the root growth of tissue culture seedlings and effectively improves the survival rate of transplanted *Rhizophora repens* tissue culture seedlings. Attached Figure Description

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] Figure 1 For MS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 The results of culture in the culture medium.

[0040] Figure 2 For MS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 +0.5 mg·L -1 Culture results in 6-BA medium.

[0041] Figure 3 For MS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 +1.0 mg·L -1 Culture results in 6-BA medium.

[0042] Figure 4 YMS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 The results of culture in the culture medium.

[0043] Figure 5 YMS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 +0.5 mg·L -1 Culture results in 6-BA medium.

[0044] Figure 6 YMS + NAA 0.3 mg·L in Table 1 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 +1.0 mg·L -1 Culture results in 6-BA medium.

[0045] Figure 7 For YMS+NAA 0.3 mg·L in Table 2 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 The results of culture in the culture medium.

[0046] Figure 8 YMS+NAA 0.3 mg·L in Table 2 -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 +0.5 mg·L -1 Culture results in 6-BA medium.

[0047] Figure 9 The results of rooting culture of tissue culture seedlings.

[0048] Figure 10 The results of transplanting tissue culture seedlings. Detailed Implementation

[0049] Example 1

[0050] To address the problems of low reproductive efficiency and whitening in the Nioh-ko crown buck teeth, this invention studies a rapid propagation system for the Nioh-ko crown buck teeth, aiming to establish an efficient tissue culture propagation technology system to propagate a large number of seedlings with uniform specifications and stable traits in a short period of time, laying the foundation for its large-scale production.

[0051] Specifically, the following steps are included:

[0052] (1) Preparation of explants

[0053] Select healthy, disease-free young *Rhizophora nigra* explants, remove withered leaves, cut off the roots, and rinse under running water for 20-30 minutes. Then, place the rinsed material in an ultrasonic cleaner for 15 minutes. Immerse the cleaned explants in 75% alcohol with shaking for 30 seconds, disinfect with 2% NaClO for 8 minutes, and then disinfect with 0.1% mercuric chloride solution for 12 minutes each time. Finally, rinse 4-6 times with sterile water and blot dry with sterile filter paper to obtain *Rhizophora nigra* explants.

[0054] (2) Tissue culture induction medium

[0055] Under aseptic conditions, sterilized explants were inoculated into different induction media, MS medium and modified MS medium, with different concentrations of plant growth hormone (0, 0.5, 1.0 mg·L⁻¹) added respectively. -1 6-BA was used to investigate the effects of different culture media and hormone concentrations on the germination of protruding teeth in *Rhizophora repens*.

[0056] Modified MS medium: Based on MS medium, the macroelements and iron salts in MS medium are adjusted, while the amounts of other elements in MS medium remain unchanged. The modified basal medium is abbreviated as YMS.

[0057] The standard MS medium formulation is as follows:

[0058] Macroelements: KNO3 1900 mg·L -1 NH4NO3 1650mg·L -1 KH2PO4 170mg·L -1 MgSO4 370 mg·L -1 CaCl2·2H2O 440mg·L -1 ;

[0059] Trace element: KI 0.83 mg·L -1 H3BO3 6.2 mg·L -1 MnSO4·4H2O 22.3 mg·L -1 ZnSO4·7H2O 8.6 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CuSO4·5H2O 0.025 mg·L -1 CoCl2·6H2O 0.025 mg·L -1 ;

[0060] Iron salt: FeSO4·7H2O 27.8 mg·L -1 Na2-EDTA·2H2O 37.3 mg·L -1 ;

[0061] Organic ingredient: Inositol 100mg / L -1 Niacin 0.5 mg·L -1 pyridoxine hydrochloride 0.5 mg·L -1 Thiamine hydrochloride 0.5 mg·L -1 glycine mg·L -1 .

[0062] The components of YMS are as follows:

[0063] Macroelements: KNO3 1700 mg·L -1 NH4NO3 1430mg·L -1 KH2PO4 340mg·L -1 MgSO4 370 mg·L -1 CaCl2 220 mg·L -1 Ca(NO3)2 220 mg·L -1 ;

[0064] Trace element: KI 0.83 mg·L -1 H3BO3 6.2 mg·L -1 MnSO4·4H2O 22.3 mg·L -1 ZnSO4·7H2O 8.6 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L⁻¹ -1 CuSO4·5H2O 0.030 mg·L -1 CoCl2·6H2O 0.025 mg·L -1 ;

[0065] Iron salt: FeSO4·7H2O 55.6 mg·L -1 Na2-EDTA·2H2O 74.6 mg·L -1 ;

[0066] Organic ingredient: Inositol 100mg / L -1 Niacin 0.5 mg·L -1 pyridoxine hydrochloride 0.5 mg·L -1 Thiamine hydrochloride 0.5 mg·L -1 Glycine 2.0 mg·L -1 ;

[0067] 0.3 mg / L NAA was added to both the MS and YMS induction media. -1 30g / L of sucrose -1 and carrageenan 8.0 g·L -1Adjust the pH to 5.8.

[0068] The incubation room was set at a temperature of 24-28℃ and a light intensity of 2500 lx, with a light exposure period of 12 h / d.

[0069] Table 1. Effects of different culture media and hormone concentrations on the induction of buck teeth in Renwang crown.

[0070]

[0071] Note: The results of the ANOVA in the list are represented by letters, and different lowercase letters indicate significant differences (P<0.05).

[0072] This invention increases the iron content in the culture medium (1). During the growth of tissue culture seedlings, iron salts promote chlorophyll formation, thereby improving photosynthetic efficiency and enabling normal plant growth and development. In the tissue culture of *Rhinoceros chinensis*, the applicant found that appropriately increasing the iron content can alleviate chlorosis in tissue culture seedlings. (2) The applicant found that the concentration of 6-BA has a significant effect on the induction of variegation in variegated plants. High concentrations of 6-BA are not conducive to the formation of variegation. Therefore, the use of 6-BA should be avoided in the tissue culture of *Rhinoceros chinensis*.

[0073] (4) Subsidiary training

[0074] The induced buck-toothed material was inoculated into different subculture proliferation media, and adventitious buds grew after subculture.

[0075] The subculture medium in step (4) is YMS + NAA 0.3 mg·L⁻¹. -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 , pH=5.8; YMS+6-BA0.5mg·L -1 +NAA 0.3 mg·L -1 + 30g / L of sucrose -1 + Carrageenan 8.0g·L -1 pH = 5.8.

[0076] Table 2 Results of 6-BA on proliferative trials of buck teeth

[0077]

[0078] Note: The results of the ANOVA in the list are represented by letters, and different lowercase letters indicate significant differences (P<0.05).

[0079] (5) Rooting culture

[0080] Select adventitious buds with uniform variegation, a height of over 2.5 cm, and 4-5 leaves from the propagation culture, and transfer them to rooting medium (1 / 2 YMS + IBA 1.0 mg·L⁻¹).-1 +NAA 0.4 mg·L -1 +20g / L sucrose -1 + Carrageenan 8.0g·L -1 Rooting culture is carried out in ) for 35 days.

[0081] See image Figure 9 .

[0082] (6) Transplanting of tissue culture seedlings

[0083] Select robust tissue culture seedlings that are over 4cm tall, with uniform variegation and at least 4 clean white roots. Place them in a greenhouse with diffused light or in a location with ample natural light but no direct sunlight for hardening off. After hardening off, remove the seedlings from the culture bottles and wash off the culture medium with clean water, taking care not to damage the young leaves and roots. After washing, immerse the roots of the seedlings in a 1000-fold diluted solution of hymexazol for 30 seconds, and then place them in a cool, ventilated place to air dry for 5 days. Then, transplant the seedlings into plug trays. The transplanting substrate should be peat moss: coconut coir: coarse sand = 2:2:1 (by volume). This substrate has good moisture retention, aeration, and is rich in nutrients. After transplanting, place them in a seedling shed for cultivation. When the transplanted seedlings have developed well in terms of leaves and roots and have reached a height of over 10cm, they can be planted outdoors or in pots. Spring (April-May) or autumn (September-October) are the best planting times. Avoid planting during periods of continuous rain or hot and humid weather.

[0084] See image Figure 10

[0085] (7) Management of transplanted seedlings

[0086] 1. Temperature, humidity and light management for transplanted seedlings of the Nioh Crown Protruding Teeth variety.

[0087] Avoid direct sunlight for 15 days after transplanting, keep the temperature between 22 and 27°C, and avoid excessively high relative humidity. Adjust the humidity by spraying or ventilation, generally keeping it between 40% and 50% to prevent disease. After 15 days, full sunlight can be used for seedling cultivation. If the sunlight is too strong for the next 1 to 5 days, provide appropriate shading at midday.

[0088] 2. Water and fertilizer management for the protruding teeth of the King of Nioh.

[0089] The Agave 'Nioh Crown' has poor tolerance to waterlogging, so watering must be strictly controlled. Water only until the soil or substrate is moist, not excessively. Water again 2-3 days after watering, or wait until the soil or substrate is slightly dry, to prevent root rot caused by overwatering. Reduce watering or stop watering in winter depending on the weather. Spring and autumn are the peak growing seasons for Agave, so timely fertilization is necessary. Apply a light fertilizer to the roots once in May and once in September, primarily using a nitrogen, phosphorus, and potassium compound fertilizer, or organic fertilizer. The fertilizer should be applied 10-15cm away from the roots, avoiding the branches and leaves. For potted plants, slow-release fertilizer can be used appropriately to avoid fertilizer burn, wilting, and other fertilizer damage caused by excessive concentration.

[0090] 3. Disease Management of Transplanted Seedlings of the Niohaku Crown with Protruding Teeth

[0091] Common diseases affecting *Agave davidii* include anthracnose, leaf spot, and gray mold. These are primarily infectious fungal diseases that mainly damage the leaves, causing lesions and spots, eventually leading to the rotting of the entire leaf. These diseases are often caused by a humid growing environment, poor ventilation, or excessive nitrogen fertilizer. During planting, ensure good ventilation and control temperature and humidity. The nitrogen content in nitrogen, phosphorus, and potassium compound fertilizers should not be too high; increase phosphorus and potassium to enhance the agave's disease resistance. Spray with 800 times diluted methyl thiophanate for prevention. For infected plants, promptly remove rotten leaves to prevent further spread of the pathogen. In the early stages of disease, spray with fungicides every 7-10 days, for three consecutive applications. Use different fungicides each time to avoid developing resistance. Suitable options include 70% methyl thiophanate at 800 times dilution, 75% chlorothalonil at 800 times dilution, anthracnose fungicide at 800 times dilution, or copper oxychloride at 1000 times dilution.

[0092] Common pests affecting agave, such as scale insects, include mealybugs, shield scales, and root mealybugs. Mealybugs and shield scales primarily damage agave leaves. Mealybugs mainly cause yellowing of leaves, which can lead to plant death in severe cases. Shield scales cause chlorotic spots on leaves, stunted growth, and in severe cases, entire leaves fall off. When mealybugs or shield scales are found, remove the insects promptly and spray the affected plant with a 1000-fold dilution of 80% dichlorvos emulsifiable concentrate. Collect, remove, and burn fallen leaves to eliminate overwintering pests. Root mealybugs primarily damage the agave root system, ultimately causing leaf wilting and plant death. When root mealybugs are found, change the outdoor cultivation location or replace the planting substrate immediately. The new substrate must be sterilized by high temperature or fumigation before use. Wipe away the insects from the roots of the agave with 70% isopropanol, cut off the affected roots, then soak the roots in a 1000-fold dilution of 40% acetamiprid emulsifiable concentrate for 1-2 hours, then remove and dry them before replanting.

[0093] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for culturing tissue from protruding teeth of the Emperor Nioh, comprising the following steps: (1) Preparation of explants Select healthy, disease-free young plants of *Gynostemma pentaphyllum*, remove the withered leaves, cut off the roots, disinfect, and obtain *Gynostemma pentaphyllum* explants. (2) Tissue culture induction Under aseptic conditions, sterilized explants were inoculated into an induction medium supplemented with 0.3 mg / L NAA. -1 30 g·L of sucrose -1 and carrageenan 8.0 g·L -1 The YMS medium, with pH adjusted to 5.8, contains the following macroelements: KNO3 1700 mg·L⁻¹. -1 NH4NO3 1430 mg·L -1 340 mg·L KH2PO4 -1 MgSO4 370 mg·L -1 220 mg·L⁻¹ CaCl₂ -1 and Ca(NO3)2 220 mg·L -1 ; The trace element is: KI 0.83 mg·L. -1 H3BO3 6.2 mg·L -1 MnSO4·4H2O 22.3 mg·L -1 ZnSO4·7H2O 8.6 mg·L -1 Na₂MoO₄·2H₂O 0.25 mg·L -1 CuSO4·5H2O 0.030 mg·L -1 and CoCl2·6H2O 0.025 mg·L -1 ; The iron salt concentration is: FeSO4·7H2O 55.6 mg·L. -1 and Na2-EDTA·2H2O 74.6 mg·L -1 ; The organic component is: inositol 100 mg·L -1 Niacin 0.5 mg·L -1 pyridoxine hydrochloride 0.5 mg·L -1 Thiamine hydrochloride 0.5 mg·L -1 and glycine 2.0 mg·L -1 ; (3) Subgeneration The induced protruding teeth material was inoculated into a subculture proliferation medium, and adventitious buds grew after subculture. The subculture medium in step (3) was supplemented with NAA 0.3 mg·L⁻¹. -1 30 g·L of sucrose -1 and carrageenan 8.0 g·L -1 YMS medium with a pH of 5.8; (4) Rooting culture Adventitious buds with uniform variegation, a height of over 2.5 cm, and 4-5 leaves, obtained from propagation culture, were transferred to a rooting medium for rooting culture for 30-40 days. The rooting medium was supplemented with IBA 1.0 mg·L⁻¹. -1 NAA 0.4 mg·L -1 20 g·L sucrose -1 and carrageenan 8.0 g·L -1 1 / 2 YMS medium; (5) Transplanting of tissue culture seedlings Select robust tissue culture seedlings that are over 4 cm tall, have uniform variegation, and have at least 4 clean white roots. Place them in a greenhouse with diffused light or in a place with sufficient natural light but no direct sunlight for hardening. After hardening, transplant the tissue culture seedlings into plug trays. The transplanting substrate and its volume ratio are: peat moss: coconut coir: coarse sand = 2:2:

1. After transplanting, place them in a seedling shed for cultivation. When the transplanted seedlings have good leaf and root growth and reach a height of over 10 cm, they can be planted in the open ground or in pots. (6) Management of transplanted seedlings; in, In both steps (2) and (3), 6-BA should be avoided.

2. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 1, characterized in that: In step (1), the disinfection method is as follows: after cutting off the roots of the young protruding tooth plant, rinse it under running water for 20-30 minutes, put the cleaned material into an ultrasonic cleaner for 15 minutes; put the cleaned explant into 75% alcohol and shake for 30 seconds, disinfect with 2% NaClO for 8 minutes, then disinfect with 0.1% mercuric chloride solution for 12 minutes, and finally rinse with sterile water 4-6 times, and dry with sterile filter paper to obtain the protruding tooth explant of Renwangguan.

3. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 1, characterized in that: In step (4), the culture period is 34~36 days.

4. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 1, characterized in that: In step (5), after the seedling hardening is completed, the tissue culture seedlings are taken out from the culture bottle and the culture medium on the tissue culture seedlings is cleaned with water. After cleaning, the roots of the tissue culture seedlings are soaked in a 1000-fold diluted solution of hymexazol for 30 seconds and placed in a cool and ventilated place to dry for 5 days. Then the tissue culture seedlings are transplanted into the seedling trays and placed in the seedling shed for cultivation after transplanting.

5. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 4, characterized in that: In step (5), the best planting time is in spring (April-May) or autumn (September-October).

6. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 1, characterized in that: In step (6), the temperature, humidity and light management of the transplanted seedlings are as follows: avoid direct sunlight within 15 days after transplanting, control the temperature at 22~27℃, and the relative humidity of the air should not be too high. Maintain it at 40%~50% by spraying or ventilation to avoid disease. After 15 days, use full light for seedling cultivation. If the sunlight is too strong within 1~5 days thereafter, provide appropriate shading at noon.

7. The method for tissue culture of protruding teeth from the Niohō-no-Tsū (a type of crown) according to claim 6, characterized in that: In step (6), water and fertilizer management is as follows: Water the roots until the soil or substrate is moist, but do not overwater. Water again 2-3 days after watering or when the soil or substrate is slightly dry. Reduce watering or stop watering in winter depending on the weather. Apply fertilizer in spring and autumn each year, with a light fertilizer applied to the roots in May and September, mainly nitrogen, phosphorus and potassium compound fertilizer. The fertilizer should be applied 10-15 cm away from the roots and avoid branches and leaves.

8. The method for tissue culture of protruding teeth from the "Ninja Crown" according to claim 6, characterized in that: In step (6), disease management is as follows: If root rot occurs, the substrate should be dried in time, and the seedlings with milder symptoms should be removed and the rotten roots removed; if leaf rot occurs, watering should be increased to make the substrate completely moist, and yellow and wilted leaves should be removed to reduce water loss and allow the seedlings to recover; if light management is poor, sunburn may occur, causing brown spots that gradually enlarge and become sunken on the surface of new leaves. At this time, shade nets should be added in time to avoid direct sunlight, ventilation in the greenhouse should be strengthened, and water should be sprayed appropriately to prevent the disease from worsening.

Images