Method for efficient rapid propagation of rhododendron simsii leaf embroidery

Abstract

The application discloses a kind of methods for efficient rapid propagation of gem slippers oakleaf hydrangea, belong to the technical field of plant rapid propagation.The current year's healthy stem section with axillary bud or apical bud of gem slippers oakleaf hydrangea is used as explant material, after surface disinfection, inoculated in adventitious bud induction and proliferation medium to carry out induction and proliferation culture of adventitious bud;Then the single bud body cluster after proliferation is separated and transferred to synchronous medium to carry out elongation, seedling and rooting synchronous culture of adventitious bud, after domestication and transplanting, finally obtain the healthy gem slippers oakleaf hydrangea regenerated plant.The beneficial effects of the application are: a kind of gem slippers oakleaf hydrangea propagation method is provided, which is fast, efficient and can maintain the excellent characteristics of the mother plant, and provides certain technical support for germplasm conservation, seedling scale propagation and later genetic improvement of gem slippers oakleaf hydrangea.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant biotechnology, and particularly relates to a method for high-efficiency rapid propagation of Hydrangea quercifolia Ruby Slippers. BACKGROUND

[0002] Hydrangea quercifolia, a perennial shrub of Saxifragaceae and Hydrangea, is a perennial landscape plant with flower, leaf and shape. It is originally from the United States, with a long flowering period (May-August), large flower ball (the largest conical inflorescence can reach 30 cm), and generally white flower ball. The color of the flower ball changes with the seasons, showing white, light green, green, pink, and the withered branches in autumn and winter remain brown and dry. At the same time, the leaf color also changes with the seasons, showing yellow-green to dark green in spring and summer, and red-brown, copper, bronze and purple in autumn, which can increase the level of landscape and color change, and can be used for landscaping and environmental greening. In addition, H. quercifolia is extremely cold-resistant, and can withstand -15℃ in winter, and is suitable for cultivation in the southern part of North China. At present, H. quercifolia has been introduced to other countries and regions as an ornamental plant, and has broad market application prospects.

[0003] As a variety of H. quercifolia that can produce pink or red flowers, Ruby Slippers has attracted widespread attention at home and abroad. At present, the main propagation method of Ruby Slippers H. quercifolia in production is cutting, but it has problems such as difficult rooting, low propagation efficiency, and propagation limited by season and material. Plant tissue culture technology has the advantages of less material, high propagation efficiency, high propagation coefficient, no limitation of season and material, and easy large-scale production of seedlings in a short period. However, there is no report on the use of tissue culture technology for the propagation of H. quercifolia.

[0004] In addition, plant variety type, genotype, explant type, explant sampling site, sampling time, and the type and concentration of plant growth regulators directly affect the in vitro regeneration ability of plants. Due to the great difference between Ruby Slippers H. quercifolia and existing H. quercifolia varieties, the difficulty and conditions of in vitro regeneration are also very different. The existing H. quercifolia tissue culture propagation technology cannot be directly applied to the industrialized propagation of Ruby Slippers H. quercifolia. The above problems seriously limit the large-scale propagation, popularization and later variety improvement of Ruby Slippers H. quercifolia. Therefore, the development of a method for high-efficiency rapid propagation of Ruby Slippers H. quercifolia is of great significance for the tissue culture industrialized propagation and later variety improvement of Ruby Slippers seedlings.

[0005] CN115812451A discloses a high-survival-rate quercus leaf hydrangea cutting method, which belongs to the technical field of quercus leaf hydrangea cutting. The cutting method comprises the following steps: cutting: cutting the shoots from the current-year semi-lignified branches of quercus leaf hydrangea, removing the lower leaves and flower buds, and cutting the upper end flat and the lower end obliquely; pretreatment: soaking the collected cutting in water, and sterilizing the seedbed and substrate; cutting: after sterilizing the cutting, performing hormone treatment, and then starting cutting. The cutting substrate of the patent is a mixture of perlite and coconut husk in a mass ratio of 3:1. The perlite has good air permeability and water drainage, but the humus content is not high, which limits the elongation of the root system. The coconut husk has good water retention, which can cause excessive water to be unable to drain, resulting in rotting of the base of the cutting. In the patent, the mixture of perlite and coconut husk ensures water retention and permeability, and also contains certain nutrients for root growth. Finally, the rooting rate of quercus leaf hydrangea can reach 95.72%. However, the propagation method of the patent has a low propagation coefficient, and only one plant can be produced from one cutting. In addition, the propagation of quercus leaf hydrangea in the patent is limited by season and material, and cannot achieve year-round large-scale production of quercus leaf hydrangea. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a method for efficient rapid propagation of quercus leaf hydrangea by using plant tissue culture technology.

[0007] The present application solves the above technical problems by the following technical means:

[0008] A method for efficient rapid propagation of quercus leaf hydrangea, comprising the following steps:

[0009] (1) Stem segment explant material and surface sterilization: using the current-year tender stems of the excellent strain of quercus leaf hydrangea as explant material, and performing surface sterilization;

[0010] (2) Induction culture of adventitious buds: cutting the explant material sterilized in step (1) into stem segments of appropriate size with axillary buds or terminal buds as explants, inoculating into the adventitious bud induction medium, and performing induction culture of adventitious buds;

[0011] (3) Proliferation culture of adventitious buds: transferring the stem segments with induced adventitious bud clusters in step (2) to the proliferation medium, and performing proliferation culture of adventitious buds;

[0012] (4) Synchronous culture of elongation, seedling strengthening and rooting of adventitious buds: separating the adventitious buds in step (3) from the bud clusters and transferring to the synchronous culture medium for elongation, seedling strengthening and adventitious root induction, and obtaining complete regenerated plants;

[0013] (5) Transplanting of the regenerated plant: the whole regenerated plant obtained in step (4) is acclimatized, washed and surface sterilized, and then transplanted into a seedling tray filled with mixed nutrient medium for seedling culture.

[0014] Preferably, the surface sterilization of the explant in step (1) refers to that the young tender stems with axillary buds or terminal buds of the current year of the healthy strain of Rhododendron calophytum leaves in the greenhouse are used as the explant material; after being washed with running water for 10 min, the stems are surface wiped with 75% anhydrous ethanol; then the stems are placed in a culture bottle in a sterile operation table, washed with sterile water for 3-5 times, and then surface sterilized with 75% anhydrous ethanol for 30 s; then the stems are washed with sterile water for 3-5 times, and then surface sterilized with 0.1% (w / v) mercury chloride solution for 2-4 min; finally, the stems are washed with sterile water for 4-6 times.

[0015] Preferably, the stems with axillary buds or terminal buds in step (2) are cut into 0.8-1.5 cm.

[0016] Preferably, the adventitious bud induction medium in step (2) is MS+0.02-0.5 mg / L TDZ+1.0-3.0 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar, pH=5.8.

[0017] Preferably, the adventitious bud proliferation medium in step (3) is MS+0.02-0.1 mg / L TDZ+0.5-2.0 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar, pH=5.8.

[0018] Preferably, the simultaneous elongation, strengthening and rooting medium for the adventitious buds in step (4) is MS+0.5-2.0 mg / L ZT+0.2-1.0 mg / L 6-BA+0.05-0.5 mg / L IAA+2.0% (w / v) sucrose+0.7% (w / v) agar, pH=5.8.

[0019] Preferably, the acclimatization, washing and surface sterilization in step (5) are specifically that the bottle seedlings of the regenerated plant obtained in step (4) are placed in natural conditions for acclimatization for 2 weeks, the regenerated plant is taken out, washed with running water to remove the agar on the roots of the plant, and then placed in an aqueous solution added with 0.05%-0.2% potassium permanganate for immersion for 10-20 min.

[0020] Preferably, the mixed nutrient medium in step (5) is mixed from Pinus nutrient soil: vermiculite: garden soil (volume ratio)=1-3: 1-2: 1-4.

[0021] Preferably, the conditions for inducing adventitious buds, proliferation, elongation, seedling strengthening and adventitious root culture in the step (2), step (3), step (4) are as follows: temperature is 25±2℃, light intensity is 2500-3000lx, and photoperiod is 16 / 8h (light / dark).

[0022] Preferably, the transplanting conditions in the step (5) are as follows: temperature is 20-28℃, humidity is 75%-85%, and light intensity is 1800-4000lx.

[0023] The present application has the advantages of:

[0024] 1. The method for inducing the plant regeneration of Rhododendron calophytum by organ direct generation approach provided by the present application can truly reflect the excellent characteristics and stable genetic traits of the mother plant, since it is by directly inducing adventitious buds.

[0025] 2. The regeneration method provided by the present application has the advantages of high propagation efficiency and high propagation coefficient (41.8 adventitious buds can be generated from each explant after one time of proliferation), and provides important technical support for the rapid propagation and genetic improvement of Rhododendron calophytum. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure of the stem segment adventitious bud cluster obtained after 4 weeks of culture on the induction medium in Example 2 of the present application;

[0027] Figure 2 Figure of the proliferated stem segment adventitious bud cluster obtained after 4 weeks of culture on the proliferation medium in Example 2 of the present application;

[0028] Figure 3 Figure of the complete regenerated plant obtained after 4 weeks of culture on the elongation, seedling strengthening and adventitious root synchronous induction medium in Example 2 of the present application;

[0029] Figure 4 Figure of the Rhododendron calophytum regenerated plant obtained after one month of domestication and transplanting in Example 2 of the present application. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in connection with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present application.

[0031] In the following examples, the test materials and reagents used, etc. can be obtained from commercial channels if not otherwise specified.

[0032] Unless otherwise specified, the specific techniques or conditions in the examples are in accordance with the techniques or conditions described in the literature or in accordance with the product manual.

[0033] The excellent healthy strain of Rhododendron calophytum used in the present application is provided by Henan Furora Agricultural Technology Co., Ltd.

[0034] Example 1

[0035] A method for efficient rapid propagation of Rhododendron calophytum, the specific operation is as follows:

[0036] (1) The tender stems with axillary buds or terminal buds of the excellent healthy strain of Rhododendron calophytum cultured in the greenhouse were used as explant materials. After being washed with running water for 10 min, the explants were surface-sterilized with 75% ethanol. Then, the explants were washed with sterile water for 3-5 times in a sterile operation table, and then sterilized with 75% ethanol for 30 s. Then, the explants were washed with sterile water for 3-5 times, and then sterilized with 0.1% (w / v) mercury chloride solution for 2 min. Finally, the explants were washed with sterile water for 4 times. After the surface water was absorbed with sterile filter paper, the explants were cut into 0.8 cm long sections with axillary buds or terminal buds, and were ready for use.

[0037] (2) The sterilized stem sections in step (1) were inoculated into the adventitious bud induction medium MS+0.02 mg / L TDZ+1.0 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8) for induction culture of adventitious buds in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a light cycle of 16 / 8 h (light / dark). After 4 weeks of light culture, the contamination rate of the stem sections was 2.4%, the induction rate of adventitious buds of the stem sections was 92.3%, and an average of 7.6 adventitious buds were produced per explant.

[0038] (3) The adventitious bud clusters obtained in step (2) were transferred to the adventitious bud proliferation medium for proliferation culture of adventitious buds; wherein the adventitious bud proliferation medium was MS+0.02 mg / L TDZ+0.5 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8), and the proliferation culture of adventitious buds was carried out in a constant temperature culture room with a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a light cycle of 16 / 8 h (light / dark). After 4 weeks of light culture, the proliferation rate of adventitious buds was 89.3%, an average of 15.1 adventitious buds were produced per explant, and the average height of the adventitious buds was 1.1 cm.

[0039] (4) The adventitious buds in step (3) are separated from the bud cluster and transferred to the adventitious bud elongation, seedling strengthening and adventitious root induction synchronization medium MS+0.5 mg / L ZT+0.2 mg / L 6-BA+0.05 mg / L IAA+2.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8) for the adventitious bud elongation, seedling strengthening and adventitious root induction synchronization culture at a temperature of 25±2°C, a light intensity of 2500-3000 lx and a light cycle of 16 / 8 h (light / dark). After 4 weeks of light culture, the adventitious buds achieve the synchronization of seedling strengthening, elongation and rooting at the same time. The induction rate of adventitious roots is as high as 92.4%, the average number of adventitious roots generated from each explant is 3.8, and the average height of the regenerated plants is 2.6 cm.

[0040] (5) The bottle seedlings of the complete regenerated plants obtained in step (4) are placed in a natural condition for 2 weeks, the regenerated plants are taken out, washed with running water to remove the agar on the roots of the plants, then soaked in an aqueous solution added with 0.05% (mass fraction) potassium permanganate for 10 min, and then transplanted into a seedling raising tray filled with a mixed nutrient medium (Pin's nutrient soil: vermiculite: garden soil (volume ratio)=1:1:4) and covered with a plastic cover for seedling culture in a plastic greenhouse at a temperature of 20-28°C, a humidity of 75%-85% and a light intensity of 1800-4000 lx. After 4 weeks of culture, the healthy complete regenerated plants are obtained, and the transplanting survival rate of the regenerated plants is 87.3%.

[0041] Example 2

[0042] A method for efficiently and rapidly propagating a red gem slipper oak leaf hydrangea is specifically implemented as follows:

[0043] (1) The tender stems with axillary buds or terminal buds of the red gem slipper oak leaf hydrangea of a healthy and excellent strain cultured in a greenhouse are used as the explant material. After being washed with running water for 10 min, the explants are surface-sterilized with 75% anhydrous ethanol, then washed with sterile water for 3-5 times in a sterile operation table, surface-sterilized with 75% anhydrous ethanol for 30 s, then washed with sterile water for 3-5 times, surface-sterilized with 0.1% (w / v) mercury chloride solution for 3 min, and finally washed with sterile water for 5 times. After the surface water is absorbed with sterile filter paper, the explants are cut into segments with a length of 1.0 cm with axillary buds or terminal buds, and are ready for use.

[0044] (2) The stems segments sterilized in step (1) were cut and inoculated on the adventitious bud induction medium MS + 0.1 mg / L TDZ + 2.0 mg / L 6-BA + 3.0% (w / v) sucrose + 0.7% (w / v) agar (pH = 5.8) for the induction of adventitious buds in a constant temperature culture room with temperature of 25 ± 2°C, light intensity of 2500-3000 lx, and light cycle of 16 / 8 h (light / dark). After 4 weeks of illumination culture, the contamination rate of the stems segments was 0.3%, the induction rate of adventitious buds of the stems segments was 100%, and the average number of adventitious buds produced by each explant was 22.4 (as shown in Figure 1 ).

[0045] (3) The adventitious bud clusters obtained in step (2) were transferred to the adventitious bud proliferation medium for the proliferation culture of adventitious buds; wherein the adventitious bud proliferation medium was MS + 0.05 mg / L TDZ + 1.0 mg / L 6-BA + 3.0% (w / v) sucrose + 0.7% (w / v) agar (pH = 5.8), and the proliferation culture of adventitious buds was carried out in a constant temperature culture room with temperature of 25 ± 2°C, light intensity of 2500-3000 lx, and light cycle of 16 / 8 h (light / dark). After 4 weeks of illumination culture, the proliferation rate of adventitious buds was 100%, the average number of adventitious buds produced by each explant was 57.8, and the average height of adventitious buds was 1.3 cm (as shown in Figure 2 ).

[0046] (4) The adventitious buds in step (3) were separated from the bud clusters and transferred to the adventitious bud elongation, seedling strengthening, and adventitious root induction simultaneous medium MS + 1.0 mg / L ZT + 0.5 mg / L 6-BA + 0.2 mg / L IAA + 2.0% (w / v) sucrose + 0.7% (w / v) agar (pH = 5.8) for the simultaneous culture of adventitious bud elongation, seedling strengthening, and adventitious root induction. After 4 weeks of illumination culture, the adventitious buds realized the synchronization of seedling strengthening, elongation, and rooting. The induction rate of adventitious roots was as high as 100%, the average number of adventitious roots produced by each explant was 6.2, and the average height of the regenerated plants was 3.1 cm (as shown in Figure 3 ).

[0047] (5) The complete regenerated plantlets obtained in step (4) are placed in a natural condition for 2 weeks to domesticate, and the regenerated plants are taken out, washed with running water to wash off the agar on the roots of the plants, and then placed in an aqueous solution added with 0.1% (mass fraction) potassium permanganate for 15 min, and then transplanted into a seedling tray filled with mixed nutrient medium (Pindu nutrient soil: vermiculite: garden soil (volume ratio) = 2:2:2) and covered with a plastic cover, and cultured in a plastic greenhouse with a temperature of 20-28°C, a humidity of 75%-85%, and a light intensity of 1800-4000 lx. After 4 weeks of culture, healthy complete regenerated plants are obtained, and the survival rate of the transplanted regenerated plants is 97.4% (as shown in FIG. 2). Figure 4

[0048] Implementation column 3:

[0049] A method for efficient rapid propagation of a red shoe oak leaf hydrangea, the specific operation being as follows:

[0050] (1) The tender stems with axillary buds or terminal buds of the red shoe oak leaf hydrangea healthy strain cultured in the greenhouse are used as explant materials; after being washed with running water for 10 min, the surfaces are wiped with 75% anhydrous ethanol; then the stems are placed in a 250 ml culture bottle on a sterile operation table, washed with sterile water for 3-5 times, and then disinfected with 75% anhydrous ethanol for 30 s; then washed with sterile water for 3-5 times, disinfected with 0.1% (w / v) mercury chloride solution for 4 min, and finally washed with sterile water for 6 times. After the surface water is absorbed with sterile filter paper, the stems are cut into 1.5 cm long sections with axillary buds or terminal buds, and are ready for use.

[0051] (2) The disinfected stem sections in step (1) are inoculated into the adventitious bud induction medium MS+0.5 mg / L TDZ+3.0 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8) to induce adventitious buds in a constant temperature culture room with a temperature of 25±2°C, a light intensity of 2500-3000 lx, and a light cycle of 16 / 8 h (light / dark). After 4 weeks of light culture, the contamination rate of the stems is 0.0%, the induction rate of the adventitious buds of the stems is 88.1%, and an average of 11.2 adventitious buds are generated per explant.

[0052] ​(3) The adventitious bud clusters obtained in step (2) are transferred to an adventitious bud proliferation medium for proliferation culture of adventitious buds; wherein the adventitious bud proliferation medium is MS+0.1 mg / L TDZ+2.0 mg / L 6-BA+3.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8), and the proliferation culture of adventitious buds is carried out at a temperature of 25±2℃, a light intensity of 2500-3000 lx, and a light cycle of 16 / 8 h (light / dark). After 4 weeks of light culture, the proliferation rate of adventitious buds is 97.2%, with an average of 21.5 adventitious buds generated per explant, and the average height of adventitious buds is 0.7 cm.

[0053] (4) The adventitious buds in step (3) are separated from the bud clusters and transferred to an adventitious bud elongation, seedling strengthening, and adventitious root induction synchronous medium MS+2.0 mg / L ZT+1.0 mg / L 6-BA+0.5 mg / L IAA+2.0% (w / v) sucrose+0.7% (w / v) agar (pH=5.8) for synchronous culture of adventitious bud elongation, seedling strengthening, and adventitious root induction. After 4 weeks of light culture, the adventitious buds achieve simultaneous seedling strengthening, elongation, and rooting synchronization. The induction rate of adventitious roots is as high as 100%, with an average of 5.4 adventitious roots generated per explant, and the average height of regenerated plants is 3.0 cm.

[0054] (5) The complete regenerated plants obtained in step (4) are placed in a natural condition for 2 weeks, and the regenerated plants are taken out, washed with running water to remove the agar on the roots of the plants, and then soaked in an aqueous solution added with 0.2% (mass fraction) potassium permanganate for 20 min, and then transplanted into a seedling raising tray filled with a mixed nutrient medium (Pin's nutrient soil: vermiculite: garden soil (volume ratio)=3:2:4) and covered with a plastic cover for seedling culture in a plastic greenhouse at a temperature of 20-28℃, a humidity of 75%-85%, and a light intensity of 1800-4000 lx. After 4 weeks of culture, healthy and complete regenerated plants are obtained, and the transplanted survival rate of regenerated plants is 93.6%.

[0055] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea, characterized in that, Includes the following steps: (1) Stem segment explant collection and surface disinfection: The current year's young stem segments of the superior strain of Ruby Slipper Oak Leaf Hydrangea were used as explant materials and the surface was disinfected. (2) Induction culture of adventitious buds: The explant material sterilized in step (1) was cut into stem segments of appropriate size with axillary or apical buds as explants and inoculated into adventitious bud induction medium for induction culture of adventitious buds; the adventitious bud induction medium was MS + 0.02-0.5 mg / L TDZ + 1.0-3.0 mg / L 6-BA + 3.0% sucrose + 0.7% agar, pH = 5.8; (3) Adventitious bud proliferation culture: The stem segments from which adventitious bud clusters were induced in step (2) were transferred to adventitious bud proliferation culture medium for adventitious bud proliferation culture; the adventitious bud proliferation culture medium was MS + 0.02-0.1 mg / L TDZ + 0.5-2.0 mg / L 6-BA + 3.0% sucrose + 0.7% agar, pH = 5.8; (4) Simultaneous culture of adventitious bud elongation, seedling strengthening and rooting: The adventitious buds in step (3) were separated from the bud cluster and transferred to the adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture medium. The adventitious bud elongation, seedling strengthening and adventitious root induction synchronous culture was carried out at a temperature of 25±2℃, a light intensity of 2500~3000lx and a photoperiod of 16 light / 8h darkness to obtain complete regenerated plants; The adventitious bud elongation, seedling strengthening and rooting synchronous culture medium was MS + 0.5~2.0mg / L ZT + 0.2~1.0mg / L 6-BA + 0.05~0.5mg / L IAA + 2.0% sucrose + 0.7% agar, pH=5.8; (5) Transplanting of regenerated plants: After acclimatization, cleaning and surface disinfection, the complete regenerated plants obtained in step (4) are transplanted into seedling trays containing mixed nutrient substrate for seedling cultivation.

2. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, The explant selection and surface disinfection in step (1) refers to using young, tender stem segments with axillary or terminal buds from healthy, superior strains of Ruby Slipper Oak Hydrangea grown in a greenhouse as explant materials; after rinsing with running water for 10 minutes, the surface is wiped with 75% anhydrous ethanol; then placed in a culture bottle on a sterile operating table, rinsed with sterile water 3-5 times, and disinfected with 75% anhydrous ethanol for 30 seconds; then rinsed with sterile water 3-5 times, and then disinfected with 0.1% mercuric chloride solution for 2-4 minutes; finally, rinsed with sterile water 4-6 times.

3. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, In step (2), the stem is cut into 0.8-1.5cm segments with axillary or terminal buds.

4. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, In step (2), the adventitious bud induction medium is MS + 0.02 mg / L TDZ + 1.0 mg / L 6-BA + 3.0% sucrose + 0.7% agar, pH = 5.

8.

5. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, In step (3), the adventitious bud proliferation medium is MS + 0.02 mg / L TDZ + 0.5 mg / L 6-BA + 3.0% sucrose + 0.7% agar, pH = 5.

8.

6. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, In step (4), the culture medium for adventitious bud elongation, seedling strengthening and rooting is MS + 0.5 mg / L ZT + 0.2 mg / L 6-BA + 0.05 mg / L IAA + 2.0% sucrose + 0.7% agar, pH = 5.

8.

7. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, The acclimatization, cleaning and surface disinfection in step (5) specifically involves placing the complete regenerated plant seedlings obtained in step (4) under natural conditions for acclimatization for 2 weeks, taking out the regenerated plants, washing away the agar from the plant roots with running water, and then soaking them in an aqueous solution containing 0.05% to 0.2% potassium permanganate for 10 to 20 minutes.

8. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, The mixed nutrient substrate in step (5) is made by mixing Pins' nutrient soil, vermiculite and garden soil in a volume ratio of 1-3:1-2:1-4.

9. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, The culture conditions for adventitious bud induction and proliferation in steps (2) and (3) are as follows: temperature 25±2℃, light intensity 2500~3000lx, and photoperiod 16 light / 8h dark.

10. The method for rapid propagation of Ruby Slipper Oak Leaf Hydrangea according to claim 1, characterized in that, The transplanting conditions in step (5) are a temperature of 20-28℃, a humidity of 75%-85%, and a light intensity of 1800-4000 lx.

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