Method suitable for industrial production of Zhongjiang salvia miltiorrhiza tissue culture seedlings

Abstract

The invention discloses a method suitable for industrial production of Salvia Zhongjiang tissue culture seedlings. The method comprises the following steps: 1) hardening and domesticating aseptic seedlings; 2) cutting and breeding production seedlings; and (3) transplanting production seedlings. The survival rate of the Zhongjiang salvia miltiorrhiza tissue culture seedlings in the transplanting and cuttage breeding process is increased, the root regeneration frequency of the Zhongjiang salvia miltiorrhiza tissue culture production seedlings is increased through the optimized matrix and nutrient solution composition, the propagation coefficient of the Zhongjiang salvia miltiorrhiza tissue culture seedlings is increased, the seedling breeding time and production procedures are shortened, and the survival rate of the Zhongjiang salvia miltiorrhiza tissue culture seedlings is increased. The production cost of the Zhongjiang salvia miltiorrhiza tissue culture seedlings is reduced, and industrialized production of the Zhongjiang salvia miltiorrhiza tissue culture seedlings is realized.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural production, and in particular to a method suitable for industrialized production of Salvia miltiorrhiza tissue culture seedlings. Background Art

[0002] Salvia miltiorrhiza Bunge is a perennial herbaceous plant of the genus Salvia in the family Lamiaceae. It is also known as blood ginseng, red salvia, red ginseng, large-leafed blood-activating danshen, etc. Its dried roots and rhizomes are used as medicine. It has the effects of promoting blood circulation and removing blood stasis, relieving pain from menstruation, clearing the heart and eliminating vexation, cooling blood and eliminating carbuncle. It is mainly used to treat cardiovascular and cerebrovascular diseases in clinical practice and is one of the commonly used bulk Chinese medicinal materials. At present, the source of Salvia miltiorrhiza mainly comes from artificial cultivation. Sichuan, Shandong, Shaanxi, Henan, Hebei and other places are its main cultivation areas. Among them, Zhongjiang Salvia miltiorrhiza produced in Zhongjiang County, Deyang City, Sichuan Province has excellent quality, significant efficacy, high unit yield, high drug efficacy, good root color, and beautiful appearance. It is one of the geographical indication authentic medicinal materials and enjoys a high degree of popularity and market recognition.

[0003] Plant in vitro culture is the cultivation of plant cells, tissues and organs under highly controlled artificial environment and nutritional conditions. Using in vitro culture technology to build a plant in vitro regeneration system can cultivate high-quality seedlings without pests and diseases, and achieve rapid and efficient seedling reproduction. There are currently a large number of related studies on the in vitro culture of Salvia miltiorrhiza. For example, CN110178728A and CN111280058B use explants such as root segments, stem tips, sterile leaves, and sterile petioles to establish a Zhongjiang Salvia miltiorrhiza regeneration system and cultivate Zhongjiang Salvia miltiorrhiza tissue culture seedlings. However, tissue culture breeding uses sterile solid culture medium to induce organ regeneration of buds and roots, and the production process is cumbersome, the production cost is high, and the transplant survival rate is low, which cannot meet the large-scale breeding and application promotion of Zhongjiang Salvia miltiorrhiza tissue culture seedlings. CN114946660A proposes a method for cultivating Salvia miltiorrhiza tissue culture seedlings suitable for mechanical planting, but the use of colchicine and amoxicillin solution therein leads to a potential risk of mutation in the regenerated Salvia miltiorrhiza seedlings, and the genetic stability of the Salvia miltiorrhiza seedlings cannot be guaranteed. In addition, it has been found through experiments that the survival rate of the mechanically cultivated tissue culture seedlings after transplanting to the field is not high.

[0004] Therefore, exploring multi-channel breeding methods for Zhongjiang Salvia miltiorrhiza tissue culture seedlings, simplifying the seedling production procedures, reducing the seedling production costs, and improving the survival rate of seedlings in the field, while ensuring the genetic stability of Salvia miltiorrhiza germplasm are the key and difficulty to the factory-based, standardized, and intensive production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings. Summary of the invention

[0005] To achieve the above object, the present invention provides a method suitable for industrial production of Salvia miltiorrhiza tissue culture seedlings, which comprises the following steps:

[0006] 1) Take sterile tissue culture seedlings of Zhongjiang Salvia miltiorrhiza, transfer them to a greenhouse, spray nutrient solution 1, acclimate them in a greenhouse environment, take tissue culture seedlings with strong root systems and transplant them into sterile substrates, water them to fix the roots, and spray foliar fertilizer after new leaves grow out;

[0007] 2) When the tissue culture seedlings of step 1) grow to 30-40 cm in height, take the tender shoots or stem segments, soak them in nutrient solution 2, and then insert them into a sterile substrate for cultivation. After the seedling roots are produced and new leaves grow, spray foliar fertilizer and continue to cultivate to obtain production seedlings;

[0008] 3) taking the healthy production seedlings obtained in step 2), soaking them in nutrient solution 2, and transplanting them to the field for cultivation;

[0009] The nutrient solution 1 is a solution containing potassium dihydrogen phosphate, brassinolide and indoleacetic acid;

[0010] The nutrient solution 2 is a solution containing brassinolide, indoleacetic acid and thiophanate-methyl;

[0011] The matrix is ​​a mixture of medical stone, coal slag and river sand;

[0012] The foliage fertilizer is an aqueous solution containing autumn fertilizer or medium-element fertilizer.

[0013] Furthermore, the nutrient solution 1 is a solution containing 3 g / L potassium dihydrogen phosphate, 0.25 mg / L brassinolide and 0.5 mg / L indoleacetic acid.

[0014] Furthermore, the nutrient solution 2 is a solution containing 0.25 mg / L brassinolide, 0.5 mg / L indoleacetic acid and 1 g / L thiophanate-methyl.

[0015] Furthermore, the matrix is ​​a mixture of medical stone, coal slag and river sand in a mass ratio of 7:1:2; the pH value of the mixture is 6.5-7.5.

[0016] Furthermore, the foliar fertilizer is an aqueous solution containing 5g / L of radix spatholobi and medium element fertilizer.

[0017] Furthermore, the mass ratio of the Wangqiu fertilizer to the medium element fertilizer is 1:1.

[0018] Furthermore, the acclimation time of step 1) is 3 days; and the fixed plant spacing of the tissue culture seedlings is 8 cm×8 cm.

[0019] Furthermore, in step 2), the tender shoots or stem segments are tender shoots or stem segments with 6-8 leaves; the soaking time is 10 minutes; and the culture conditions are: temperature 18-26° C., humidity 75-85%, and avoid direct sunlight.

[0020] Furthermore, the soaking time in step 3) is 10-15s.

[0021] Furthermore, the foliar fertilizer in step 1) and step 2) is sprayed once every 14 days.

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

[0023] The invention can promote the growth of seedling root system and leaf development and improve the survival rate of transplanting and cutting by spraying the specific nutrient solution 1 on the original tissue culture seedlings of Zhongjiang salvia miltiorrhiza and soaking the young tender shoots or stem segments of the domesticated tissue culture seedlings with the specific nutrient solution 2; the matrix formed by compounding multiple materials such as medical stone and coal slag has the characteristics of water and fertilizer retention, looseness and air permeability, and strong buffering capacity compared with the traditional compound nutrient soil such as peat soil, perlite, vermiculite, etc., and can improve the quality of the seedlings, and is more suitable for the complex ecological environment of the field after transplanting, and improves the survival rate of the seedlings after transplanting; the young tender shoots or stem segments with 6-8 leaves have vigorous growth, strong rooting ability and high seedling rate; the preferred fungicide thiophanate-methyl can effectively inhibit the occurrence of diseases and insect pests in the breeding process of salvia miltiorrhiza tissue culture seedlings.

[0024] The factory-based production method of Zhongjiang Salvia miltiorrhiza tissue culture seedlings of the present invention improves the survival rate of Zhongjiang Salvia miltiorrhiza tissue culture seedlings during transplanting and cutting propagation, optimizes the composition of the matrix and nutrient solution to improve the root regeneration frequency of Zhongjiang Salvia miltiorrhiza tissue culture seedlings, improves the propagation coefficient of Zhongjiang Salvia miltiorrhiza tissue culture seedlings, shortens the seedling propagation time and production procedures, reduces the production cost of Zhongjiang Salvia miltiorrhiza tissue culture seedlings, and realizes the factory-based production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the sterile seedling hardening of Salvia miltiorrhiza tissue culture in Zhongjiang (top: laboratory; bottom: greenhouse);

[0028] Figure 2 Illustration of the transplantation of sterile seedlings of Zhongjiang Salvia miltiorrhiza tissue culture (left) and the growth of sterile seedlings of Zhongjiang Salvia miltiorrhiza tissue culture (right);

[0029] Figure 3 Schematic diagram of the cutting production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings and the growth diagram of the cutting seedlings;

[0030] Figure 4Illustration of the growth of Zhongjiang Salvia miltiorrhiza tissue culture seedlings (left) and the growth of Zhongjiang Salvia miltiorrhiza tissue culture seedlings after transplantation (right);

[0031] Figure 5 Illustration of transplanting and growing the seedlings of Zhongjiang Salvia miltiorrhiza tissue culture production DETAILED DESCRIPTION

[0032] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all known products and can be purchased commercially.

[0033] Example 1 Production method of Salvia miltiorrhiza tissue culture seedlings of the present invention

[0034] 1. Preparation of production solution, substrate and seedbed

[0035] Nutrient solution 1: Mix potassium dihydrogen phosphate, brassinolide and indoleacetic acid, and add water to dissolve to obtain an aqueous solution with a final concentration of 3g / L potassium dihydrogen phosphate, 0.25mg / L brassinolide and 0.5mg / L indoleacetic acid;

[0036] Nutrient solution 2: Mix brassinolide and indoleacetic acid, add water to dissolve to obtain an aqueous solution with a final concentration of 0.25 mg / L brassinolide and 0.5 mg / L indoleacetic acid; mix and dissolve the aqueous solution and chlorothalonil in a ratio of 1000 ml: 1 g to obtain;

[0037] Matrix: Take medical stone, coal slag and river sand, mix them in a mass ratio of 7:1:2;

[0038] Foliar fertilizer: Take Wangqiu and medium-element fertilizer in a mass ratio of 1:1, add water to dissolve and obtain a mixed solution with a final concentration of 5g / L.

[0039] Seedbed: Take the substrate, sterilize it to obtain a sterile substrate, adjust the pH value of the sterile substrate to 6.5 with potassium dihydrogen phosphate solution, lay the seedbed with the sterile substrate with a pH value of 6.5, set the seedbed ridge width to 125 cm, height to 10 cm, and ridge furrow width to 30 cm. After the seedbed is laid, water it to keep the humidity of the seedbed at 15%;

[0040] Rooting water: clean tap water or drinking water.

[0041] 2. Production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings

[0042] 1) Sterile seedling hardening and domestication

[0043] Transfer the healthy and robust Zhongjiang Salvia miltiorrhiza tissue culture sterile seedlings from the culture room to the net room greenhouse with a temperature of 15-30℃, a light intensity of 1800-2300lux, and a humidity of 75%-85%. Immediately open the bottle cap or film, and spray nutrient solution 1 until the liquid drips down the leaf edge to promote the root growth of the tissue culture seedlings. After three days of acclimatization ( Figure 1), select Zhongjiang Salvia miltiorrhiza tissue culture seedlings with good root growth and transplant them into the seedbed with sterile substrate according to the plant spacing of 8cm×8cm. After fixing the roots with the substrate, water the roots until water seeps out from the bottom of the substrate. After the new leaves grow out, spray foliar fertilizer once every 14 days, and each time spray until the liquid drips down from the edge of the leaves. After 25 days of transplanting the sterile seedlings, the statistical survival rate is 98% ( Figure 2 Left);

[0044] 2) Propagation of production seedlings by cuttings

[0045] When the tissue culture seedlings obtained in step 1) grow to 30-40 cm in height, cut a young shoot or stem segment with 6-8 leaves with a knife, soak it in nutrient solution 2 for 10 minutes to promote rooting, and then vertically insert the shoot or stem segment into a seedbed covered with sterile substrate ( Figure 2 Right), water thoroughly, control the ambient temperature to 18-26℃, humidity to 75-85%, avoid direct sunlight, spray foliar fertilizer once every 14 days after the seedling roots are produced and new leaves grow, and each time the liquid drips down from the edge of the leaves to promote plant growth. After 25 days of cutting, the roots of the Zhongjiang Salvia miltiorrhiza tissue culture seedlings are evenly distributed in the substrate, the rooting rate is 95.8%, and the survival rate is 100% ( Figure 3 );

[0046] 3) Transplanting of production seedlings

[0047] Select healthy plants without diseases and insect pests, carefully remove the production seedlings from the substrate with roots using a small bamboo pry, place them in a low-temperature foam box, and send them to the transplanting field within 6 hours; before transplanting, immerse the transplanted seedlings completely in nutrient solution 2 for 10-15 seconds, take them out and transplant them to the field. After the production seedlings are transplanted to the field, the survival rate is statistically 95% (100 days later). Figure 3 ).

[0048] Among the above steps, step 1) and step 2) are implemented in the production base in Zhongjiang County, Sichuan Province, and step 3) is implemented in Liangshan Prefecture, Sichuan Province.

[0049] Example 2 Production method of Salvia miltiorrhiza tissue culture seedlings of the present invention

[0050] The specific cultivation method steps are the same as those in Example 1, except that: Step 1) and Step 2) are carried out at the production base in Ya'an City, Sichuan Province ( Figure 4 ), step 3) is implemented at the production base in Deyang City, Sichuan Province ( Figure 5 );

[0051] The pH of the sterile substrate laid when preparing the seedbed is 7.5.

[0052] During the sterile seedling hardening and acclimatization stage, 25 days after transplanting, the survival rate was 96.83%

[0053] During the seedling cutting propagation stage, 25 days after cutting, the roots of the Zhongjiang Salvia miltiorrhiza tissue culture seedlings were evenly distributed in the substrate, with a rooting rate of 98.66% and a survival rate of 100%.

[0054] During the transplanting stage of production seedlings, the production seedlings were transplanted to the field, and the survival rate reached 98.75% after 100 days.

[0055] The beneficial effects of the present invention are described below by means of test examples:

[0056] Experimental Example 1 Screening of culture medium suitable for industrial production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings

[0057] According to the method and steps of Example 1, the components and proportions of the sterile substrate, nutrient solution 1, nutrient solution 2 and foliar fertilizer were adjusted respectively, and Zhongjiang Salvia miltiorrhiza tissue culture seedlings were produced at the Zhongjiang County production base in Sichuan Province. Statistics were collected on the sterile seedling hardening, sterile seedling transplant survival and cutting propagation during the production process.

[0058] 1. The influence of cultivation medium on the survival rate of sterile seedlings during the seedling acclimatization stage

[0059] 1.1 Effect of transplanting medium on survival rate of seedlings

[0060] In the acclimation stage of the sterile seedlings of Zhongjiang Salvia miltiorrhiza tissue culture, the nutrient solution 1 sprayed was 0.5 mg / L indoleacetic acid, the formula of the sterile matrix transplanted was the matrix in Table 1, and no foliar fertilizer was sprayed during the entire cultivation stage. Other cultivation methods were the same as in Example 1. After 3 days of hardening, the seedlings were transplanted to the matrix, and the survival rate was calculated 30 days after transplanting. The effects of transplanting matrices such as nutrient soil, perlite, vermiculite and medical stone cinder on the transplanting of sterile seedlings of Zhongjiang Salvia miltiorrhiza were compared, and the results are shown in Table 1.

[0061] Table 1 Survival statistics of sterile seedlings of Salvia miltiorrhiza transplanted in different transplanting media

[0062]

[0063] As can be seen from Table 1: in combinations 2-5, after spraying 0.50 mg / L indoleacetic acid and transplanting into the substrate, the survival rate of sterile seedlings of Zhongjiang Salvia miltiorrhiza was 77.67%-85.41%, which was higher than the survival rate of 72.42% in the control combination 1, indicating that spraying 0.50 mg / L indoleacetic acid can effectively promote the survival of sterile seedlings of Zhongjiang Salvia miltiorrhiza transplanted.

[0064] After spraying indoleacetic acid, the survival rates of sterile seedlings of Zhongjiang Salvia miltiorrhiza in different matrices were compared. The results showed that the highest survival rate was 85.41% in the transplanting matrix with a particle mass ratio of 7:1:2 of medical stone, coal slag and river sand, which was significantly higher than the combination of perlite and vermiculite, and the combination of nutrient soil perlite and vermiculite. Therefore, a mixture of medical stone, coal slag and river sand with a mass ratio of 7:1:2 was selected as the transplanting matrix for sterile seedlings of Zhongjiang Salvia miltiorrhiza.

[0065] 1.2 Effect of growth regulators on survival rate of seedling hardening

[0066] In the acclimation stage of the aseptic seedlings of Zhongjiang Salvia miltiorrhiza tissue culture, the growth regulator formula in Table 2 was used as nutrient solution 1 and foliar fertilizer, and medical stone, coal slag and river sand in a mass ratio of 7:1:2 were compounded as the substrate for transplanting the aseptic seedlings of Zhongjiang Salvia miltiorrhiza tissue culture after acclimation, and other cultivation methods were the same as in Example 1. After 3 days of hardening, the seedlings were transplanted to the substrate, and the survival rate was calculated 30 days after transplanting to compare the effects of the growth regulators on the transplanting of aseptic seedlings of Zhongjiang Salvia miltiorrhiza. The results are shown in Table 2.

[0067] Table 2 Statistics on hardening of sterile seedlings of Zhongjiang Salvia miltiorrhiza under different growth regulator treatments

[0068]

[0069] As can be seen from Table 2: within the spraying range of potassium dihydrogen phosphate concentration of 0.1%-0.4% (combinations 2-5), the survival rate of sterile seedlings of Zhongjiang Salvia miltiorrhiza showed a trend of first increasing and then decreasing, and the spraying effect of 0.3% potassium dihydrogen phosphate was the best, which was 83.90% (combination 4). Adding brassinolide to 0.3% potassium dihydrogen phosphate can significantly improve the survival rate of transplanting (combinations 6 and 7). On this basis, when indoleacetic acid is further added, that is, combination 9 (Wangqiu, medium element fertilizer, 0.3% potassium dihydrogen phosphate, 0.5mg / L indoleacetic acid, 0.25mg / L brassinolide), the survival rate of sterile seedlings is the highest, which is 96.86. Therefore, combination 9 was selected as the growth regulator for hardening of Zhongjiang Salvia miltiorrhiza tissue culture seedlings.

[0070] 2. Effect of growth regulators on the survival rate after cutting propagation

[0071] In the production seedling cutting propagation stage, medical stone, coal slag and river sand with a mass ratio of 7:1:2 were used as the cutting medium for 30-40cm high tissue culture seedlings, and the growth of the young shoots / stem segments was observed after 30d of cutting, and the survival rate of the expansion was calculated (survival rate (%) = number of survivors / total number of transplants (cuttings)), and the effects of the size of the young shoots and the types and concentrations of growth regulators on the expansion of the production seedlings of Zhongjiang Salvia miltiorrhiza tissue culture were compared, and the types and ratios of the regulators of the expansion nutrient solution 2 were optimized. Other cultivation methods are the same as in Example 1, and the results are shown in Table 3.

[0072] Table 3 Statistics on the expansion of Zhongjiang Salvia miltiorrhiza seedlings under different growth regulator treatments

[0073]

[0074] From Table 3, it can be seen that under the same cultivation conditions, the survival rate of the cuttings of the young shoots / stem segments of Zhongjiang Salvia miltiorrhiza tissue culture seedlings with 6-8 leaves was 71.08%, which was higher than the survival rate of the young shoots / stem segments with 2-4 leaves (65.58%).

[0075] The effects of spraying indoleacetic acid, potassium dihydrogen phosphate and brassinolide on the survival of 6-8 leaf Zhongjiang Salvia miltiorrhiza tissue culture seedlings were compared. The results showed that when the nutrient solution lacked plant growth regulators, it was not conducive to cutting propagation. The combination of plant growth regulators indoleacetic acid and brassinolide did not always improve the survival rate of propagation compared with either of them alone. Only in the case of combination 5 and combination 6, the survival rate of the combination of indoleacetic acid and brassinolide was significantly higher than that of either of them alone. In particular, when spraying with combination 6 (0.5 mg / L indoleacetic acid, 0.25 mg / L brassinolide, and thiophanate-methyl combination), the survival rate of Zhongjiang Salvia miltiorrhiza tissue culture seedlings was the highest, at 96.48%.

[0076] Therefore, the young shoots / stem segments of Zhongjiang Salvia miltiorrhiza tissue culture seedlings with 6-8 leaves were selected as materials, and spraying with Wangqiu, medium-element fertilizer, 0.5 mg / L indoleacetic acid, 0.25 mg / L brassinolide and thiophanate-methyl (combination 6) was used as the method for expanding the production of Zhongjiang Salvia miltiorrhiza tissue culture seedlings.

[0077] In summary, the present invention can promote the growth of the seedling root system and leaf development, and improve the survival rate of transplanting and cuttings by spraying the specific nutrient solution 1 on the Zhongjiang Salvia miltiorrhiza tissue culture original seedlings and soaking the young tender buds or stem segments with 6-8 leaves of the domesticated tissue culture seedlings with the specific nutrient solution 2; the matrix compounded with multiple materials such as medical stone and coal slag can improve the quality of the seedlings, make them more suitable for the complex ecological environment of the field after transplanting, and improve the survival rate of the seedlings after transplanting.

Claims

1. A method suitable for industrial production of Salvia miltiorrhiza tissue culture seedlings, characterized in that: It includes the following steps: 1) Take sterile tissue culture seedlings of Zhongjiang Salvia miltiorrhiza, transfer them to a greenhouse, spray nutrient solution 1, acclimate them in a greenhouse environment, take tissue culture seedlings with strong root systems and transplant them into sterile substrates, water them to fix the roots, and spray foliar fertilizer after new leaves grow out; 2) When the tissue culture seedlings of step 1) grow to 30-40 cm in height, take the tender shoots or stem segments, soak them in nutrient solution 2, and then insert them into a sterile matrix for cultivation. After the seedling roots are produced and new leaves grow, spray foliar fertilizer and continue to cultivate to obtain production seedlings; 3) taking the healthy production seedlings obtained in step 2), soaking them in nutrient solution 2, and transplanting them to the field for cultivation; The nutrient solution 1 is a solution containing potassium dihydrogen phosphate, brassinolide and indoleacetic acid; The nutrient solution 2 is a solution containing brassinolide, indoleacetic acid and thiophanate-methyl; The matrix is ​​a mixture of medical stone, coal slag and river sand; The foliage fertilizer is an aqueous solution containing radix spatholobi and medium element fertilizer.

2. The method according to claim 1, characterized in that: The nutrient solution 1 is a solution containing 3 g / L potassium dihydrogen phosphate, 0.25 mg / L brassinolide and 0.5 mg / L indoleacetic acid.

3. The method according to claim 1, characterized in that: The nutrient solution 2 is a solution containing 0.25 mg / L brassinolide, 0.5 mg / L indoleacetic acid and 1 g / L thiophanate-methyl.

4. The method according to claim 1, characterized in that: The matrix is ​​a mixture of medical stone, coal slag and river sand in a mass ratio of 7:1:2; the pH value of the mixture is 6.5-7.

5.

5. The method according to claim 1, characterized in that: The foliar fertilizer is an aqueous solution containing 5g / L of mulberry and medium element fertilizer.

6. The method according to claim 5, characterized in that: The mass ratio of the Wangqiu fertilizer to the medium-element fertilizer is 1:

1.

7. The method according to claim 1, characterized in that: Step 1) The acclimation time is 3 days; the spacing between the transplanted tissue culture seedlings is 8 cm×8 cm.

8. The method according to claim 1, characterized in that: Step 2) the tender shoots or stem segments are tender shoots or stem segments with 6-8 leaves; the soaking time is 10 minutes; the culture conditions are: temperature 18-26° C., humidity 75-85%, and avoid direct sunlight.

9. The method according to claim 1, characterized in that: The soaking time in step 3) is 10-15 seconds.

10. The method according to claim 1, characterized in that: The foliar fertilizer in step 1) and step 2) is sprayed once every 14 days.

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