Method for culturing bud tissue of haliotis diversicolor

Abstract

The tissue culture method comprises the following steps: (1) preparing an explant; (2) preparing a tissue culture induction culture medium; (4) subculture; (5) rooting culture; (6) transplanting tissue culture seedlings and the like. According to the method disclosed by the invention, through a series of working procedures such as disinfection, basic culture, multiplication culture and rooting culture on the euonymus japonicus tissue culture seedlings, a large number of euonymus japonicus bud tissue culture seedlings with stable quality can be quickly obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and more specifically to a breeding and cultivation method of Renwangguan abalone buds. Background Art

[0002] Agave titanota, also known as Yanliu, is native to tropical arid areas of America, mainly distributed in Oaxaca, Mexico. It is a perennial plant of the genus Agave in the Agave family. The plant has no obvious main stem, and the fleshy leaves are wide and thick, clustered radially and arranged in a rosette shape. The maximum plant width can reach 35-40cm. The upper half of the leaves are narrowly elliptical, with a sharp tip and a sharp black-brown thick thorn. Agave titanota has the function of absorbing harmful gases, such as formaldehyde, benzene, trichloroethylene, etc. It has important application value in gardening. It can be placed on indoor tables and desks for viewing, and can also be used to arrange desert plant landscapes, urban landscapes or gardens, public green spaces. It has a high ability to resist pollution and purify the air. It is a common ornamental plant in parks, green belts and homes.

[0003] Baoya is one of the variegated variants of Renwangguan. It has significant yellow stripes on both edges of its leaves, bright colors, beautiful appearance, and a small and delicate plant with higher ornamental value. However, its maintenance and breeding are also difficult. It is easy to bleach during the breeding process, leading to death. Baoya, like most agaves, only blooms once in its life, and the plant gradually dies after flowering, so it is difficult to collect seeds. Baoya grows slowly, generally only growing 1-2 cm per year. It needs sufficient sunlight and a dry environment to grow healthily. It relies on traditional methods such as division and cuttings for breeding. The seedlings grow slowly and the breeding efficiency is extremely low. At present, traditional breeding methods can no longer meet the market demand for Baoya seedlings and the development of its planting industry.

[0004] Prior art, a method for inducing agave bulbils (Yang Zhijian et al., Jiangsu Agricultural Sciences, 2019) discloses that agave stem segments are used as culture materials, and different culture media are obtained by treating with different concentrations of hormone combinations, thereby establishing a tissue culture system for Agave tequilae. However, the inventors found in the experiment that when the above method was used to tissue culture the Renwangguan abalone buds, the tissue culture seedlings showed severe whitening, resulting in low efficiency of tissue culture propagation. In view of this, the present invention uses a tissue culture rapid propagation technology system for research, with the aim of providing a breeding and cultivation method for Renwangguan abalone buds, providing a theoretical basis for the efficient cultivation and promotion of abalone buds, and laying the foundation for their large-scale production. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for culturing abalone bud tissue.

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

[0007] A method for culturing Renwangguan abalone bud tissue, comprising the following steps:

[0008] (1) Preparation of explants

[0009] Select healthy and disease-free young abalone bud plants, remove dry leaves from the plants, cut off roots, and sterilize to obtain Renwangguan abalone bud explants;

[0010] (2) Tissue culture induction

[0011] Under sterile conditions, the sterilized explants were inoculated into YMS medium with the following macroelements: KNO3 1700 mg·L -1 、NH4NO3 1430mg·L -1 、KH2PO4 340mg·L -1 、MgSO4 370mg·L -1 、CaCl2 220mg·L -1 ,Ca(NO3)2220mg·L -1 ;

[0012] Trace elements: KI 0.83mg·L -1 、H3BO3 6.2mg·L -1 、MnSO4·4H2O 22.3mg·L -1 、ZnSO4·7H2O 8.6mg·L -1 、Na2MoO4·2H2O 0.25mg·L -1 、CuSO4·5H2O 0.025mg·L -1 、CoCl2·6H2O0.025mg·L -1 ;

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

[0014] Organic ingredients: Inositol 100mg·L -1 , niacin 0.5mg·L -1 , Pyridoxine hydrochloride 0.5mg·L -1 , Thiamine hydrochloride 0.5mg·L -1 , Glycine 2.0mg·L -1 ;

[0015] The above induction medium was also supplemented with NAA 0.3 mg·L -1, sucrose 30g·L -1 , Carrageenan 8.0g·L -1 , pH = 5.8;

[0016] (3) Subculture

[0017] The induced abalone bud materials are inoculated into different subculture proliferation mediums, and adventitious buds grow out after subculture.

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

[0019] (4) Rooting culture

[0020] The adventitious buds with uniform variegation, a height of more than 2.5 cm, and 4-5 leaves were selected and transferred into a rooting medium for rooting culture for 30-40 days; the rooting medium was 1 / 2YMS+IBA1.0mg·L -1 +NAA0.4mg·L -1 + sucrose 20g·L -1 + Carrageenan 8.0g·L -1 ;

[0021] (5) Transplantation of tissue culture seedlings

[0022] Select healthy tissue culture seedlings with a plant height of more than 4 cm, uniform variegation, and more than 4 white roots and place them in a greenhouse with scattered light or a place with sufficient natural light and no direct light for seedling hardening; after completing the seedling hardening, transplant the tissue culture seedlings into a plug tray, the transplanting matrix is ​​peat soil: coconut bran: coarse sand=2:2:1 in volume ratio, and after the transplanting is completed, place them in a seedling shed for cultivation; when the leaves and root system of the transplanted seedlings grow well and the seedling height reaches more than 10 cm, they can be planted in the open field or in a pot;

[0023] (6) Management of transplanted seedlings.

[0024] Furthermore, in step (1), the disinfection method is as follows: after cutting off the roots of the young abalone bud plants, rinse them under running water for 20-30 minutes, and place the washed materials in an ultrasonic cleaner for cleaning for 15 minutes; place the cleaned explants in 75% alcohol and oscillate and soak them for 30 seconds, disinfect them with 2% NaClO for 8 minutes, and then disinfect them with 0.1% mercuric chloride solution for 12 minutes respectively, and finally rinse them with sterile water for 4-6 times, and dry them with sterile filter paper to obtain the Renwangguan abalone bud explants.

[0025] Furthermore, in step (2), the use of 6-BA is avoided.

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

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

[0028] Furthermore, in step (5), after completing the seedling hardening, the tissue culture seedlings are taken out from the culture bottle, the culture medium on the tissue culture seedlings is cleaned with clean water, and after cleaning, the roots of the tissue culture seedlings are immersed in a 1000-fold diluted solution of carbendazim for 30 seconds, placed in a cool and ventilated place for drying for 5 days, and then the tissue culture seedlings are transplanted into the plug tray. After transplanting, they are placed in a seedling shed for cultivation.

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

[0030] Furthermore, 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°C, and the relative humidity of the air should not be too high. It should be adjusted by spraying or ventilation, generally maintained at 40%-50% to avoid diseases. After 15 days, full light can be used for seedling cultivation. If the sunlight is too strong within 1-5 days thereafter, appropriate shading should be performed at noon.

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

[0032] Water the roots until the soil or substrate is moist, but not excessively; water again 2 to 3 days after watering or when the soil or substrate is slightly dry; in winter, reduce the amount of watering or do not water depending on the climate; add fertilizer in time in spring and autumn every year, apply a thin layer of fertilizer to the roots once in May and September respectively, mainly nitrogen, phosphorus and potassium compound fertilizers, and the applied fertilizer should be 10 to 15 cm away from the roots, avoiding branches and leaves.

[0033] Furthermore, in step (6), the 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 should be removed; if leaf atrophy occurs, the amount of water should be supplemented to moisten the substrate, and the yellow and wilted leaves should be removed to reduce water loss and resume the growth of the seedlings. If the light management is not good, it may cause sunburn, causing the new leaves to have brown spots that gradually expand and sink. At this time, the shade net should be covered in time to avoid direct sunlight, strengthen ventilation in the shed, and spray water appropriately to prevent the disease from getting worse.

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

[0035] (1) The present invention can quickly obtain a large number of Renwangguan abalone bud tissue culture seedlings with stable quality by performing a series of processes such as disinfection, basic culture, proliferation culture and rooting culture on the Renwangguan abalone bud tissue culture seedlings.

[0036] (2) The present invention adjusts the macroelements and iron salt elements in MS to obtain the improved YMS medium. The present invention found that the use of MS medium and YMS medium has little effect on the induction rate, but has a greater impact on the growth of the material. The use of YMS medium can effectively alleviate the whitening phenomenon that occurs during the tissue culture of Renwangguan abalone buds and reduce the whitening rate. At the same time, the present invention found that 6-BA easily causes mutations and deformities in tissue culture seedlings. Removing 6-BA during the tissue culture process can maintain its stable properties.

[0037] (3) The present invention found that during transplantation, the ratio of peat soil: coconut husk: coarse sand in the matrix is ​​2:2:1, which can significantly increase the survival rate of transplantation. Peat soil has rich nutrition and good water retention; coconut husk has good water retention and air permeability, and is also rich in organic matter, which is suitable for the growth of tissue culture seedlings; coarse sand particles are large, which can increase the air content in the soil, which is beneficial to the respiration and growth of plants. The combination of peat soil, coconut husk and coarse sand is beneficial to the growth of the root system of tissue culture seedlings, and effectively improves the survival rate of transplantation of Renwangguan Baoya tissue culture seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0039] Figure 1 =MS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 Culture results of the culture medium.

[0040] Figure 2 =MS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 +0.5mg·L -1 Culture results of 6-BA medium.

[0041] Figure 3 MS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 +1.0mg·L -1 Culture results of 6-BA medium.

[0042] Figure 4 YMS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 Culture results of the culture medium.

[0043] Figure 5 YMS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 +0.5mg·L -1 Culture results of 6-BA medium.

[0044] Figure 6 YMS+NAA0.3mg·L in Table 1 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 +1.0mg·L -1 Culture results of 6-BA medium.

[0045] Figure 7 YMS+NAA0.3mg·L in Table 2 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 Culture results of the culture medium.

[0046] Figure 8 YMS+NAA0.3mg·L in Table 2 -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 +0.5mg·L -1 Culture results of 6-BA medium.

[0047] Fig. 9 The results of rooting and cultivation of tissue culture seedlings.

[0048] Fig.10 These are the results of transplanting tissue culture seedlings. DETAILED DESCRIPTION

[0049] Example 1

[0050] In order to solve the problems of low reproduction efficiency and albinism of Renwangguan abalone buds, the present invention studies the rapid reproduction system of Renwangguan abalone buds, in order to establish an efficient tissue culture breeding technology system, reproduce a large number of seedlings with consistent specifications and stable species in a short period of time, and lay the foundation for its large-scale production.

[0051] The specific steps include:

[0052] (1) Preparation of explants

[0053] Select healthy, disease-free and insect-free young abalone plants, remove the dry leaves on the plants, cut off the roots, rinse under running water for 20-30 minutes, and put the washed materials into an ultrasonic cleaner for 15 minutes. Put the washed explants into 75% alcohol and oscillate and soak for 30 seconds, disinfect with 2% NaCIO for 8 minutes, and then disinfect with 0.1% mercuric chloride solution for 12 minutes, and finally rinse with sterile water for 4-6 times, and dry with sterile filter paper to obtain Renwangguan abalone explants.

[0054] (2) Tissue culture induction medium

[0055] Under sterile conditions, the sterilized explants were inoculated into different induction mediums, MS medium and modified MS medium, and different concentrations of plant growth hormone (0, 0.5, 1.0) mg·L were added. -1 6-BA, to explore the effects of different culture media and hormone concentrations on the germination induction of Abalone buds.

[0056] Improved MS medium: Using MS as the basic medium, adjust the macroelements and iron salts in MS, while keeping the amounts of other elements in MS medium unchanged. The improved basic medium is referred to as YMS.

[0057] The formula of conventional MS medium is:

[0058] Macroelements: KNO3 1900mg·L -1 、NH4NO3 1650mg·L -1 、KH2PO4 170mg·L -1 、MgSO4370mg·L -1 、CaCl2·2H2O 440mg·L -1 ;

[0059] Trace elements: KI 0.83mg·L -1 、H3BO3 6.2mg·L -1 、MnSO4·4H2O 22.3mg·L -1 、ZnSO4·7H2O 8.6mg·L -1 、Na2MoO4·2H2O 0.25mg·L -1 、CuSO4·5H2O 0.025mg·L -1 、CoCl2·6H2O0.025mg·L -1 ;

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

[0061] Organic ingredients: Inositol 100mg·L -1 , niacin 0.5mg·L -1 , Pyridoxine hydrochloride 0.5mg·L -1 , Thiamine hydrochloride 0.5mg·L -1 , glycine mg·L -1 .

[0062] The ingredients of YMS are as follows:

[0063] Macroelements: KNO3 1700mg·L -1 、NH4NO3 1430mg·L -1 、KH2PO4 340mg·L -1 、MgSO4370mg·L -1 、CaCl2 220mg·L -1 , Ca(NO3)2 220mg·L -1 ;

[0064] Trace elements: KI 0.83mg·L -1 、H3BO3 6.2mg·L -1 、MnSO4·4H2O 22.3mg·L -1 、ZnSO4·7H2O 8.6mg·L -1 、Na2MoO4·2H2O 0.25mg·L -1 、CuSO4·5H2O 0.030mg·L -1 、CoCl2·6H2O0.025mg·L -1 ;

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

[0066] Organic ingredients: Inositol 100mg·L -1 , niacin 0.5mg·L -1 , Pyridoxine hydrochloride 0.5mg·L -1 , Thiamine hydrochloride 0.5mg·L -1 , Glycine 2.0mg·L -1 ;

[0067] NAA 0.3 mg·L was added to the above MS and YMS induction medium. -1 , sucrose 30g·L -1 and carrageenan 8.0 g·L -1, pH was adjusted to 5.8.

[0068] The temperature in the culture room is 24-28℃, the light intensity is 2500lx, and the lighting test is 12h / d.

[0069] Table 1 Effects of different culture media and hormone concentrations on the induction of Abalone buds

[0070]

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

[0072] The present invention (1) increases the iron content in the culture medium. During the growth of tissue culture seedlings, iron salts promote the formation of chlorophyll, thereby improving the efficiency of photosynthesis and allowing the plants to grow and develop normally. During the tissue culture of Renwangguan abalone buds, the applicant found that appropriately increasing the iron content can alleviate the whitening of tissue culture seedlings. (2) The applicant found that the concentration of 6-BA has a significant effect on the induction of differentiation of variegated species, and high concentrations of 6-BA are not conducive to the formation of variegated species. Therefore, the use of 6-BA should be avoided during the tissue culture of Renwangguan abalone buds.

[0073] (4) Subculture

[0074] The induced abalone bud materials are inoculated into different subculture proliferation mediums, and adventitious buds grow out after subculture.

[0075] The subculture medium in step (4) is YMS + NAA 0.3 mg·L -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 , pH=5.8; YMS+6-BA0.5mg·L -1 +NAA0.3mg·L -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 , pH = 5.8.

[0076] Table 2 Results of the 6-BA test on abalone sprout proliferation

[0077]

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

[0079] (5) Rooting culture

[0080] Select the adventitious buds with uniform patterns, a height of more than 2.5 cm, and 4-5 leaves that have been cultured and transferred to the rooting medium (1 / 2YMS + IBA 1.0 mg·L-1 +NAA0.4mg·L -1 + sucrose 20g·L -1 + Carrageenan 8.0g·L -1 ) for rooting culture with a cycle of 35 days.

[0081] See the pictures Fig. 9 .

[0082] (6) Transplantation of tissue culture seedlings

[0083] Select a healthy tissue culture seedling with a plant height of more than 4 cm, uniform variegation, and more than 4 white roots to be placed in a greenhouse with scattered light or a place with sufficient natural light and no direct light for hardening. After completing hardening, take the tissue culture seedling out of the culture bottle, clean the culture medium on the tissue culture seedling with clean water, pay attention not to hurt the young leaves and roots during cleaning, immerse the roots of the tissue culture seedlings in a 1000-fold dilution of oxadipazole for 30 seconds after cleaning, place it in a cool and ventilated place to air the seedlings for 5 days, and then transplant the tissue culture seedlings to the plug tray, the transplanting matrix is ​​peat soil: coconut husk: coarse sand = 2:2:1 (volume ratio), the matrix is ​​well ventilated and nutritious, and is placed in a nursery shed for cultivation after transplanting. When the transplanted seedling leaves and root system grow well and the seedling height reaches more than 10 cm, open field planting or pot planting can be carried out, spring (April-May) or autumn (September-October) is the best planting period, and it is important to avoid continuous rainy or high temperature and high humidity weather.

[0084] See the pictures Fig.10

[0085] (7) Transplant seedling management

[0086] 1. Temperature, humidity and light management of Renwangguan abalone seedlings

[0087] Avoid direct sunlight within 15 days after transplanting, control the temperature at 22-27℃, and keep the relative humidity of the air at a low level. Use spraying or ventilation to adjust the humidity, generally at 40%-50%, to avoid diseases. After 15 days, full light can be used for seedling cultivation. If the sunlight is too strong within 1-5 days, shade the area at noon.

[0088] 2. Water and fertilizer management of Renwangguan abalone

[0089] The waterlogging tolerance of the Renwangguan Baoya is poor, so the amount of watering needs to be strictly controlled. Water the roots until the soil or substrate is moist, but not excessively. Water again 2 to 3 days after watering or when the soil or substrate is slightly dry, so as to avoid excessive water causing root rot. In winter, the amount of watering can be reduced or not watered depending on the climate. Spring and autumn are the peak seasons for the growth of Agave every year, so fertilizers need to be added in time. Thin fertilizers should be applied to the roots once in May and September, mainly nitrogen, phosphorus, and potassium compound fertilizers. Organic fertilizers can also be applied. The fertilizers should be 10 to 15 cm away from the roots, avoiding branches and leaves. Potted plants can also use slow-release fertilizers in a timely and appropriate manner to avoid fertilizer damage such as root burn and wilting caused by excessive fertilizer concentration.

[0090] 3. Disease management of transplanted Renwangguan abalone seedlings

[0091] Common diseases of Renwangguan Baoya include anthracnose, leaf spot and gray mold, which are mainly infectious fungal diseases. These diseases mainly harm the leaves of Renwangguan Baoya, causing lesions and spots on the leaves, which may eventually cause the entire leaf to rot. Most of these diseases are caused by a relatively humid cultivation environment, poor ventilation or excessive use of nitrogen fertilizers. When planting, attention should be paid to ventilation and control of temperature and humidity. The proportion of nitrogen in nitrogen, phosphorus and potassium compound fertilizers should not be too high. Phosphorus and potassium elements should be increased to enhance the disease resistance of agave, and 800 times of methyl thiophanate should be sprayed for prevention. For plants that have already fallen ill, rotten leaves should be removed in time to prevent the further spread of pathogens. In the early stage of the disease, spray the pesticide every 7 to 10 days for prevention and control, and spray continuously for 3 times. Different pesticides should be replaced each time to avoid drug resistance. You can choose 800 times of 70% methyl thiophanate, 800 times of 75% thiophanate-methyl, 800 times of anthracnose or 1000 times of green milk copper.

[0092] Common insect pests of Renwangguan Baoya include scale insect pests: mainly mealybugs, shield scales and root mealybugs. Mealybugs and shield scales mainly harm the leaves of agave. Among them, the main symptom of mealybugs is yellowing of leaves, which can cause plant death in severe cases; the main symptom of shield scales is chlorotic spots on leaves, declining growth, and in severe cases, the whole leaf falls off. When mealybugs and shield scales are found to be harmful, the insects should be removed in time, and then 1000 times of 80% dichlorvos emulsifiable concentrate should be sprayed on the diseased plants. When the old leaves fall off, the fallen leaves should be collected, removed and burned in time to eliminate the overwintering insect sources. Root mealybugs mainly harm the roots of agave, eventually causing the leaves to wilt and the plants to die. When root mealybugs are found to be harmful, the open-field cultivation site or the planting substrate should be replaced in time. The new substrate must be sterilized by high temperature or drug fumigation before use. Use 70% isopropyl alcohol to wipe off the insects on the roots of agave, cut off the damaged roots, and then soak the roots in 1000 times diluted 40% cyproconazole emulsion. After 1 to 2 hours, take out and dry them, and replant them.

[0093] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for culturing abalone bud tissue, comprising the following steps: (1) Preparation of explants Select healthy and disease-free young abalone bud plants, remove dry leaves from the plants, cut off roots, and sterilize to obtain Renwangguan abalone bud explants; (2) Tissue culture induction Under sterile conditions, the sterilized explants were inoculated into YMS medium with the following macroelements: KNO3 1700 mg·L -1 、NH4NO3 1430mg·L -1 、KH2PO4 340mg·L -1 、MgSO4 370mg·L -1 、CaCl2 220mg·L -1 ,Ca(NO3)2220mg·L -1 ; Trace elements: KI 0.83mg·L -1 、H3BO3 6.2mg·L -1 、MnSO4·4H2O 22.3mg·L -1 、ZnSO4·7H2O8.6mg·L -1 、Na2MoO4·2H2O 0.25mg·L -1 、CuSO4·5H2O 0.025mg·L -1 、CoCl2·6H2O0.025mg·L -1 ; Iron salt: FeSO4·7H2O 55.6mg·L -1 、Na2-EDTA·2H2O 74.6mg·L -1 ; Organic ingredients: Inositol 100mg·L -1 , niacin 0.5mg·L -1 , Pyridoxine hydrochloride 0.5mg·L -1 , Thiamine hydrochloride 0.5mg·L -1 , Glycine 2.0mg·L -1 ; NAA 0.3 mg·L was also added to the above induction medium. -1 , sucrose 30g·L -1 , Carrageenan 8.0g·L -1 , pH = 5.8; (3) Subculture The induced abalone bud materials are inoculated into different subculture proliferation mediums, and adventitious buds grow out after subculture. The subculture medium in step (3) is YMS + NAA 0.3 mg·L -1 + sucrose 30g·L -1 + Carrageenan 8.0g·L -1 , pH = 5.8; (4) Rooting culture The adventitious buds with uniform variegation, a height of more than 2.5 cm, and 4-5 leaves were selected and transferred into a rooting medium for rooting culture for 30-40 days; the rooting medium was 1 / 2YMS+IBA1.0mg·L -1 +NAA0.4mg·L -1 + sucrose 20g·L -1 + Carrageenan 8.0g·L -1 ; (5) Transplantation of tissue culture seedlings Select healthy tissue culture seedlings with a plant height of more than 4 cm, uniform variegation, and more than 4 white roots and place them in a greenhouse with scattered light or a place with sufficient natural light and no direct light for seedling hardening; after completing the seedling hardening, transplant the tissue culture seedlings into a plug tray, the transplanting matrix is ​​peat soil: coconut bran: coarse sand=2:2:1 in volume ratio, and after the transplanting is completed, place them in a seedling shed for cultivation; when the leaves and root system of the transplanted seedlings grow well and the seedling height reaches more than 10 cm, they can be planted in the open field or in a pot; (6) Management of transplanted seedlings.

2. The method for tissue culture of Abalone buds of Renwangguan according to claim 1, characterized in that: In step (1), the disinfection method is as follows: after cutting off the roots of the young abalone bud plants, rinse them under running water for 20-30 minutes, and place the washed materials in an ultrasonic cleaner for cleaning for 15 minutes; place the cleaned explants in 75% alcohol and oscillate and soak them for 30 seconds, disinfect them with 2% NaClO for 8 minutes, and then disinfect them with 0.1% mercuric chloride solution for 12 minutes respectively, and finally rinse them with sterile water for 4-6 times, and dry them with sterile filter paper to obtain the Renwangguan abalone bud explants.

3. The method for tissue culture of Abalone buds of Renwangguan according to claim 2, characterized in that: In step (2), avoid using 6-BA in the induction medium.

4. The method for tissue culture of Abalone buds of Renwangguan according to claim 1, characterized in that: In step (3) of subculture, avoid using 6-BA.

5. The method for tissue culture of Abalone buds of Renwangguan according to claim 1, characterized in that: In step (4), the culture period is 34 to 36 days.

6. The method for tissue culture of Abalone buds of Renwangguan 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, the culture medium on the tissue culture seedlings is cleaned with clean water, and after cleaning, the roots of the tissue culture seedlings are immersed in a 1000-fold diluted solution of carbendazim for 30 seconds, placed in a cool and ventilated place for drying for 5 days, and then the tissue culture seedlings are transplanted into the plug tray. After the transplanting is completed, they are placed in a seedling shed for cultivation.

7. The method for tissue culture of Abalone buds of Renwangguan according to claim 6, characterized in that: In step (5), the best planting period is April to May in spring or September to October in autumn.

8. The method for tissue culture of Abalone buds of Renwangguan 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°C, and the relative humidity of the air should not be too high, and should be adjusted by spraying or ventilation to maintain at 40%-50% to avoid diseases; after 15 days, full light can be used for seedling cultivation. If the sunlight is too strong within 1-5 days thereafter, appropriate shading should be performed at noon.

9. The method for tissue culture of Abalone buds of Renwangguan according to claim 8, characterized in that: In step (6), water and fertilizer management is: Water the roots until the soil or substrate is moist, but not excessively; water again 2 to 3 days after watering or when the soil or substrate is slightly dry; in winter, reduce the amount of watering or do not water depending on the climate; add fertilizer in time in spring and autumn every year, apply a thin layer of fertilizer to the roots once in May and September respectively, mainly nitrogen, phosphorus and potassium compound fertilizers, and the applied fertilizer should be 10 to 15 cm away from the roots, avoiding branches and leaves.

10. The method for tissue culture of Abalone buds of Renwangguan according to claim 8, characterized in that: In step (6), the disease management is as follows: if root rot occurs, the substrate should be dried in time, and the seedlings with milder symptoms should be taken out and the rotten roots should be removed; if leaf atrophy occurs, it is necessary to add water to make the substrate completely moist, and remove the yellow and wilted leaves to reduce water loss and allow the seedlings to resume growth; if light management is poor, it may cause sunburn, causing the new leaves to have brown patches that gradually expand and sink. At this time, the shade net should be added in time to avoid direct sunlight, strengthen ventilation in the shed, and spray water appropriately to prevent the disease from getting worse.

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