Method for inducing adventitious buds of hydrangea tissue culture seedling leaves at high frequency to regenerate plants

Abstract

The invention provides a method for high-frequency induction of adventitious buds of hydrangea tissue culture seedling leaves to regenerate plants, and belongs to the technical field of plant tissue culture. The method comprises the following steps: step 1, taking sterile leaves of tissue culture seedlings of 'Zuozhongfeng' as explants, carrying out dark culture on the explants in an induction culture medium to induce calluses, and differentiating to form adventitious buds; 2, transferring the formed adventitious buds into a proliferation culture medium, and culturing to obtain cluster buds; 3, transferring the obtained buds into a strong seedling culture medium for strong seedling growth to form seedlings; 4, transferring the seedlings into a rooting culture medium, and inducing root systems to obtain complete plantlets; according to the method, the callus induction and differentiation culture medium is regulated and controlled, so that the adventitious bud induction efficiency 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 very good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for high-frequency induction of adventitious buds and regeneration of plants from leaves of hydrangea tissue culture seedlings. Background Art

[0002] Hydrangea in a broad sense is a general term for plants of the genus Hydrangea (Hydrangea L.) of the family Hydrangeaceae. Hydrangea in a narrow sense refers to the most cultivated species in the genus Hydrangea, commonly known as large-flowered hydrangea or large-leafed hydrangea. Hydrangea in a narrow sense [Hydrangea macrophylla (Thunb) Ser.] is also known as hydrangea, hydrangea, etc. It has a wide range of uses in garden landscaping, home gardening, fresh cut flowers and dried flowers. In addition, hydrangea is acid-resistant and aluminum-resistant, and also plays an important role in ecology and medicine, with broad development prospects. At present, research on hydrangea focuses on cultivation technology, physiology and biochemistry, molecular mechanisms, and flower color regulation. Due to the poor fruiting performance of hydrangea under natural conditions and the extremely low seed germination rate, the production mainly uses cuttings, layering, division and other propagation methods for seedling cultivation; however, such methods are highly restricted by seasons and have a low propagation coefficient, which makes it difficult to meet the needs of large-scale production. Therefore, rapid propagation of hydrangea seedlings through tissue culture is currently one of the most efficient methods to obtain high-quality and high-yield seedlings. At the same time, with the in-depth research of genetic engineering technology, the use of transgenic or gene editing technology based on tissue culture to improve plant traits has become a very effective breeding method.

[0003] 'Chikushi no Kaze' is a flat-topped, double-petaled large-leafed hydrangea variety introduced from Japan. It has a short plant shape, with a height of only about 40 cm, and has a good ornamental effect in potted plants. It has excellent uprightness and branching, and can be naturally formed without artificial pruning, showing a dense branched and leafy form. It blooms on old branches, with cymes, and is sensitive to pH, and can be adjusted to pink, blue, purple, etc. 'Chikushi no Kaze' is also a typical late-flowering variety with a long flowering period. It begins to bloom in late spring every year and can bloom until autumn. Summer is its peak flowering period. It is also a new variety that is very popular in home gardening and balcony landscaping. Therefore, it is urgent to adopt effective methods to rapidly propagate the hydrangea 'Chikushi no Kaze' to meet the needs of germplasm preservation, large-scale seedling production, and genetic improvement.

[0004] At present, different varieties, explants and experimental methods have been applied to the study of the regeneration system of large-leaf hydrangea. The prior art discloses a method for rapidly breeding large-flowered hydrangea inexhaustible summer using tissue culture seedling leaves. The large-flowered hydrangea inexhaustible summer tissue culture rapid propagation method provided by the method can significantly improve the callus induction rate and rooting rate of large-flowered hydrangea inexhaustible summer during the seedling period. However, the dark culture time in the induction process of this method is as long as 20 days, which greatly prolongs the seedling period; and the differentiation efficiency of the best medium for inducing callus in this method is very low, and the medium with different hormone ratios needs to be replaced to promote the regeneration of adventitious buds, which not only increases the difficulty of rapid propagation technology and reduces the seedling efficiency, but also easily causes pollution, reduces the survival rate and increases the production cost. Therefore, it is necessary to provide a method for inducing adventitious bud regeneration plants that improves the efficiency of inducing adventitious buds, shortens the seedling period, and has a simpler operation method, lower cost and higher survival rate. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a method for inducing adventitious buds and regenerating plants at a high frequency using leaves of hydrangea tissue culture seedlings; the method comprises: step 1: using sterile leaves of 'Chikushi no Kaze' tissue culture seedlings as explants, inducing callus tissue from the explants after dark culture in an induction medium, and differentiating to form adventitious buds; step 2: transferring the formed adventitious buds to a proliferation medium for culture to obtain clustered buds; step 3: transferring the obtained buds to a seedling medium for seedling growth to form seedlings; step 4: transferring the seedlings to a rooting medium to induce roots to obtain complete plantlets; the present invention improves the efficiency of inducing adventitious buds and shortens the seedling raising period by regulating the callus induction and differentiation medium, and the operation method is simpler, the cost is lower, the survival rate is higher, and the method has good practicality.

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

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

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

[0009] The present invention provides a method for inducing adventitious buds to regenerate plants at a high frequency using leaves of hydrangea tissue culture seedlings, the method comprising:

[0010] Step 1: using sterile leaves of hydrangea tissue culture seedlings as explants, inducing callus tissue after culturing them in the above-mentioned callus induction and differentiation medium under darkness + normal light, and differentiating them into adventitious buds;

[0011] Step 2: Transfer the obtained adventitious buds to a proliferation medium for further culture to form secondary buds;

[0012] Step 3: transferring the obtained sprouts to a seedling medium for seedling growth to form seedlings;

[0013] Step 4: Transfer the obtained seedlings to rooting medium to induce root system to obtain complete plantlets.

[0014] Preferably, in step 1, the method for obtaining hydrangea tissue culture seedlings comprises: cutting the robust stem segments with buds of the current year of hydrangea potted seedlings, and obtaining sterile hydrangea tissue culture seedlings after disinfection and sterilization and in vitro germination.

[0015] Preferably, the hydrangea includes Chikushi no Kaze.

[0016] Preferably, in step 1, the conditions of the dark + normal light culture are: culture in a dark environment for 7 to 14 days, and then switch to normal light culture until callus tissue is induced.

[0017] Preferably, the culture temperature in the dark environment is 25±2°C; the conditions for normal light culture are: the culture temperature is 25±2°C, the light intensity is 1800-2000Lux, and the light time is 14h / d.

[0018] Preferably, in step 2, the proliferation medium comprises: MS+1.0-3.0 mg / L 6-BA+0.01-0.05 mg / L 1 BA+0.01-0.05 mg / L 2,4-D.

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

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

[0021] Preferably, in steps 2 to 4, the culture conditions in the proliferation medium, seedling medium, seedling medium and rooting medium are: culture temperature is 25±2° C., light intensity is 1800-2000 Lux, and illumination time is 14 h / d.

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

[0023] The present invention uses sterile leaves of tissue culture seedlings of bigleaf hydrangea 'Chikushi no Kaze' as explants, induces callus formation and can promote 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 can also provide support for Agrobacterium-mediated stable genetic transformation technology of bigleaf hydrangea, laying a foundation for the cultivation of new and superior varieties of hydrangea.

[0024] The present invention uses sterile leaves of 'Chikushi no Kaze' tissue culture seedlings as materials, which are easy to obtain and avoid the problems of sterilization of explants and high contamination rate in the culture process during tissue culture. The seedling raising cycle is short, the production cost is low, the operation is simple, the propagation coefficient is high, and it is convenient for the factory-based seedling breeding.

[0025] In the prior art, the hydrangea tissue culture technology uses stem segments with buds obtained by cutting as explant materials, and after inducing callus with N-(2-chloro-4-pyridyl)-N'-phenylurea, transfers the callus to a culture medium containing 6-benzylaminopurine BAP to induce adventitious buds. The adventitious bud induction rate is 74.3%, and the average number of adventitious buds is 5. The callus induction cycle is 20 days of dark culture and 40 days of light culture, and the proliferation coefficient is 3-5. The present invention uses N-(2-chloro-4-pyridyl)-N'-phenylurea to induce callus and regenerate adventitious buds from the leaves of hydrangea sterile seedlings, the regeneration rate can reach 93.3%, and the average number of adventitious buds can reach 6.9; the period of callus induction and regeneration of adventitious buds is only 40 days (including 10 days of dark culture), achieving high-frequency induction of adventitious buds; culture in a proliferation medium containing 6-benzylaminopurine BAP, the proliferation coefficient can reach 8.7; the seedling raising period is greatly shortened, pollution is reduced, and the degree of practicality is achieved, providing a new technology for carrying out factory-based production of hydrangea test tube seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of adventitious bud regeneration.

[0027] Figure 2 Schematic diagram of cluster bud formation.

[0028] Figure 3 This is a schematic diagram of the growth of tissue culture seedlings.

[0029] Figure 4 Schematic diagram of rooting culture of tissue culture seedlings. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0032] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the product.

[0033] The tissue culture seedlings of Hydrangea macrophylla 'Tsukushi no Kaze' used in the present invention were provided by the Hydrangea tissue culture room of the Leisure Agriculture Research Institute of Jiangsu Academy of Agricultural Sciences.

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

[0035] Embodiment 1:

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

[0037] Cut the strong stem segments with buds of the current year from the hydrangea potted seedlings, obtain sterile hydrangea tissue culture seedlings through disinfection and sterilization, and in vitro germination. Select the leaves of the 'Chikushi no Kaze' tissue culture seedlings that have been propagated in the laboratory for more than 3 times and have grown healthily as explant materials, cut off the leaf tips and petioles, make 3 vertical cross cuts along the veins, and inoculate them on the induction and differentiation medium.

[0038] (2) Culture conditions

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

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

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

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

[0043] The culture conditions in the proliferation medium, seedling medium, seedling medium and rooting medium are: culture temperature is 25±2°C, light intensity is 1800-2000Lux, and light time is 14h / d.

[0044] (3) Inoculation and cultivation:

[0045] Seven explants were inoculated in each culture dish and cultured in the dark for 10 days. Then, they were cultured under normal light conditions for 30 days. Callus tissue and adventitious buds would be directly produced at the wounds of the leaves. The induced adventitious buds would be rapidly propagated on the proliferation medium. After 30 days, the clustered buds in the proliferation medium would be transferred to the seedling medium for seedling growth. After 40 days, the seedlings would be transferred to the rooting medium until a complete plant was formed.

[0047] Embodiment 2:

[0048] This example investigates the effects of different concentrations of hormones and different dark culture times on the induction of adventitious buds from leaf callus in step 1. The specific steps are as follows:

[0049] (1) Effects of different hormone concentration ratios on the induction of adventitious bud regeneration from leaf callus

[0050] 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. The culture conditions were the same as in Example 1. The adventitious bud regeneration rate and the average number of adventitious buds were calculated and the state of the adventitious buds was observed. The results are shown in Table 1:

[0051] Table 1 Effects of different hormone concentration ratios on the induction of adventitious buds from leaf callus of 'Chikushi no Kaze'

[0052] deal with CPPU(mg / L) 2,4-D (mg / L) Regeneration rate (%) Average number of adventitious buds Adventitious bud state 1 1.0 0.05 46.7±6.7gh 2.2±0.6cd Adventitious buds are few and small, and the growth is weak 2 2.0 0.05 56.7±8.8fg 1.6±0.3d Adventitious buds are few and small, and the growth is weak 3 3.0 0.05 58.8±5.1ef 2.8±0.4cd Fewer adventitious buds and weak growth 4 4.0 0.05 63.3±3.3ef 3.1±0.5c There are many adventitious buds and the growth is weak. 5 1.0 0.1 68.8±3.8de 3.4±0.5c The adventitious buds are large and weak in growth 6 2.0 0.1 74.4±3.8cd 3.1±1.4c There are many adventitious buds and the growth is weak. 7 3.0 0.1 93.3±6.7a 6.9±0.9a Adventitious buds are numerous and large, and the growth is strong. 8 4.0 0.1 87.7±5.1ab 5.3±0.6b Adventitious buds are numerous and large, and the growth is strong. 9 1.0 0.15 82.2±5.1bc 3.6±0.8c The adventitious buds are large and grow robustly. 10 2.0 0.15 61.1±5.1ef 2.1±1.0cd Adventitious buds are few and small, and the growth is weak 11 3.0 0.15 55.6±8.4fg 2.8±0.7cd Adventitious buds are few and small, and the growth is weak 12 4.0 0.15 36.7±8.8h 1.4±0.7d Adventitious buds are few and small, and the growth is weak

[0053] Note: Data in the same column followed by different lowercase letters indicate significant differences (p < 0.05).

[0054] 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%, with an average of 6.9 adventitious buds; the adventitious bud state was better than other treatments. Therefore, with MS as the basic culture medium, 3.0 mg / L CPPU and 0.1 mg / L 2,4-D were the choices with the highest efficiency of adventitious bud regeneration in the leaves of 'Chikushi no Kaze' tissue culture seedlings.

[0055] In the prior art, after the callus is induced, the leaves need to be transferred to the adventitious bud differentiation medium to achieve the regeneration of adventitious buds, and the highest regeneration rate of adventitious buds is only 74.3%, and the average number of adventitious buds is 5.3; the combination of 6-BA and KT can only increase the average number of adventitious buds to 5. Compared with the prior art, the present invention selects CPPU 2.5-4.0mg / L and 2,4-D 0.05-0.15mg / L, which can not only significantly improve the efficiency of inducing adventitious buds, but also its operation method is simpler than the prior art, with low cost and higher survival rate.

[0056] (2) Effects of different dark culture times on the induction of adventitious bud regeneration from leaf callus:

[0057] According to the optimized conditions in step (1), the explants in Example 1 were transferred to MS medium containing 3.0 mg / L CPPU and 0.1 mg / L 2,4-D for dark culture. The dark culture time was set to 0, 7, 10, 14, 21, and 28 days for a total of 6 time points. The subsequent light time was increased or shortened accordingly until the induction of regeneration of adventitious buds. The other culture conditions were the same as in Example 1. The adventitious bud regeneration rate and the average number of adventitious buds were calculated. The results are shown in Table 2:

[0058] Table 2. Effects of different dark culture times on adventitious bud regeneration from leaves

[0059]

[0060]

[0061] Note: Data in the same column followed by different lowercase letters indicate significant differences (p < 0.05).

[0062] As can be seen from Table 2, when the dark culture time is 0 days, the explant cannot induce callus differentiation into adventitious buds, indicating that a certain dark condition is very necessary for the high-frequency induction of adventitious buds and regeneration of plants from leaves; when the dark culture is 10 days, the regeneration rate of leaves 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 leaves also turn green and yellow due to insufficient light, and their regeneration rate is also reduced. The above results show that when the dark culture is 10 days, the efficiency of regenerating adventitious buds from the leaves of the 'Chikushi no Kaze' tissue culture seedlings is the highest. Compared with the dark culture of 15 to 25 days used in the prior art, the method of the present invention further shortens the seedling cycle on the basis of improving the efficiency of inducing adventitious buds, and is more convenient for factory seedling cultivation.

[0063] Embodiment 3:

[0064] This example investigates the effects of different concentrations of 6-BA on the proliferation of adventitious buds of 'Chikushi no Kaze'. The specific steps are as follows:

[0065] The adventitious buds obtained 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 and cultured for 40 days. The culture conditions were the same as in Example 1, and the proliferation coefficient of the adventitious buds was calculated. The results are shown in Table 3:

[0066] Table 3. Effects of different concentrations of 6-BA and IBA on the proliferation of adventitious buds of 'Chikushi no Kaze'

[0067] deal with 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

[0068] Note: Data in the same column followed by different lowercase letters indicate significant differences (p < 0.05).

[0069] 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 clustered buds grow well; but when the concentration of 6-BA is too high (3.0 mg / L), the leaves will curl and vitrify; when the concentration of 6-BA is too low, the leaves will turn yellow and the proliferation coefficient will be low (<3.5); the proliferation coefficient of adventitious buds in treatment 2 is the highest and the growth state is good. Therefore, the most suitable culture 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.

[0070] Embodiment 4:

[0071] This example investigates the effects of different concentrations of 6-BA on the growth of 'Chikushi no Kaze' tissue culture seedlings. The specific steps are as follows:

[0072] The clustered shoots obtained in Example 3 were separated into individual 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 for 40 days. The culture conditions were the same as those in Example 1. The stem growth was measured and the growth status of the seedlings was observed. The results are shown in Table 4:

[0073] Table 4. Effects of different concentrations of 6-BA on the growth of 'Chikushi no Kaze' tissue culture seedlings

[0074] deal with 6-BA(mg / L) 2,4-D (mg / L) Stem growth (cm) Seedling growth status 1 0 0.1 1.2±0.12d Dwarf, partially yellow 2 0.5 0.1 2.3±0.25c Good, partially yellowed 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 Leaves slightly curled, partially yellowed 6 1.5 0.2 2.3±0.22c Good, partially yellowed 7 1.8 0.2 1.3±0.20d Short, partially water-soaked

[0075] Note: Data in the same column followed by different lowercase letters indicate significant differences (p < 0.05).

[0076] 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 rising and then falling, and there are obvious differences in its growth state; when the concentration of 6-BA is 1.0 mg / L, the stem growth is the highest, the plant is strong and the stem is the thickest (Table 4); when the 6-BA concentration is too low or too high, it will affect the normal growth of the plant, showing the phenomenon of short plant, yellowing and even waterlogging. Therefore, the most suitable culture medium for the growth of strong seedlings of 'Chikushi no Kaze' tissue culture seedlings is MS+1.0 mg / L6-BA+0.1 mg / L2,4-D. Compared with the average height of the regenerated plants after strong seedling cultivation in the prior art of 3.1 cm, the present invention increases the stem growth of the regenerated plants by 1.5 times under the optimized formula. It is possible that the selection of the culture medium formula of MS+1.0 mg / L6-BA+0.1 mg / L2,4-D in the present invention can make the regenerated plants stronger and more conducive to survival.

[0077] Embodiment 5:

[0078] This example investigates the effects of different concentrations of IBA and 2,4-D on the rooting of 'Chikushi no Kaze' tissue culture seedlings. The specific steps are as follows:

[0079] The seedlings obtained in Example 4 were 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 IBA and 0.1 mg / L, 0.2 mg / L 2,4-D, and cultured for 40 days. The culture conditions were the same as in Example 1, and the number of roots, root length and rooting rate were measured. The results are shown in Table 5:

[0080] Table 5 Effects of different concentrations of IBA on the rooting of 'Chikushi no Kaze' tissue culture seedlings

[0081]

[0082]

[0083] Note: Data in the same column followed by different lowercase letters indicate significant differences (p < 0.05).

[0084] As can be seen from Table 5, when IBA was not added to the culture medium, the rooting rate could reach 40.9%, but the number of roots was small and the root system was short, with a root length of only 1.8 cm; with the increase of IBA concentration, the number of roots and root length of the plant also increased significantly; however, when the concentration of IBA was higher than 0.3 mg / L or even added in multiples, the number of roots of the plant decreased significantly and the root length gradually shortened. Therefore, the effect was best when the concentration of IBA was 0.3 mg / L, the number of roots and root length of the plant were the highest, and the rooting rate could reach 88.4%; therefore, MS+0.3 mg / L IBA+0.1 mg / L 2,4-D was selected as the rooting culture medium formula. After acclimatization and transplantation of the rooted 'Chikushi no Kaze' tissue culture seedlings, the survival rate could reach more than 90%. Compared with the prior art in which an average of 6.2 adventitious roots are produced per explant during rooting culture, the present invention increases the number of roots by 2.8 times under an optimized formula, greatly improving the survival rate of the plants and providing a guarantee for the survival of subsequent transplanting.

[0085] In summary, the present invention uses sterile leaves of tissue culture seedlings of Hydrangea macrophylla 'Chikushi no Kaze' as explants to induce callus formation and promote the regeneration of adventitious buds, maintaining the characteristics of continuous proliferation and seedling formation, and can provide a large number of high-quality seedlings for large-scale production. It can also provide support for the Agrobacterium-mediated stable genetic transformation technology of Hydrangea macrophylla, laying a foundation for the cultivation of new and superior varieties of hydrangea.

[0086] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A callus induction and differentiation medium, characterized in that: The culture medium comprises: MS culture medium, N-(2-chloro-4-pyridyl)-N'-phenylurea CPPU and 2,4-dichlorophenoxyacetic acid 2,4-D.

2. The callus induction and differentiation medium according to claim 1, characterized in that The final concentration of the CPPU is 2.5-4.0 mg / L, and the final concentration of the 2,4-D is 0.05-0.15 mg / L.

3. A method for inducing adventitious buds to regenerate plants at a high frequency from leaves of hydrangea tissue culture seedlings, characterized in that: The method comprises: Step 1: using sterile leaves of hydrangea tissue culture seedlings as explants, inducing callus tissue after culturing them in the callus induction and differentiation medium of claim 1 or 2 under darkness + normal light, and differentiating them to form adventitious buds; Step 2: Transfer the obtained adventitious buds to a proliferation medium for further culture to form secondary buds; Step 3: transferring the obtained sprouts to a seedling medium for seedling growth to form seedlings; Step 4: Transfer the obtained seedlings to rooting medium to induce root system to obtain complete plantlets.

4. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 3, characterized in that: In step 1, the method for obtaining hydrangea tissue culture seedlings includes: cutting the strong stem segments with buds of the current year of hydrangea potted seedlings, and obtaining sterile hydrangea tissue culture seedlings after disinfection and sterilization and in vitro germination.

5. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 4, characterized in that: The hydrangeas include Chikushi no Kaze.

6. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 3, characterized in that: In step 1, the conditions of the dark + normal light culture are: culture in a dark environment for 7 to 14 days, and then switch to normal light culture until callus tissue is induced.

7. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 6, characterized in that: The culture temperature in the dark environment is 25±2°C; the conditions for normal light culture are: the culture temperature is 25±2°C, the light intensity is 1800-2000Lux, and the light time is 14h / d.

8. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 3, characterized in that: In step 2, the proliferation medium includes: 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.

9. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 3, characterized in that: In step 3, the seedling medium comprises: MS+0.5-1.5 mg / L 6-BA+0.1-0.2 mg / L 2,4-D; In step 4, the rooting medium includes: MS+0.1-0.5 mg / LIBA+0.1-0.2 mg / L 2,4-D.

10. The method for inducing adventitious buds and regenerating plants at a high frequency from leaves of hydrangea tissue culture seedlings according to claim 3, characterized in that: In steps 2 to 4, the culture conditions in the proliferation medium, seedling medium, seedling medium and rooting medium are: culture temperature is 25±2° C., light intensity is 1800-2000 Lux, and light time is 14 h / d.

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