Tissue culture and rapid propagation method for rhododendron simsii seedlings

Abstract

The invention discloses a tissue culture and rapid propagation method for rhododendron simsii seedlings, and belongs to the technical field of seedling propagation. The rapid propagation method comprises the steps of explant disinfection, induction culture, axillary bud subculture multiplication culture, strong seedling culture, rooting culture, acclimatization and transplantation and the like. According to the tissue culture and rapid propagation method for the rhododendron simsii seedlings, the germination rate can reach 97% or above, the rooting rate can reach 74% or above, the survival rate can reach 90%, the proliferation rate, the strong seedling rate and the survival rate of clustered seedlings are further improved, a theoretical basis and a matched technical means are provided for large-scale industrial development of the rhododendron simsii, and the method is suitable for large-scale industrial production of the rhododendron simsii. An efficient cultivation mode and a management technology are established, excellent characters are reserved, and meanwhile the seedling raising cost is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of seedling propagation, and in particular to a method for rapid propagation of 'vermicelli' azalea seedlings through tissue culture. Background Art

[0002] Rhododendron simsii Planch. is an evergreen or deciduous shrub of the genus Rhododendron (Rhododendron L.) in the family Ericaeae. It is also known as azalea, mountain red, mountain red, mountain pomegranate, and mountain azalea. It is one of the three major natural famous flowers in the world and one of the ten traditional famous flowers in China.

[0003] The Encore azalea series is a variety group bred in the United States in the 1980s. There are more than 30 varieties on the market. They are hybrids of Taiwan's Rhododendron oldhamii and other azalea varieties with better stress resistance. The advantage of this variety group is that it blooms in multiple seasons, mainly in spring and autumn, and some varieties even bloom until December. Encore azalea is cold-resistant, evergreen, does not require frequent pruning, and is easy to maintain. It is an excellent azalea cultivar and the best choice for planting. At present, the best-selling variety is 'Pink Ribbons' (R. hybrid Pink Ribbons), which is a good variety for landscaping and is known as the best-selling azalea variety group in the 21st century; however, there are few reports on the breeding technology related to the "Pink Ribbons" azalea in the existing technology.

[0004] Chinese patent document CN117356396A discloses a cultivation matrix and preparation method for Azalea anku. The present invention provides a cultivation matrix for Azalea anku, and the raw materials for its preparation are, by volume percentage, 5-80% earthworm castings, 5-80% peat, and 10-20% porous materials. The soilless cultivation matrix in the present invention has environmentally friendly raw materials, simple ingredients, and resource recycling. The physical and chemical properties of the soilless cultivation matrix in the present invention, such as pH value, organic matter content, EC value, and ventilation porosity, are close to the physical and chemical properties of the root growth of Azalea, and can improve the plant height, ground diameter, and survival rate of Azalea. The present invention only involves the optimization of the soilless cultivation matrix, and does not involve the entire tissue culture propagation process. Summary of the invention

[0005] The main purpose of the present invention is to propose a tissue culture rapid propagation method for 'vermicelli belt' azalea, including explant disinfection, induction culture, axillary bud subculture proliferation culture, seedling culture, rooting culture, and seedling hardening and transplanting. By screening the culture medium formulas for cultivation at various stages, the best culture medium formula is screened out, which significantly improves the survival rate of the seedlings of 'vermicelli belt' azalea, the rooting rate can reach more than 74%, and the survival rate can reach 90%, which is beneficial to the reproduction of 'vermicelli belt' azalea.

[0006] To achieve the above object, the present invention proposes a method for rapid propagation of 'Fan Belt' azalea seedlings through tissue culture, comprising the following steps:

[0007] S1. Explant disinfection: Select fresh "'Fan Dai'" azalea stem segments with axillary buds as explants, wash the explants and disinfect them;

[0008] S2. Induction culture

[0009] Inoculate the sterilized stem segments with axillary buds into the induction culture medium; incubate at 23-27°C with light for 10-12 hours / day and a light intensity of 1000-2000 lx; ​​after culturing for 18-20 days, count the number of axillary buds;

[0010] S3. Subculture and proliferation of axillary buds

[0011] The induced sterile axillary buds were inoculated into a proliferation medium, and the light intensity was 1000-2000 lx at a temperature of 23-27°C for 10-12 h / d. After culturing for 45-55 days, clustered buds grew out of the stem segments.

[0012] S4. Seedling cultivation

[0013] The axillary buds obtained by proliferation culture are inoculated into a seedling medium, and the light intensity is 1000-2000 lx at a temperature of 23-27°C for 10-12 hours / day. After culturing for 35-45 days, tissue culture seedlings with broad leaves and strong growth are obtained.

[0014] S5. Rooting culture

[0015] The healthy tissue culture seedlings are transferred to the rooting medium for rooting culture, and the light intensity is 1000-2000lx at a temperature of 23-27°C for 10-12h / d. After culturing for 50-70 days, rooted seedlings with root systems are obtained;

[0016] S6. Hardening and transplanting

[0017] Before transplanting, harden the seedlings indoors for 5-6 days, then gently take out the seedlings, wash the culture medium on the roots with tap water, and then place them in clean water to keep moist, soak the washed roots of the tissue culture seedlings in 0.1-0.2% carbendazim solution for 3-5 minutes, and then transplant them into the substrate.

[0018] Preferably, the S1 step is as follows: select fresh 'vermicelli' azalea stem segments with axillary buds as explants, cut off the leaves from the petiole, trim them into small segments with bud points of 2.0-3.0 cm in length, soak them in a detergent solution for 1-2 hours, then rinse them under running water for 20-30 minutes, first soak them in 75% alcohol for 20-30 seconds, and rinse them with sterile water for 2-3 times; then disinfect them with a 1-3wt% sodium hypochlorite aqueous solution for 6-10 minutes; then rinse them with a 1-2wt% calcium chloride solution for 2-3 times, and finally rinse them with sterile water for 3-5 times.

[0019] Further preferably, in step S1, the concentration of the sodium hypochlorite aqueous solution is 2%, and the disinfection time is 8 minutes.

[0020] Preferably, the induction medium formula in S2 is WPM medium + 0.5-3.0 mg·L -1 ZT, 0.1~1.0mg·L -1 NAA; wherein, ZT is zeatin and NAA is naphthylacetic acid.

[0021] Preferably, the proliferation medium formula in step S3 is WPM medium + 1.0-3.0 mg·L -1 ZT+0.1~0.5mg·L -1 NAA.

[0022] Preferably, the seedling medium formula in step S4 is WPM medium + 0.1-0.5 mg·L -1 ZT+0.1~0.5mg·L -1 NAA.

[0023] Preferably, the rooting medium formula in step S5 is WPM medium + 1-3 mg·L -1 IBA + 0.1~1.0g·L -1 AC; wherein, IBA is indolebutyric acid and AC is activated carbon.

[0024] Preferably, the length of the tissue culture seedlings in step S5 is 1.5-2.5 cm.

[0025] Preferably, in step S6, the substrate is at least one of peat soil, perlite and river sand.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention establishes a method for propagating 'Fan Belt' azalea by tissue culture technology, and selects and optimizes the induction medium, proliferation medium, rooting medium, and seedling medium involved in the method. The method has the advantages of high bud induction rate, high proliferation coefficient, high rooting rate, fast plant growth, normal leaf shape and color, and a developed root system. It can meet the market demand for 'Fan Belt' azalea red, successfully solves technical problems such as difficult sterilization and easy browning of explants, is not affected by changes in time and seasonal environment, greatly improves the propagation rate, and improves the ornamental value and commercial value of 'Fan Belt' azalea, which is of great significance for protecting excellent germplasm resources and exploring and creating new germplasm;

[0028] (2) The present invention can quickly obtain azaleas with a high survival rate by performing a series of processes such as disinfection, induction, proliferation and rooting culture on the azaleas. In the disinfection process, 2% sodium hypochlorite is selected as a disinfectant, which is more conducive to preventing browning;

[0029] (3) When the present invention uses WPM as the basic culture medium, plant growth regulators ZT and NAA at appropriate concentrations are added thereto. The synergistic effect of the two increases the proliferation rate of axillary buds and shortens the proliferation time. During the rooting induction process, the synergistic effect of IBA and AC promotes more rooting of bottle seedlings, rapid and luxuriant growth;

[0030] (4) The tissue culture rapid propagation method of the 'vermicelli belt' azalea seedlings of the present invention can achieve a germination rate of 97%, a rooting rate of more than 70%, and a survival rate of 90%. It also improves the proliferation rate, seedling rate and survival rate of the clump seedlings, providing a theoretical basis and supporting technical means for the large-scale industrial development of the 'vermicelli belt' azalea, establishing efficient cultivation models and management technologies, and reducing seedling costs while retaining excellent traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 This is a graph showing the growth of axillary buds of the 'vermicelli strip' of the present invention after being sterilized with a 2wt% sodium hypochlorite aqueous solution for 8 minutes and inoculated into a culture medium for 5 days;

[0033] Figure 2 This is a graph showing the growth of axillary buds of the 'vermicelli strip' of the present invention after being sterilized with a 2wt% sodium hypochlorite aqueous solution for 10 minutes and inoculated into a culture medium for 5 days;

[0034] Figure 3 The 'vermicelli strip' of the present invention is in the culture medium formula WPM, 3.0mg·L -1 ZT, 0.5mg·L -1 Figure 2 shows the results of proliferation culture under NAA conditions. DETAILED DESCRIPTION

[0035] To avoid redundancy, the items used in the following examples are all commercially available products unless otherwise specified, and the methods used are all conventional methods unless otherwise specified.

[0036] Some of the raw materials of the present invention come from the following sources:

[0037] The stem segments with axillary buds of Rhododendron 'Fan Dai' were obtained from Hunan Botanical Garden.

[0038] Example 1

[0039] A method for rapid propagation of 'Fan Band' azalea seedling tissue culture, comprising the following steps:

[0040] S1. Explant disinfection: Select fresh 'Fan Dai' azalea stem segments with axillary buds as explants, cut off the leaves from the petiole, trim them into small segments with buds of 2.0-3.0 cm in length, soak them in a detergent solution for 1 hour, rinse them under running water for 30 minutes, soak them in 75% alcohol for 30 seconds, rinse them with sterile water for 3 times; then disinfect them with 2wt% sodium hypochlorite aqueous solution for 8 minutes; then rinse them with 2wt% calcium chloride solution for 3 times, and finally rinse them with sterile water for 5 times; inoculate the disinfected stem segments with axillary buds in WPM+1.0mg·L -1 ZT+0.1mg·L -1 The pH value of the medium was 5.4. Three explants were inoculated in each bottle and repeated three times. After 5 days of inoculation, the contamination rate of the stem segments was 23.61%, the browning rate was 13.89%, and the axillary buds grew rapidly and robustly.

[0041] S2. Induction culture

[0042] The sterilized stem segments with axillary buds were inoculated into an induction medium for induction culture; the induction medium formula was WPM medium + 2.5 mg / L ZT + 0.1 mg / L NAA, with a total of 8 bottles. The time of axillary bud germination was observed and recorded at 7.67 days after inoculation. After 20 days, the number of axillary buds was counted and the germination rate was calculated. The average number of axillary buds was 23.33 (pieces), and the average germination rate was 97%;

[0043] S3. Subculture and proliferation of axillary buds

[0044] The induced sterile axillary buds were transferred to WPM medium + 3.0 mg·L -1 ZT+0.5mg·L -1The buds were cultured in a proliferation medium composed of NAA, with a total of 10 bottles, 3 buds in each bottle, and the proliferation and growth of buds were observed after 50 days of culture. Buds larger than 1 cm were considered effective buds; the proliferation coefficient was measured to be 6.00±1.00a; Note: The proliferation coefficient data are mean ± standard deviation; the same index data are marked with different lowercase letters to indicate significant differences between the data (P<0.05);

[0045] S4. Seedling cultivation

[0046] The axillary buds obtained through proliferation culture were inoculated into the seedling medium, the seedling medium formula of which was WPM basal medium + 0.5 mg·L -1 ZT+0.5mg·L -1 NAA; 8 bottles in total, 3 seedlings in each bottle, 12h / d at 25℃, light intensity 2000lx, 45d after cultivation, the results of seedling cultivation treatment are thicker and longer stems, wider leaves;

[0047] S5. Rooting culture

[0048] The healthy sterile seedlings with a length of 1.5 to 2.5 cm were transferred into the rooting medium, wherein the rooting medium formula was WPM medium + 3 mg·L -1 IBA+1.0g·L -1 AC, 8 bottles in total, 2 seedlings in each bottle, observed and counted the rooting situation after 60 days, the rooting culture treatment results showed that there were many roots and the seedlings were strong, with a rooting rate of 74.63±2.12a(%); Note: The rooting rate data are mean ± standard deviation; the same indicator data are marked with different lowercase letters to indicate significant differences between the data (P<0.05).

[0049] S6. Hardening and transplanting

[0050] Before transplanting, harden the seedlings indoors for 6 days, then use tweezers to gently remove the rooted seedlings with complete root systems, wash the culture medium on the roots with tap water, and then place them in clean water to keep moist, soak the washed roots of the tissue culture seedlings in 0.2% carbendazim solution for 3 minutes, and then transplant them into a matrix composed of peat soil, perlite, and river sand in a mass ratio of 2:1:1. Water them thoroughly once, and then water them once a week. The survival rate was counted after 60 days, and the survival rate was 90%.

[0051] Example 2

[0052] A method for rapid propagation of 'Fan Band' azalea seedling tissue culture, comprising the following steps:

[0053] S1. Explant disinfection: Select fresh 'Fan Dai' azalea stem segments with axillary buds as explants, cut off the leaves from the petiole, trim them into small segments with buds of 2.0-3.0 cm in length, soak them in a detergent solution for 2 hours, rinse them under running water for 30 minutes, soak them in 75% alcohol for 20 seconds, rinse them twice with sterile water; then disinfect them with 1wt% sodium hypochlorite aqueous solution for 10 minutes; then rinse them with 1wt% calcium chloride solution for 3 times, and finally rinse them with sterile water for 5 times; inoculate the disinfected stem segments with axillary buds in WPM+1.0mg·L -1 ZT+0.1mg·L -1 On the medium composed of NAA, the pH value of the medium was 5.4. Three explants were inoculated in each bottle, and the process was repeated three times. Five days after inoculation, the contamination rate of the stem segments was 8.33%, the browning rate was 30.56%, and the axillary buds grew rapidly but were thin and weak.

[0054] S2. Induction culture

[0055] The sterilized stem segments with axillary buds were inoculated into an induction medium for induction culture; the induction medium formula was WPM medium + 2.5 mg / L ZT + 0.1 mg / L NAA, with a total of 8 bottles. The time of axillary bud germination was observed and recorded at 7.67 days after inoculation. After 20 days, the number of axillary buds was counted and the germination rate was calculated. The average number of axillary buds was 23.33 (pieces), and the average germination rate was 97%;

[0056] S3. Subculture and proliferation of axillary buds

[0057] The induced sterile axillary buds were transferred to WPM medium + 3.0 mg·L -1 ZT+0.5mg·L -1 The buds were cultured in a proliferation medium composed of NAA, with a total of 10 bottles, 3 buds in each bottle, and the proliferation and growth of buds were observed after 50 days of culture. Buds larger than 1 cm were considered effective buds; the proliferation coefficient was measured to be 6.00±1.00a; Note: The proliferation coefficient data are mean ± standard deviation; the same index data are marked with different lowercase letters to indicate significant differences between the data (P<0.05);

[0058] S4. Seedling cultivation

[0059] The axillary buds obtained through proliferation culture were inoculated into the seedling medium, the seedling medium formula of which was WPM basal medium + 0.5 mg·L -1 ZT+0.5mg·L -1 NAA; 8 bottles in total, 3 seedlings in each bottle, 12h / d at 25℃, light intensity 2000lx, 45d after cultivation, the results of seedling cultivation treatment are thicker and longer stems, wider leaves;

[0060] S5. Rooting culture

[0061] The healthy sterile seedlings with a length of 1.5 to 2.5 cm were transferred into the rooting medium, wherein the rooting medium formula was WPM medium + 3 mg·L -1 IBA+1.0g·L -1 AC, 8 bottles in total, 2 seedlings in each bottle, the rooting situation was observed and counted after 60 days, the result of rooting culture treatment was few roots, and the rooting rate was 3.24±0.80g(%); Note: The rooting rate data are mean±standard deviation; The same indicator data are marked with different lowercase letters to indicate significant differences between the data (P<0.05).

[0062] S6. Hardening and transplanting

[0063] Before transplanting, harden the seedlings indoors for 5 days, then use tweezers to gently remove the rooted seedlings with complete root systems, wash the culture medium on the roots with tap water, and then place them in clean water to keep moist, soak the washed roots of the tissue culture seedlings in 0.2% carbendazim solution for 3 minutes, and then transplant them into a matrix composed of peat soil and perlite in a mass ratio of 2:1, water them thoroughly once, and then water them once a week. Statistical analysis of the survival rate after 60 days showed that the survival rate was 40%.

[0064] Example 3

[0065] A method for rapid propagation of 'Fan Band' azalea seedling tissue culture, comprising the following steps:

[0066] S1. Explant disinfection: Select fresh 'Fan Dai' azalea stem segments with axillary buds as explants, cut off the leaves from the petiole, trim them into small segments with buds of 2.0-3.0 cm in length, soak them in a detergent solution for 1 hour, rinse them under running water for 30 minutes, soak them in 75% alcohol for 20 seconds, rinse them with sterile water for 3 times; then disinfect them with 2wt% sodium hypochlorite aqueous solution for 6 minutes; then rinse them with 1wt% calcium chloride solution for 3 times, and finally rinse them with sterile water for 5 times; inoculate the disinfected stem segments with axillary buds in WPM+1.0mg·L -1 ZT+0.1mg·L -1 On the medium composed of NAA, the pH value of the medium was 5.4. Three explants were inoculated in each bottle, and repeated three times. Five days after inoculation, the contamination rate of the stem segments was 43.06%, the browning rate was 2.78%, and the axillary buds grew rapidly and robustly.

[0067] S2. Induction culture

[0068] The sterilized stem segments with axillary buds were inoculated into an induction medium for induction culture; the induction medium formula was WPM medium + 2.5 mg / L ZT + 1 mg / L NAA, with a total of 8 bottles. The time of axillary bud germination was observed and recorded at 12.67 days after inoculation. After 20 days, the number of axillary buds was counted and the germination rate was calculated. The average number of axillary buds was 15.33 (pieces), and the average germination rate was 64%;

[0069] S3. Subculture and proliferation of axillary buds

[0070] The induced sterile axillary buds were transferred to WPM medium + 2.0 mg·L -1 +, 0.5mg·L -1 The buds were cultured in a proliferation medium composed of NAA, with a total of 10 bottles, and 3 buds were inoculated in each bottle. After 50 days of culture, the proliferation and growth of the buds were observed, and those larger than 1 cm were considered effective buds; the proliferation coefficient was measured to be 3.67±0.67c; Note: The proliferation coefficient data are mean ± standard deviation; the same index data are marked with different lowercase letters to indicate significant differences between the data (P<0.05);

[0071] S4. Seedling cultivation

[0072] The axillary buds obtained through proliferation culture were inoculated into the seedling medium, the seedling medium formula of which was WPM basal medium + 0.1 mg·L -1 ZT+0.1mg·L -1 NAA; 8 bottles in total, 3 seedlings in each bottle, 12h / d at 25℃, light intensity 2000lx, 45d after cultivation, the result of seedling cultivation treatment was thickened stems, but not elongated, small leaves;

[0073] S5. Rooting culture

[0074] The healthy sterile seedlings with a length of 1.5 to 2.5 cm were transferred into the rooting medium, wherein the rooting medium formula was WPM medium + 2 mg·L -1 IBA+1.0g·L -1 AC, 8 bottles in total, 2 seedlings in each bottle, the rooting situation was observed and counted after 60 days, the rooting culture treatment results showed that there were more roots, the seedlings were strong, and the rooting rate was 50.00±1.39b(%); Note: The rooting rate data are mean±standard deviation; The same indicator data are marked with different lowercase letters to indicate significant differences between the data (P<0.05).

[0075] S6. Hardening and transplanting

[0076] Before transplanting, harden the seedlings indoors for 6 days, then use tweezers to gently remove the rooted seedlings with complete root systems, wash the culture medium on the roots with tap water, and then place them in clean water to keep moist, soak the washed roots of the tissue culture seedlings in 0.2% carbendazim solution for 5 minutes, and then transplant them into a matrix composed of peat soil and river sand in a mass ratio of 2:1, water them thoroughly once, and then water them once a week. The survival rate was counted after 60 days, and the survival rate was 65%.

[0077] From the above results, it can be concluded that the special culture medium and tissue culture rapid propagation method of the present invention have completeness, speciality, specificity and comprehensive effects on the tissue culture rapid propagation of the 'Fan Belt' azalea variety. The invention can make the germination rate reach 97%, the rooting rate can reach more than 74%, and the survival rate can reach 90%, providing a theoretical basis and supporting technical means for the large-scale industrial development of 'Fan Belt' azalea, establishing efficient cultivation models and management technologies, retaining excellent traits while reducing seedling costs.

[0078] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for rapid propagation of 'Fan Belt' azalea seedling tissue culture, characterized in that: The following steps are involved: S1. Explant disinfection: Select fresh stem segments of azalea with axillary buds as explants, wash and disinfect the explants; S2. Induction Culture Inoculate the sterilized stem segments with axillary buds into the induction culture medium; incubate at 23-27°C with light for 10-12 hours / day and a light intensity of 1000-2000 lx; ​​after culturing for 18-20 days, count the number of axillary buds; S3. Subculture and proliferation of axillary buds The induced sterile axillary buds were inoculated into a proliferation medium, and the light intensity was 1000-2000 lx at a temperature of 23-27°C for 10-12 h / d. After 45-55 days of culture, clustered buds grew out of the stem segments. S4. Seedling cultivation The axillary buds obtained by proliferation culture are inoculated into a seedling medium, and the light intensity is 1000-2000 lx at a temperature of 23-27°C for 10-12 hours / day. After culturing for 35-45 days, tissue culture seedlings with broad leaves and strong growth are obtained. S5. Rooting culture The healthy tissue culture seedlings are transferred to the rooting medium for rooting culture, and the light intensity is 1000-2000lx at a temperature of 23-27°C for 10-12h / d. After culturing for 50-70 days, rooted seedlings with root systems are obtained; S6. Hardening and transplanting Before transplanting, harden the seedlings indoors for 5-6 days, then gently take out the seedlings, wash the culture medium on the roots with tap water, and then place them in clean water to keep moist, soak the washed roots of the tissue culture seedlings in 0.1-0.2% carbendazim solution for 3-5 minutes, and then transplant them into the substrate.

2. The rapid propagation method according to claim 1, characterized in that: The S1 step is specifically as follows: select fresh 'vermicelli' azalea stem segments with axillary buds as explants, cut off the leaves from the petiole, trim them into small segments with bud points of 2.0-3.0 cm in length, soak them in a detergent solution for 1-2 hours, then rinse them under running water for 20-30 minutes, first soak them in 75% alcohol for 20-30 seconds, rinse them with sterile water 2-3 times; then disinfect them with a 1-3wt% sodium hypochlorite aqueous solution for 6-10 minutes; then rinse them with a 1-2wt% calcium chloride solution for 2-3 times, and finally rinse them with sterile water 3-5 times.

3. The rapid propagation method according to claim 2, characterized in that: In step S1, the concentration of the sodium hypochlorite aqueous solution is 2%, and the disinfection time is 8 minutes.

4. The rapid propagation method according to claim 1, characterized in that: The induction medium formula in S2 is WPM medium + 0.5-3.0 mg·L -1 ZT+0.1~1.0mg·L -1 NAA.

5. The rapid propagation method according to claim 1, characterized in that: The proliferation culture medium formula in step S3 is WPM culture medium + 1.0-3.0 mg·L -1 ZT+0.1~0.5mg·L -1 NAA.

6. The rapid propagation method according to claim 1, characterized in that: The formula of the seedling medium in step S4 is WPM medium + 0.1-0.5 mg·L -1 ZT+0.1~0.5mg·L -1 NAA.

7. The rapid propagation method according to claim 1, characterized in that: The rooting medium formula in step S5 is WPM medium + 1-3 mg·L -1 IBA + 0.1~1.0g·L -1 AC.

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