A culture medium, method and application of siler

Abstract

The present invention provides a kind of windproof rapid propagation culture medium, method and application thereof, and belongs to the field of plant tissue culture technology. The windproof rapid propagation culture medium provided by the present invention includes callus induction culture medium, subculture culture medium and rooting culture medium, and the callus induction culture medium is based on MS culture medium and also includes the following mass concentration components: NAA 0.2~0.4mg / L, 6-BA 0.4~0.6mg / L, IBA 0.2~0.4 mg / L, sucrose 30~32g / L and agar 7.0~7.2g / L. The windproof rapid propagation culture medium of the present invention can greatly improve the callus emergence rate, seedling emergence rate, rooting rate and transplant survival rate of windproof tissue culture, so that the callus emergence rate reaches more than 94.5% in the tissue culture process, the seedling emergence rate reaches more than 83.5%, the rooting rate reaches more than 90%, and the rooted seedling transplant survival rate reaches 100%.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a siler rapid propagation culture medium, a method and an application thereof. Background Art

[0002] The quality of the Umbelliferae plant, Saposhnikovia divaricata, is a commonly used medicinal ingredient in Traditional Chinese Medicine (TCM). This quality is crucial for the effectiveness of clinical medications and the development of the TCM industry, and is therefore of particular significance to the modernization and internationalization of TCM. Currently, in-depth research on Saposhnikovia divaricata has been conducted, and related studies have found that it primarily contains polysaccharides, trace elements, organic acids, and other chemical components that play an important role in human immune regulation. Plant tissue culture technology allows for rapid propagation, freeing it from the constraints of natural growth and enabling the improvement of varieties and detoxification of plants. Studies have shown that the explants commonly used for tissue culture of Saposhnikovia divaricata plants are roots and stems, and the ratio of raw materials in the culture medium results in differences in the induction of differentiation. Related reports have found that current Saposhnikovia divaricata plants suffer from a series of problems, including low rooting rates and transplant survival rates. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a rapid propagation culture medium for Saposhnikovia divaricata with high callus emergence rate, seedling emergence rate, rooting rate and transplant survival rate.

[0004] Another object of the present invention is to provide a rapid propagation method for windproof with high callus rate, seedling emergence rate, rooting rate and transplant survival rate.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a rapid propagation culture medium for Saposhnikovia divaricata, comprising a callus induction culture medium, a subculture culture medium and a rooting culture medium. The callus induction culture medium uses an MS culture medium as a basal culture medium and further comprises the following components in mass concentrations: 0.2-0.4 mg / L of naphthaleneacetic acid (NAA), 0.4-0.6 mg / L of 6-benzylaminopurine (6-BA), 0.2-0.4 mg / L of indolebutyric acid (IBA), 30-32 g / L of sucrose and 7.0-7.2 g / L of agar. The pH value of the callus induction culture medium is 6.0-6.2.

[0007] In the present invention, the callus induction culture medium is based on MS medium, and preferably further comprises the following components in mass concentrations: NAA 0.25-0.35 mg / L, 6-BA 0.45-0.55 mg / L, IBA 0.25-0.35 mg / L, sucrose 30 g / L and agar 7.2 g / L, and the pH of the callus induction culture medium is 6.0-6.2.

[0008] In the present invention, the subculture medium is based on MS medium, and preferably further includes the following mass concentration components: NAA 0.2~0.4 mg / L, 6-BA 0.4~0.6 mg / L, IBA 0.2~0.4 mg / L, sucrose 30~32 g / L and agar 7.0~7.2 g / L, and the pH of the subculture medium is 6.0-6.2. It is more preferably further included in the following mass concentration components: NAA 0.25~0.35 mg / L, 6-BA 0.45~0.55 mg / L, IBA 0.25~0.35 mg / L, sucrose 30 g / L and agar 7.2 g / L, and the pH of the subculture medium is 6.0-6.2. In the present invention, the rooting medium uses 1 / 2 MS medium as the basal medium, and preferably further includes the following components by mass concentration: NAA 0.4-0.6 mg / L, IBA 0.4-0.6 mg / L, sucrose 30-32 g / L, and agar 7.0-7.2 g / L. The pH of the rooting medium is 6.0-6.2. More preferably, the rooting medium further includes the following components by mass concentration: NAA 0.45-0.55 mg / L, IBA 0.45-0.55 mg / L, sucrose 30 g / L, and agar 7.2 g / L. The pH of the rooting medium is 6.0-6.2. The present invention does not specifically limit the basal medium and the specific sources of the components in the above-mentioned culture media.

[0009] The present invention also provides application of the above-mentioned siler rapid propagation culture medium in siler rapid propagation.

[0010] The present invention also provides a method for rapid propagation of Saposhnikovia divaricata based on the above-mentioned Saposhnikovia divaricata rapid propagation culture medium, comprising the following steps: using Saposhnikovia divaricata leaves as explants, inoculating them into the induction callus tissue culture medium, and culturing to obtain callus tissue; inoculating the callus tissue into the subculture culture medium, and culturing to obtain regenerated seedlings; inoculating the regenerated seedlings onto the rooting culture medium, and culturing to obtain rooted seedlings.

[0011] In the present invention, the method for obtaining the windproof leaves is preferably: sowing windproof seeds in nutrient soil, taking leaves for cleaning and disinfection after the plants grow, and the cleaning and disinfection method is preferably placing the windproof leaves under running water for 30 minutes to 1 hour, then placing them on a clean bench, and after sterilization, soaking them in 75% ethanol solution for 3 minutes, rinsing them with sterile water for 3-5 times, and then adding 1% sodium hypochlorite solution to soak them for 5 minutes, and then rinsing them with sterile water for 5-6 times.

[0012] In the present invention, the culture conditions are preferably: daytime temperature 25°C, nighttime temperature 15°C, relative humidity 60%, light intensity 600 μmol / (m 2s). The light / dark cycle for culturing callus to obtain callus tissue is preferably 16 h / 8 h, the light / dark cycle for culturing regenerated seedlings is preferably 14 h / 10 h, and the light / dark cycle for culturing rooted seedlings is preferably 14 h / 10 h. During the process of culturing regenerated seedlings of the present invention, the effects of different subculture cycles on callus proliferation are recorded. Depending on the growth status, subculture can be repeated 2-3 times, with subculture every 20 days.

[0013] The method for rapid propagation of Saposhnikovia divaricata of the present invention preferably further comprises the step of transplanting the rooted seedlings into nutrient soil to obtain transplanted seedlings. The present invention has no particular limitation on the specific source of the nutrient soil.

[0014] Beneficial effects of the present invention:

[0015] The use of the windproof rapid propagation culture medium of the present invention can greatly improve the callus emergence rate, seedling emergence rate, rooting rate and transplant survival rate of the windproof tissue culture, so that the callus emergence rate in the tissue culture process reaches more than 94.5%, the seedling emergence rate reaches more than 83.5%, the rooting rate reaches more than 90%, and the rooted seedling transplant survival rate (referring to the survival rate of the transplanted seedlings after the rooted seedlings are transplanted into nutrient soil) reaches 100%.

[0016] The present invention uses radix fangfeng leaves as explant material, combined with the radix fangfeng rapid propagation culture medium provided by the present invention, to successfully establish an efficient regeneration system for radix fangfeng tissue culture of the traditional Chinese medicine, significantly increasing its callus emergence rate, seedling emergence rate, rooting rate, and transplant survival rate. This is of great significance to the protection of radix fangfeng germplasm resources of the traditional Chinese medicine radix fangfeng, and is also conducive to the promotion and application of the radix fangfeng regeneration system of the traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figures are the result of Example 2, wherein Figure A is a schematic diagram of the windproof leaves inoculated on the callus induction culture medium for 3 days, Figure B is a schematic diagram of the windproof leaves inoculated on the callus induction culture medium for 20 days, and Figure C is a schematic diagram of the windproof leaves inoculated on the callus induction culture medium for 25 days; Figure D is a schematic diagram of the callus tissue differentiation into regenerated seedlings at 50 days; Figures E and F are effect diagrams of the regenerated seedlings differentiated from the leaves of two parallel experiments inoculated in the rooting culture medium on the 30th day; Figures G and H are schematic diagrams of the lateral roots and fibrous roots grown from the regenerated seedlings differentiated from the leaves of the two parallel experiments inoculated in the rooting culture medium on the 60th day; Figures I to L are schematic diagrams of the situation where the regenerated seedlings differentiated from the leaves of four parallel experiments were transplanted into the nutrient soil for 3 days.

[0018] Figure 2 The results are as follows: 22 days after the leaves were inoculated into different callus induction culture media, wherein Figure A shows the result of inoculation into Example 2, Figure B shows the result of inoculation into Comparative Example 1, and Figure C shows the result of inoculation into Comparative Example 2.

[0019] Figure 3 The results of Example 6 are shown in Figure 6, where A shows the callus differentiation process in the callus induction medium, with the results at the 10th, 20th, and 50th days from left to right; B shows the callus differentiation in the subculture medium, with the results at the 60th, 80th, and 100th days from left to right; and C shows the root differentiation process of the regenerated seedlings in the rooting medium, with the results at the 110th, 130th, and 150th days from left to right.

[0020] Figure 4 and Figure 5 The rooted seedlings obtained in Example 6 with different root states were selected and transplanted to the external environment for 0-24 days and 3-24 days of growth. DETAILED DESCRIPTION

[0021] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0022] In the following examples, unless otherwise specified, all methods are conventional.

[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0024] Example 1

[0025] A siler rapid propagation medium comprises a callus induction medium, a subculture medium and a rooting medium, wherein the callus induction medium uses MS medium as a basal medium and contains only the following components in mass concentrations: 0.3 mg / L NAA, 0.5 mg / L 6-BA, 0.25 mg / L IBA, 30 g / L sucrose and 7.2 g / L agar, and the pH of the callus induction medium is 6.0.

[0026] The subculture medium is based on MS medium and contains only the following components in mass concentrations: NAA 0.3 mg / L, 6-BA 0.5 mg / L, IBA 0.25 mg / L, sucrose 30 g / L, and agar 7.2 g / L. The pH of the subculture medium is 6.0.

[0027] The rooting medium uses 1 / 2 MS medium as a base medium and only contains the following components in mass concentrations: NAA 0.5 mg / L, IBA 0.5 mg / L, sucrose 30 g / L, and agar 7.2 g / L. The pH of the rooting medium is 6.0.

[0028] The MS medium in the above culture medium was purchased from Beijing Coolaibo Technology Co., Ltd., and the 1 / 2MS medium was purchased from Hangzhou Mobaite Biotechnology Co., Ltd.

[0029] Example 2

[0030] A method for rapid propagation of windbreak:

[0031] (1) Acquisition of sterile materials

[0032] Saposhnikovia seeds were sown in nutrient soil (coconut brick: nutrient soil weight ratio = 2:1) and cultured in Plant Culture Room 606 of the Life Science Building at Qiqihar University. Healthy, well-growing Saposhnikovia plants were selected and their leaves were used as explants. The leaves were rinsed under running water for 30 minutes and set aside. The rinsed leaves, laboratory equipment, sterile water, 75% ethanol solution, alcohol lamp, and culture medium were placed on a clean bench beforehand. Sterilize the leaves before handling, and disinfect your hands with 75% ethanol solution. Soak the rinsed Saposhnikovia leaves in 75% ethanol solution for 3 minutes and rinse three times with sterile water. Repeat this step once (soaking the previously rinsed Saposhnikovia leaves in 75% ethanol solution for 3 minutes and then rinsing three times with sterile water). Add 1% sodium hypochlorite solution and soak for 5 minutes. Rinse five times with sterile water after completion. Place the sterile leaves in a glass dish lined with sterile filter paper.

[0033] (2) Induction of callus tissue

[0034] Light an alcohol lamp and sterilize the tweezers and scalpel with the outer flame of the alcohol lamp. Cool them and set aside. Use a sterile scalpel to separate the windproof leaves and cut them into pieces of about 0.5cm. 2 . Scratch the leaves with a sterile scalpel. Open the pre-configured and autoclaved (121°C 20min) callus induction culture medium in Example 1, and sterilize the cap and mouth of the triangular flask by burning them in the outer flame of an alcohol lamp for 3 seconds. Use sterile tweezers to quickly pick up the cut explants, and quickly put them into the bottle without touching the inner wall. Place 3-5 leaves in each bottle. After the end, rotate the cap and mouth of the bottle around the outer flame of the alcohol lamp for 3 seconds to sterilize them, and tighten the cap. Place it in a day / night temperature of 25°C / 15°C, relative humidity of 60%, light / darkness of 16h / 8h, and light intensity of 600μmol / (m 2 .s) culture rack. The schematic diagrams of the inoculation of the wind blade leaves on the callus induction medium in Example 1 at 3d, 20d and 25d are shown as follows. Figure 1 As shown in A, B and C.

[0035] (3) Subculture of callus tissue

[0036] Place the autoclaved scalpel, tweezers, a glass dish filled with filter paper, an alcohol lamp, and a culture medium containing callus tissue in a clean bench in advance. Sterilize for 20 minutes and ventilate for 10 minutes before operating in the clean bench. After disinfecting your hands and lighting the alcohol lamp, sterilize the tweezers and scalpel with the outer flame of the alcohol lamp and cool them for later use. Use sterile tweezers to remove the windproof callus tissue induced in step (2), place the well-growing callus tissue in a pre-sterilized glass dish filled with filter paper, and place it near the alcohol lamp. Use a sterile scalpel to cut the loose callus tissue into 0.5 cm 3 Small pieces. Open the pre-configured and sterilized (121°C 20min) triangular flask containing the subculture medium in Example 1, and rotate and burn the bottle cap and bottle mouth under the outer flame of an alcohol lamp for 3 seconds. Use sterile tweezers to quickly pick up the yellow-green callus tissue, and place it on the subculture medium in Example 1 for subculture without touching the inner wall. After the end, rotate the bottle cap and bottle mouth around the outer flame of the alcohol lamp for 3 seconds to burn and sterilize, and tighten the bottle cap. Place it in a day / night temperature of 25°C / 15°C, relative humidity of 60%, light / darkness of 14h / 10h, and light intensity of 600μmol / (m 2 .s) culture rack to obtain regenerated seedlings. The schematic diagram of callus differentiation into regenerated seedlings on the 50th day after the explant was inoculated into the induced callus is shown in the figure. Figure 1 As shown in D.

[0037] (4) Rooting culture of regenerated seedlings

[0038] When the seedlings grow to 3.0-4.0cm in height, carry out rooting culture. Pre-place the high-pressure sterilized tweezers, a glass dish filled with filter paper, an alcohol lamp, a culture medium containing the regenerated seedlings of Saposhnikovia divaricata on the clean bench. After sterilization for 20 minutes, ventilate for 10 minutes and enter the clean bench for operation. Disinfect your hands, light the alcohol lamp, sterilize the tweezers with the outer flame of the alcohol lamp, and cool for use. Open the pre-configured and sterilized (121℃ 20min) triangular flask containing the rooting culture medium in Example 1. Rotate and burn the bottle cap and bottle mouth for 3 seconds under the outer flame of the alcohol lamp. Use sterile tweezers to separate the Saposhnikovia divaricata plants in the triangular flask into individual seedlings. Quickly take out the single seedling in the bottle, and transfer the well-growing seedlings into the rooting culture medium for cultivation without touching the inner wall. After the end, rotate the bottle cap and bottle mouth around the outer flame of the alcohol lamp for 3 seconds to burn and sterilize, and tighten the bottle cap. Place it in a day / night temperature of 25℃ / 15℃, relative humidity of 60%, light / darkness of 14h / 10h, and light intensity of 600μmol / (m 2 .s) culture rack to obtain rooted seedlings. The regenerated seedlings differentiated from the leaves of two parallel experiments were inoculated into the rooting medium on the 30th day. Figure 1The regenerated seedlings differentiated from the leaves of the two parallel experiments were inoculated into the rooting medium and grew lateral roots and fibrous roots on the 60th day. Figure 1 As shown in G and H.

[0039] (5) Transplantation and cultivation of rooted seedlings

[0040] Carefully remove the windbreak plants that are in good growth condition, have relatively thick main roots and relatively dense lateral roots from the rooting medium, remove the culture medium at the base of the stem, and place them under running water to continuously rinse the residual culture medium and differentiated callus tissue on the roots. After rinsing, transplant them into seedling pots filled with nutrient soil (the weight ratio of coconut bricks to nutrient soil is 2:1), and plant 1-3 plants in each pot. After transplanting, spray a little water with a watering can to maintain humidity and avoid wilting. The schematic diagram of the situation in which the regenerated seedlings differentiated from the leaves in the four parallel experiments were transplanted into the nutrient soil for 3 days is shown as follows Figure 1 As shown in I to L.

[0041] Comparative Example 1

[0042] The difference from Example 2 is that the composition ratio of the callus induction culture medium used in step (2) is MS basal medium + 0.8 mg / L NAA + 1.0 mg / L 6-BA + 0.8 / mg / L IBA + 30 g / L sucrose + 7.2 g / L agar, the pH value is 6.0, and the rest are the same as Example 2.

[0043] Comparative Example 2

[0044] The difference from Example 2 is that the composition ratio of the callus induction culture medium used in step (2) is MS basal medium + 1 mg / L 2,4-dichlorophenoxyacetic acid (2.4D) + 1 mg / L 6-BA + 1 mg / L kinetin (KT) + 30 g / L sucrose + 7.2 g / L agar, the pH value is 6.0, and the rest are the same as Example 2.

[0045] Example 3

[0046] At 20 days and 22 days after induction in step (2) of Example 2, Comparative Example 1 and Comparative Example 2, the callus induction rates of different groups were calculated. The callus induction rate = (number of differentiated callus tissues / number of inoculated tissues) × 100%. The results are shown in Table 1, indicating that the callus induction culture medium of the present invention has a better effect and the callus induction rate within two days is improved more quickly. The schematic diagrams of leaves with the same characteristics being inoculated in the callus induction culture medium of Example 2, Comparative Example 1 and Comparative Example 2 on the 22nd day are shown in Table 1. Figure 2 As shown in Figures A to C in.

[0047] Table 1 Statistical results of cure rates in different groups

[0048] Group 20-day cure rate (%) 22-day cure rate (%) Example 2 35.668 54.92 Comparative Example 1 21.5 30.92 Comparative Example 2 27.388 36.6

[0049] Comparative Example 3

[0050] The difference from Example 2 is that the composition ratio of the subculture medium used in step (3) is MS basal medium + 0.8 mg / L NAA + 1.0 mg / L 6-BA + 0.8 mg / L IBA + 30 g / L sucrose + 7.2 g / L agar, the pH value is 6.0, and the rest is the same as Example 2.

[0051] Comparative Example 4

[0052] The difference from Example 2 is that the composition ratio of the subculture medium used in step (3) is MS basal medium + 1 mg / L 2.4D + 1 mg / L 6-BA + 1 mg / L KT + 3% sucrose + 0.9% agar, the pH value is 6.0, and the rest are the same as Example 2.

[0053] Comparative Example 5

[0054] The difference from Example 2 is that the composition ratio of the rooting medium used in step (4) is MS basal medium + 0.4 mg / L NAA + 0.2 mg / L 6-BA + 30 g / L sucrose + 7.2 g / L agar, the pH value is 6.0, and the rest are the same as Example 2.

[0055] Comparative Example 6

[0056] The difference from Example 2 is that the composition ratio of the rooting medium used in step (4) is 1 / 2MS basal medium + 0.1mg / L NAA + 0.1mg / L 6-BA + 30g / L sucrose + 7.2g / L agar, the pH value is 6.0, and the rest are the same as Example 2.

[0057] Example 4

[0058] When the culture was carried out in step (4) of Example 2, Comparative Example 5 and Comparative Example 6 for 60 days, the rooting rates of different groups were counted. Rooting rate = (number of rooted adventitious buds / total number of inoculated adventitious buds) × 100%. The results are shown in Table 2.

[0059] Table 2 Rooting rate statistics of different groups

[0060] Group Rooting rate (%) Example 2 90 Comparative Example 5 23.58 Comparative Example 6 30.268

[0061] Example 5

[0062] A siler rapid propagation medium comprises a callus induction medium, a subculture medium and a rooting medium, wherein the callus induction medium uses MS medium as a basal medium and contains only the following components in mass concentrations: 0.3 mg / L NAA, 0.5 mg / L 6-BA, 0.3 mg / L IBA, 32 g / L sucrose and 7.0 g / L agar, and the pH of the callus induction medium is 6.0.

[0063] The subculture medium is based on MS medium and contains only the following components in mass concentrations: NAA 0.3 mg / L, 6-BA 0.5 mg / L, IBA 0.3 mg / L, sucrose 32 g / L, and agar 7.0 g / L. The pH of the subculture medium is 6.0.

[0064] The rooting medium uses 1 / 2 MS medium as a basic medium and only contains the following components in mass concentrations: NAA 0.6 mg / L, IBA 0.5 mg / L, sucrose 32 g / L, and agar 7.0 g / L. The pH of the rooting medium is 6.0.

[0065] The components of the MS medium and 1 / 2MS medium in the above culture medium are the same as those in Example 1.

[0066] Example 6

[0067] A method for rapid propagation of Saposhnikovia divaricata, which differs from Example 2 in that the culture medium used is the callus induction culture medium, subculture culture medium and rooting culture medium in Example 5, and the remaining steps are the same as in Example 2. The callus differentiation process using the callus induction culture medium described in Example 5 is as follows Figure 3 As shown in A in FIG, the differentiation of callus tissue subcultured using the subculture medium described in Example 5 is shown in FIG. Figure 3 As shown in B, the root differentiation process of the regenerated seedlings rooting culture using the rooting medium described in Example 5 is as follows Figure 3 As shown in C.

[0068] In the transplanting and cultivation steps of the rooted seedlings, the rooted seedlings with green roots and short root systems (three parallel tests) and the rooted seedlings with purple roots, thicker shapes and more developed root systems (three parallel tests) were selected and transplanted into the nutrient soil respectively. The other steps in the two groups were the same. The growth of the two groups of rooted seedlings transplanted into the nutrient soil was observed in each group. The results were as follows: Figure 4 (corresponding to the results of rooted seedlings with green roots and short root systems) and Figure 5 (The results for the rooted seedlings with purple-red roots, relatively thick shapes and relatively developed root systems) show that the growth states of roots with different shapes are different when transplanted into nutrient soil.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A rapid propagation culture medium for Saposhnikovia divaricata, characterized in that: The invention comprises a callus induction medium, a subculture medium and a rooting medium, wherein the callus induction medium comprises an MS medium and the following components in mass concentrations: 0.2-0.4 mg / L NAA, 0.4-0.6 mg / L 6-BA, 0.2-0.4 mg / L IBA, 30-32 g / L sucrose and 7.0-7.2 g / L agar, and the pH of the callus induction medium is 6.0-6.2; The subculture medium is composed of MS medium and the following components in mass concentrations: NAA 0.2-0.4 mg / L, 6-BA 0.4-0.6 mg / L, IBA 0.2-0.4 mg / L, sucrose 30-32 g / L, and agar 7.0-7.2 g / L, and the pH of the subculture medium is 6.0-6.2; The rooting medium consists of 1 / 2MS medium and the following components in mass concentrations: 0.4-0.6 mg / L NAA, 0.4-0.6 mg / L IBA, 30-32 g / L sucrose, and 7.0-7.2 g / L agar. The pH of the rooting medium is 6.0-6.

2.

2. Use of the rapid propagation culture medium of Saposhnikovia divaricata according to claim 1 in the rapid propagation of Saposhnikovia divaricata.

3. A method for rapid propagation of Saposhnikovia divaricata based on the Saposhnikovia divaricata rapid propagation medium according to claim 1, characterized in that: The following steps are involved: The windproof leaves are used as explants and inoculated into the callus induction culture medium to obtain callus tissue; the callus tissue is inoculated into the subculture culture medium to obtain regenerated seedlings; the regenerated seedlings are inoculated into the rooting culture medium to obtain rooted seedlings.

4. The method according to claim 3, characterized in that The culture conditions are: daytime temperature 25°C, nighttime temperature 15°C, relative humidity 60%, light intensity 600 μmol / (m 2 .s).

5. The method according to claim 3, characterized in that The light / dark conditions for obtaining callus tissue were 16 h / 8 h, the light / dark conditions for obtaining regenerated seedlings were 14 h / 10 h, and the light / dark conditions for obtaining rooted seedlings were 14 h / 10 h.

6. The method according to claim 3, characterized in that The method also includes the following steps: transplanting the rooted seedlings into nutrient soil to obtain transplanted seedlings.

7. The method according to claim 3, characterized in that The method for obtaining the windproof leaves is as follows: sowing windproof seeds in nutrient soil, and taking leaves after the plants grow into plants, washing and disinfecting them.

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