A method for high-frequency induction of adventitious bud regeneration of hosta plantlets in vitro

Abstract

The application provides a method for high-frequency induction of adventitious buds from leaves of Hosta plantaginea tissue culture seedlings and regeneration of plants, and belongs to the technical field of plant tissue culture; the method comprises the following steps: step 1: taking aseptic leaves of 'Chuzhi Zhi Feng' tissue culture seedlings as explants, and inducing callus and differentiation of the explants into adventitious buds after dark culture in an induction culture medium; step 2: transferring the formed adventitious buds into a proliferation culture medium to obtain multiple shoots; step 3: transferring the obtained shoots into a strong seedling culture medium for strong seedling growth to form seedlings; and step 4: transferring the seedlings into a rooting culture medium to induce root systems to obtain complete small plants; the application regulates callus induction and differentiation culture media, so that the efficiency of induction of adventitious buds is improved, the seedling raising cycle is shortened, the operation method is simpler, the cost is lower, the survival rate is higher, and the application has good practicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a method for high-frequency induction of adventitious bud regeneration of leaves of Hydrangea macrophylla tissue culture seedlings. BACKGROUND

[0002] In a broad sense, Hydrangea is a collective term for plants of the genus Hydrangea L. in the family Hydrangaceae, and in a narrow sense, it refers to the most commonly cultivated species of the genus Hydrangea, commonly known as large-flowered hydrangea or large-leaved hydrangea. Hydrangea macrophylla (Thunb) Ser., also known as eight immortals flower and purple sunflower, has a wide range of applications in landscape design, home gardening, fresh-cut flowers and dried flowers, etc. In addition, hydrangea is resistant to acid and aluminum, and has important roles in ecology and medicine, and has a broad development prospect. At present, research on hydrangea is focused on cultivation techniques, physiology and biochemistry, molecular mechanisms, and flower color regulation. Due to the poor seed setting performance and extremely low seed germination rate of hydrangea under natural conditions, production is mainly carried out by propagation methods such as cutting, layering and division. However, such methods are highly seasonal and have low propagation coefficients, making it difficult to meet the needs of large-scale production. Therefore, rapid propagation of hydrangea seedlings through tissue culture is one of the most efficient methods for obtaining high-quality and high-yield seedlings. At the same time, with the in-depth study of genetic engineering technology, using transgenic or gene editing technology based on tissue culture for plant trait improvement has become a very effective breeding method.

[0003] 'Chuzhi Zhifeng' is a flat, double-flowered large-leaved hydrangea variety introduced from Japan. It has a dwarf plant type with a height of about 40 cm, and is good for potted ornamental effect. It has excellent upright and branching properties and can naturally form a shape without artificial pruning, showing a dense leafy form. Old branches bloom with cymose inflorescences, and are sensitive to pH, which can be adjusted to pink, blue, purple and other colors. 'Chuzhi Zhifeng' is also a typical late-flowering variety with a long flowering period, starting in late spring and blooming until autumn. Summer is its peak flowering period, making it a popular new variety for home gardening and balcony landscaping. Therefore, it is urgent to take effective measures to rapidly propagate hydrangea 'Chuzhi Zhifeng' to meet the needs of germplasm preservation, large-scale seedling production and genetic improvement.

[0004] At present, different varieties and explants and experimental methods have been applied to the research of the regeneration system of Hydrangea macrophylla. A method for rapidly breeding Hydrangea macrophylla Endless Summer by using leaf blades of tissue culture seedlings is disclosed in the prior art. The method for rapidly breeding Hydrangea macrophylla Endless Summer can significantly improve the callus induction rate and rooting rate of Hydrangea macrophylla Endless Summer during the seedling raising period. However, the dark culture time in the induction process is as long as 20 days, which greatly prolongs the seedling raising period. In addition, the differentiation efficiency of the optimal callus induction medium in the method is very low, and different hormone ratio media need to be replaced to promote the regeneration of adventitious buds, which not only increases the difficulty of rapid propagation technology and reduces the seedling raising efficiency, but also is prone to contamination, reduces the survival rate and increases the production cost. Therefore, a method for inducing adventitious bud regeneration plants is needed, which can improve the efficiency of inducing adventitious buds, shorten the seedling raising period, and has a simpler operation method, lower cost and higher survival rate. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a method for high-frequency induction of adventitious bud regeneration plants from Hydrangea leaf blades. The method comprises the following steps: step 1: taking sterile leaves of 'Chuzhi Zifeng' tissue culture seedlings as explants, inducing callus and differentiating into adventitious buds after dark culture in an induction medium; step 2: transferring the formed adventitious buds to a proliferation medium to obtain multiple shoots; step 3: transferring the obtained shoots to a strong seedling culture medium for strong seedling growth to form seedlings; and step 4: transferring the seedlings to a rooting medium to induce root systems to obtain complete small plants. By adjusting the callus induction and differentiation medium, the efficiency of inducing adventitious buds is improved, the seedling raising period is shortened, the operation method is simpler, the cost is lower, the survival rate is higher, and the method has good practicability.

[0006] In order to achieve the above technical purposes, the present application adopts the following technical means:

[0007] The present application first provides a callus induction and differentiation medium, which comprises: MS medium, N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) and 2,4-dichlorophenoxyacetic acid (2,4-D).

[0008] Preferably, the final concentration of CPPU is 2.5-4.0 mg / L, and the final concentration of 2,4-D is 0.05-0.15 mg / L.

[0009] The present application provides a method for high-frequency induction of adventitious bud regeneration plants from Hydrangea leaf blades, which comprises:

[0010] Step 1: taking the aseptic leaf of the tissue culture seedling of Hydrangea macrophylla as an explant, culturing the explant in the above-mentioned callus induction and differentiation medium under darkness + normal light to induce callus and differentiate into adventitious buds;

[0011] Step 2: continuing to culture the obtained adventitious buds in the proliferation medium to form sprouts;

[0012] Step 3: culturing the obtained sprouts in the strong seedling medium to grow strong seedlings and form seedlings;

[0013] Step 4: transferring the obtained seedlings to the rooting medium to induce root systems to obtain complete small plants.

[0014] Preferably, in step 1, the method for obtaining the tissue culture seedling of Hydrangea macrophylla comprises: cutting the current-year healthy stem section with buds of the pot seedling of Hydrangea macrophylla, and obtaining the aseptic tissue culture seedling of Hydrangea macrophylla through disinfection and sterilization and in-vitro germination.

[0015] Preferably, the Hydrangea macrophylla comprises Chizhi Zhifeng.

[0016] Preferably, in step 1, the culture condition of darkness + normal light is that the explant is cultured in a dark environment for 7-14 days, and then is transferred to normal light culture until callus is induced.

[0017] Preferably, the culture temperature in the dark environment is 25±2℃; and the culture condition of normal light is that the culture temperature is 25±2℃, the light intensity is 1800-2000Lux, and the light time is 14h / d.

[0018] Preferably, in step 2, the proliferation medium comprises: MS+1.0-3.0mg / L 6-BA+0.01-0.05mg / L IBA+0.01-0.05mg / L 2,4-D.

[0019] Preferably, in step 3, the strong seedling medium comprises: MS+0.5-1.5mg / L 6-BA+0.1-0.2mg / L 2,4-D.

[0020] Preferably, in step 4, the rooting medium comprises: MS+0.1-0.5mg / L IBA+0.1-0.2mg / L 2,4-D.

[0021] Preferably, in steps 2-4, the culture condition in the proliferation medium, the strong seedling medium, the strong seedling medium and the rooting medium is that the culture temperature is 25±2℃, the light intensity is 1800-2000Lux, and the light time is 14h / d.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application takes aseptic leaves of tissue culture seedlings of Hydrangea macrophylla 'Chikushinozora' as explants to induce callus occurrence and promote adventitious bud regeneration, maintains the characteristics of continuous proliferation and seedling formation, can provide a large number of high-quality seedlings for large-scale production, and can also provide support for stable genetic transformation technology of Hydrangea macrophylla mediated by Agrobacterium, and lay a foundation for breeding new and excellent varieties of Hydrangea macrophylla.

[0024] The present application takes aseptic leaves of tissue culture seedlings of 'Chikushinozora' as materials, which is convenient, and avoids the problems of disinfection of explants and high contamination rate in the process of tissue culture. The seedling raising period is short, the production cost is low, the operation is simple, the propagation coefficient is high, and the factory seedling breeding is facilitated.

[0025] In the prior art, the tissue culture technology of Hydrangea macrophylla takes a stem segment with buds obtained by cutting as explant material, N-(2-chloro-4-pyridyl)-N'-phenylurea is used to induce callus, and then the callus is transferred to a culture medium containing 6-benzylaminopurine BAP to induce adventitious buds. The induction rate of adventitious buds is 74.3%, and the average number of adventitious buds is 5. The induction cycle of callus is 20 days of dark culture and 40 days of light culture, and the propagation coefficient is 3-5. In the present application, N-(2-chloro-4-pyridyl)-N'-phenylurea is selected to induce callus from aseptic seedling leaves of Hydrangea macrophylla and regenerate adventitious buds. The regeneration rate can reach 93.3%, and the average number of adventitious buds can reach 6.9. The cycle of callus induction and regeneration of adventitious buds is only 40 days (including 10 days of dark culture), and high-frequency induction of adventitious buds is realized. The propagation coefficient can reach 8.7 in the proliferation culture medium containing 6-benzylaminopurine BAP. The seedling raising period is greatly shortened, the pollution is reduced, and the practical degree is reached, which provides a new technology for the factory production of Hydrangea macrophylla test tube seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram of adventitious bud regeneration.

[0027] Figure 2 It is a schematic diagram of cluster bud formation.

[0028] Figure 3 It is a schematic diagram of strong seedling growth of tissue culture seedlings.

[0029] Figure 4 It is a schematic diagram of rooting culture of tissue culture seedlings. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with the drawings and specific embodiments, but the protection scope of the present application is not limited thereto. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0031] The test materials and reagents used in the following examples, if not specifically stated, can be obtained commercially.

[0032] If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual can be used.

[0033] The large-leaf hydrangea 'Chuzhi Zhifeng' tissue culture seedlings used in the present application were provided by the Hydrangea Tissue Culture Room of the Leisure Agriculture Research Institute of Jiangsu Academy of Agricultural Sciences. The reagents used in the following examples are as follows:

[0034] 6-BA is 6-benzylaminopurine; IBA is indole-3-butyric acid; CPPU is N-(2-chloro-4-pyridyl)-N'-phenylurea; 2,4-D is 2,4-dichlorophenoxyacetic acid;

[0035] Example 1

[0036] (1) Source and treatment of experimental materials

[0037] The current-year healthy and strong stem segments of pot-grown hydrangea plants were cut, sterilized and germinated in vitro to obtain aseptic hydrangea tissue culture seedlings. The leaf tips and petioles of the hydrangea tissue culture seedlings that had been multiplied more than 3 times in the laboratory and were healthy and strong were selected as explant materials. The leaf tips and petioles were cut along the veins and inoculated on induction and differentiation medium.

[0038] (2) Culture conditions

[0039] Callus induction and differentiation medium: MS + 2.5-4.0 mg / L CPPU + 0.05-0.15 mg / L 2,4-D; culture temperature in the dark was 25±2℃; normal light culture conditions: culture temperature was 25±2℃, light intensity was 1800-2000 Lux, and light time was 14h / d.

[0040] Proliferation medium: MS + 1.0-3.0 mg / L 6-BA + 0.01-0.05 mg / L IBA + 0.01-0.05 mg / L 2,4-D.

[0041] Strong seedling medium: MS + 0.5-1.5 mg / L 6-BA + 0.1-0.2 mg / L 2,4-D.

[0042] Rooting medium: MS + 0.1-0.5 mg / L IBA + 0.1-0.2 mg / L 2,4-D.

[0043] The culture conditions in the proliferation medium, strong seedling medium, strong seedling medium and rooting medium were as follows: culture temperature was 25±2℃, light intensity was 1800-2000 Lux, and light time was 14h / d.

[0044] (3) Inoculation and culture:

[0045] Each culture dish was inoculated with 7 explants, and cultured in the dark for 10 days, and then cultured under normal light conditions for 30 days. Callus and adventitious buds were directly produced at the wound of the leaf. The adventitious buds induced to differentiate were rapidly propagated on the proliferation medium. After 30 days, the clusters of buds in the proliferation medium were transferred to the seedling growth medium for seedling growth. After 40 days, the seedlings were transferred to the rooting medium until the complete plants were formed

[0046] Example 2:

[0047] This example investigated the effects of different concentrations of hormone combinations and different dark culture times on the induction of leaf callus and regeneration of adventitious buds. The specific steps are shown below.

[0048] (1) Effects of different concentrations of hormone combinations on the induction of leaf callus and regeneration of adventitious buds

[0049] The explants in Example 1 were transferred to MS medium containing 1.0-4.0 mg / L CPPU and 0.05-0.15 mg / L 2,4-D and cultured for 40 days under the same culture conditions as in Example 1. The regeneration rate of adventitious buds, the average number of adventitious buds, and the state of the adventitious buds were calculated. The results are shown in Table 1.

[0050] Table 1 Effects of different concentrations of hormone combinations on the induction of leaf callus and regeneration of adventitious buds of 'Tsukiyofu'

[0051] Treatment CPPU (mg / L) 2,4-D (mg / L) Regeneration rate (%) Average number of adventitious shoots State of adventitious shoots 1 1.0 0.05 46.7±6.7gh 2.2±0.6cd Few and small, weak 2 2.0 0.05 56.7±8.8fg 1.6±0.3d Few and small, weak 3 3.0 0.05 58.8±5.1ef 2.8±0.4cd Few, weak 4 4.0 0.05 63.3±3.3ef 3.1±0.5c More, weak 5 1.0 0.1 68.8±3.8de 3.4±0.5c Larger, weak 6 2.0 0.1 74.4±3.8cd 3.1±1.4c More, weak 7 3.0 0.1 93.3±6.7a 6.9±0.9a More and large, strong 8 4.0 0.1 87.7±5.1ab 5.3±0.6b More and large, strong 9 1.0 0.15 82.2±5.1bc 3.6±0.8c Larger, strong 10 2.0 0.15 61.1±5.1ef 2.1±1.0cd Few and small, weak 11 3.0 0.15 55.6±8.4fg 2.8±0.7cd Few and small, weak 12 4.0 0.15 36.7±8.8h 1.4±0.7d Few and small, weak

[0052] Note: The same column data marked with different lowercase letters indicate significant differences (p<0.05).

[0053] As can be seen from Table 1, when the concentrations of CPPU and 2,4-D were 3.0 mg / L and 0.1 mg / L, respectively, the regeneration rate was the highest, reaching 93.3%, and the average number of adventitious buds was 6.9. The state of the adventitious buds was better than that of the other treatments. Therefore, MS medium with 3.0 mg / L CPPU and 0.1 mg / L 2,4-D was the most suitable for the regeneration of adventitious buds of 'Tsukiyofu' tissue culture seedlings.

[0054] The prior art needs to be transferred to the adventitious bud differentiation medium to realize the regeneration of the adventitious bud after the leaf blade induces the callus, and the highest regeneration rate of the adventitious bud is only 74.3%, and the average number of adventitious buds is 5.3; when the combination of 6-BA and KT is selected, the average number of adventitious buds can only be increased to 5. Compared with the prior art, the method disclosed by the application can not only significantly improve the efficiency of inducing the adventitious bud, but also has the advantages of simple operation, low cost and high survival rate.

[0055] (2) Effect of different dark culture times on regeneration of leaf blade callus into adventitious buds:

[0056] According to the optimized conditions in step (1), the explants in Example 1 are transferred to the MS medium containing 3.0 mg / L CPPU and 0.1 mg / L 2,4-D for dark culture, and the dark culture time is set to 0, 7, 10, 14, 21 and 28 days, and the light time is increased or shortened accordingly until the regeneration of the adventitious bud is induced, and the other culture conditions are the same as those in Example 1. The regeneration rate of the adventitious bud and the average number of adventitious buds are calculated. The results are shown in Table 2.

[0057] Table 2. Effect of different dark culture times on regeneration of leaf blade into adventitious buds

[0058]

[0059]

[0060] Note: The same column data marked with different lowercase letters indicates significant difference (p<0.05).

[0061] As can be seen from Table 2, when the dark culture time is 0 d, the explants cannot induce the callus to differentiate into the adventitious bud, which shows that a certain dark condition is necessary for the high-frequency induction of the adventitious bud to regenerate the plant; when the dark culture time is 10 d, the regeneration rate of the leaf blade can reach 93.3%, and the average number of adventitious buds can reach 8.9; but when the dark culture time is too long, the leaf blade is yellow due to insufficient light, and the regeneration rate is also reduced. The above results show that when the dark culture time is 10 d, the efficiency of regenerating the adventitious bud of the tissue culture seedling leaf blade of 'Tsukiyofu' is the highest, compared with the dark culture time of 15-25 days in the prior art, the method disclosed by the application further shortens the seedling period on the basis of improving the efficiency of inducing the adventitious bud, and is more convenient for factory seedling.

[0062] Example 3:

[0063] In this example, the effect of different concentrations of 6-BA on the proliferation of the adventitious bud of 'Tsukiyofu' is investigated, and the specific steps are as follows:

[0064] The obtained adventitious buds in Example 2 were transferred to MS medium containing 1.0 mg / L, 2.0 mg / L, 3.0 mg / L 6-BA and 0.01 mg / L, 0.05 mg / L IBA and 0.01 mg / L, 0.05 mg / L 2,4-D, respectively, and cultured for 40 d under the same conditions as in Example 1, and the proliferation coefficient of the adventitious buds was calculated. The results are shown in Table 3:

[0065] Table 3. Effect of different concentrations of 6-BA and IBA on the proliferation of 'Chikushi Kazabha' adventitious buds

[0066] Treatment 6-BA (mg / L) IBA (mg / L) 2,4-D (mg / L) Proliferation coefficient 1 1.0 0.01 0.05 3.2±0.8c 2 2.0 0.05 0.05 8.7±1.3a 3 3.0 0.05 0.01 5.3±1.1b

[0067] Note: The same column data marked with different lowercase letters represent significant differences (p < 0.05).

[0068] As can be seen from Table 3, when the concentration of 6-BA is 2.0 mg / L, the proliferation coefficient is the highest, and the cluster buds grow well; but when the concentration of 6-BA is too high (3.0 mg / L), leaf curling and vitrification phenomenon will occur; when the concentration of 6-BA is too low, leaf yellowing will occur and the proliferation coefficient is low (<3.5); the proliferation coefficient of the adventitious buds in treatment 2 is the highest and the growth state is better. Therefore, the most suitable medium for adventitious bud proliferation is MS + 2.0 mg / L 6-BA + 0.05 mg / L IBA + 0.05 mg / L 2,4-D.

[0069] Example 4:

[0070] This example investigates the effect of different concentrations of 6-BA on the growth of 'Chikushi Kazabha' tissue culture seedlings, and the specific steps are as follows:

[0071] The cluster buds obtained in Example 3 were separated into single plants and transferred to MS medium containing 0 mg / L, 0.5 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.5 mg / L, 1.8 mg / L 6-BA and 0.1 mg / L 2,4-D, 0.2 mg / L 2,4-D, respectively, and cultured for 40 d under the same conditions as in Example 1, and the stem growth was measured and the growth state of the seedlings was observed. The results are shown in Table 4:

[0072] Table 4. Effect of different concentrations of 6-BA on the growth of 'Chikushi Kazabha' tissue culture seedlings

[0073] Treatment 6-BA (mg / L) 2,4-D (mg / L) Stem growth (cm) State of seedling growth 1 0 0.1 1.2±0.12d Dwarf, partial yellowing 2 0.5 0.1 2.3±0.25c Good, partial yellowing 3 0.8 0.1 3.4±0.17b Strong, thick stem 4 1.0 0.1 4.5±0.15a Strong, thick stem 5 1.2 0.2 3.2±0.15b Leaf slightly curled, partial yellowing 6 1.5 0.2 2.3±0.22c Good, partial yellowing 7 1.8 0.2 1.3±0.20d Dwarf, partial waterlogged

[0074] Note: The same column data marked with different lowercase letters represent significant differences (p < 0.05).

[0075] As can be seen from Table 4, with the increase of 6-BA concentration, the stem growth of the plant shows a trend of first increasing and then decreasing, and the growth state also has obvious difference; when the concentration of 6-BA is 1.0 mg / L, the stem growth is the highest, the plant is robust and the stem is the thickest (Table 4); when the concentration of 6-BA is too low or too high, it will affect the normal growth of the plant, which shows that the plant is dwarf, yellowing and even appears waterlogged and the like. Therefore, the most suitable culture medium for the robust seedling growth of 'Tsukiyofu' tissue culture seedlings is MS+1.0 mg / L 6-BA+0.1 mg / L 2,4-D. Compared with the average height of 3.1 cm of the regenerated plant after the robust seedling culture in the prior art, the stem growth of the regenerated plant is increased by 1.5 times under the optimized formula, and possibly, the culture medium formula of MS+1.0 mg / L 6-BA+0.1 mg / L 2,4-D in the present application can make the regenerated plant more robust, thereby being more conducive to survival.

[0076] Example 5:

[0077] This example investigates the influence of different concentrations of IBA and 2,4-D on the rooting of 'Tsukiyofu' tissue culture seedlings, and the specific steps are as follows:

[0078] The seedlings obtained in Example 4 are transferred to MS medium containing 0 mg / L, 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, 0.7 mg / L, 1.0 mg / L, 1.5 mg / L of IBA and 0.1 mg / L, 0.2 mg / L of 2,4-D, and cultured for 40 d, and the culture conditions are the same as those in Example 1. The number of roots, root length and rooting rate are determined. The results are shown in Table 5:

[0079] Table 5 Influence of different concentrations of IBA on the rooting of 'Tsukiyofu' tissue culture seedlings

[0080]

[0081]

[0082] Note: The same column data marked with different lowercase letters indicates significant difference (p<0.05).

[0083] As shown in Table 5, when no IBA is added in the culture medium, the rooting rate can reach 40.9%, but the number of roots is less and the root length is short, only 1.8 cm; with the increase of the concentration of IBA, the number of roots and the root length of the plant are also significantly increased; however, when the concentration of IBA is higher than 0.3 mg / L or even multiple times, the number of roots of the plant is significantly reduced, and the root length is also gradually shortened. Therefore, the effect is best when the concentration of IBA is 0.3 mg / L, the number of roots and the root length of the plant are the highest, and the rooting rate can reach 88.4%; therefore, MS+0.3 mg / L IBA+0.1 mg / L 2,4-D is selected as the rooting culture medium formula. After the 'Chuzhi Zhi Feng' tissue culture seedlings after rooting are acclimatized and transplanted, the survival rate can reach more than 90%. Compared with the prior art, the number of adventitious roots generated by each explant during rooting culture is 6.2, and the number of roots is increased by 2.8 times under the optimized formula of the present application, which greatly improves the survival rate of the plant and provides a guarantee for the survival of the later transplanting.

[0084] In summary, the present application uses the aseptic leaf of the large-leaf hydrangea 'Chuzhi Zhi Feng' tissue culture seedling as the explant, induces the occurrence of callus and promotes the regeneration of adventitious buds, maintains the characteristics of continuous proliferation and seedling formation, can provide a large number of high-quality seedlings for large-scale production, and also can provide support for the stable genetic transformation technology of Agrobacterium-mediated large-leaf hydrangea, and lays a foundation for the cultivation of new and excellent hydrangea varieties.

[0085] The above embodiments are preferred embodiments of the present application, but the present application is not limited to the above embodiments, and any obvious improvements, replacements or modifications made by those skilled in the art without departing from the essential content of the present application shall fall within the protection scope of the present application.

Claims

1. A method for high frequency induction of adventitious shoots from leaves of in vitro culture seedlings of Hydrangea macrophylla, characterized in that, The method comprises: Step 1: Taking the aseptic leaf of the hosta plantaginea tissue culture seedling as an explant, inducing callus and differentiating into adventitious buds after dark culture for 7-14 days and normal light culture in a callus induction and differentiation medium; the callus induction and differentiation medium is MS medium, N-(2-chloro-4-pyridyl)-N'-phenylurea CPPU and 2,4-dichlorophenoxyacetic acid 2,4-D; In the callus induction and differentiation medium, the final concentration of CPPU is 2.5-4.0 mg / L, the final concentration of 2,4-D is 0.1 mg / L, or the final concentration of CPPU is 1.0 mg / L, and the final concentration of 2,4-D is 0.15 mg / L; Step 2: The obtained adventitious buds are transferred to a proliferation medium for continuous culture to form sprouts; the proliferation medium is MS + 1.0-3.0 mg / L 6-BA + 0.01-0.05 mg / L IBA + 0.01-0.05 mg / L 2,4-D; Step 3: The obtained sprouts are transferred to a strong seedling culture medium for strong seedling growth to form seedlings; the strong seedling culture medium is MS + 0.5-1.5 mg / L 6-BA + 0.1-0.2 mg / L 2,4-D; Step 4: The obtained seedlings are transferred to a rooting medium to induce root systems to obtain complete small plants; In step 4, the rooting medium is MS + 0.1-0.5 mg / L IBA + 0.1-0.2 mg / L 2,4-D.

2. The method for high-frequency induction of adventitious buds to regenerate plants from hydrangea tissue culture seedlings according to claim 1, characterized in that, In step 1, the method for obtaining the hosta plantaginea tissue culture seedling comprises: cutting the current-year healthy stem segments with buds of the hosta plantaginea potted seedling, and obtaining the aseptic hosta plantaginea tissue culture seedling through disinfection and sterilization and in-vitro germination.

3. The method of claim 2, wherein the leaves of the in vitro plantlets of Hydrangea macrophylla are induced to regenerate plants at a high frequency by high frequency induction of adventitious buds. The hosta plantaginea comprises Chuzhi Zhifeng.

4. The method of claim 1, wherein the leaves of the in vitro plantlets of Hydrangea macrophylla are induced to regenerate plants at a high frequency of adventitious buds. The culture temperature in the dark environment is 25±2℃; the normal light culture conditions are that the culture temperature is 25±2℃, the light intensity is 1800-2000 Lux, and the light time is 14h / d.

5. The method of claim 1, wherein the leaves of the in vitro plantlets of Hydrangea macrophylla are induced to regenerate plants at a high frequency of adventitious buds. In steps 2-4, the culture conditions in the proliferation medium, the strong seedling culture medium, the strong seedling culture medium and the rooting medium are that the culture temperature is 25±2℃, the light intensity is 1800-2000 Lux, and the light time is 14h / d.

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