A method for tissue culture and rapid propagation of honey locust stem segments

Abstract

The application discloses a Gleditsia delavayi stem segment tissue culture and rapid propagation method and relates to the technical field of biology. The Gleditsia delavayi stem segment tissue culture and rapid propagation method comprises the following steps: after disinfection treatment of Gleditsia delavayi stem segments, obtaining the disinfection external explants; inoculating the disinfection external explants into a primary culture medium to perform cluster bud induction culture; inoculating the cluster buds obtained through the cluster bud induction culture into a subculture and proliferation culture medium to perform subculture and proliferation culture; inoculating the adventitious buds obtained through the subculture and proliferation culture into a rooting culture medium to perform rooting culture; inoculating the seedlings obtained through the rooting culture into a strong seedling culture medium to perform strong seedling culture, and obtaining Gleditsia delavayi tissue culture seedlings. The method can solve the browning problem in the Gleditsia delavayi stem segment tissue culture, and has the advantages of high cluster bud induction rate, rooting rate and seedling survival rate and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a tissue culture and rapid propagation method for honey locust stem segments. Background Art

[0002] Honey locust (Gleditsia sinensis) is a widely distributed native tree species in my country, boasting significant economic, ecological, and landscape benefits. It is a highly desirable species worthy of protection, development, and utilization. Honey locust trees can be used as ecological afforestation species, as well as cultivated as fruit trees and woody Chinese medicinal materials. They are widely used in the cultivation of carbon sink forests, ecological forests, and economic forests. Honey locust seeds contain a rich gum, with the endosperm containing up to 68.6% gum. This gum rapidly dissolves in water, forming a stable, high-viscosity solution. This gum can be used as a thickener, stabilizer, or adhesive, and is widely used in industries such as oil, gas, textiles, and papermaking. Currently, domestic research on honey locust (Gleditsia sinensis) primarily focuses on its medicinal properties, chemical composition analysis, and primary utilization value. However, few reports exist on tissue culture of honey locust stem segments. In recent years, tissue culture of honey locust (Gleditsia sinensis) has primarily used immature or mature embryos, cotyledons, young stem segments, or axillary buds to induce bud germination, proliferation, and rooting. However, successful results have been limited, primarily due to the severe browning problem. The frequency of adventitious buds produced by culturing honey locust stem segments as explants is low, the stems easily turn black and brown, and the survival rate is low. The browning of woody plants in tissue culture is mainly due to phenol contamination. Phenolic compounds secreted from the vacuoles at the wound of the explant come into contact with polyphenol oxidase in the cytoplasm. After an oxidation reaction occurs under the action of polyphenol oxidase, quinone compounds are formed. Under the action of tyrosinase, they polymerize with proteins, leading to metabolic disorders in other enzyme systems, thereby affecting plant growth; the second is due to unfavorable environmental conditions (such as temperature, pH value, etc.). How to solve the problem of high browning rate in tissue culture of honey locust stem segments has become an important direction of current research and development. Summary of the Invention

[0003] The present invention aims to provide a method for rapid propagation of honey locust stem segments by tissue culture to solve the problems of the prior art. The method can solve the browning problem in tissue culture of honey locust stem segments and has the advantages of high bud induction rate, rooting rate and seedling survival rate.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a tissue culture and rapid propagation method for honey locust stem segments, comprising the following steps:

[0006] After sterilizing the stem segments of honey locust, sterilized explants are obtained;

[0007] Inoculating the sterilized explants into a primary culture medium for cluster bud induction culture;

[0008] Inoculating the clustered buds obtained by the clustered bud induction culture into a subculture proliferation culture medium for subculture proliferation culture;

[0009] inoculating the adventitious buds obtained from the subculture proliferation culture into a rooting medium for rooting culture;

[0010] Inoculating the seedlings obtained through the rooting culture into a seedling-strengthening culture medium for seedling-strengthening culture to obtain Gleditsia sinensis tissue culture seedlings;

[0011] The primary culture medium is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+IBA 0.5 mg / L;

[0012] The secondary proliferation medium is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+GA3 0.8 mg / L;

[0013] The rooting medium is 1 / 2MS+IBA 1.2 mg / L+zeatin 0.15 mg / L.

[0014] Furthermore, the seedling-strengthening culture medium is MS+6-BA 1.5 mg / L+IBA 0.1 mg / L.

[0015] Furthermore, the honey locust stem segment is a honey locust stem segment with axillary buds.

[0016] Furthermore, the disinfection method includes the step of disinfecting with 0.1% mercuric chloride solution for 10 minutes.

[0017] Furthermore, the culture temperature of the cluster bud induction culture is 25° C., the light intensity is 15 h / d, and the culture time is 30 days.

[0018] Furthermore, the culture temperature of the subculture proliferation culture is 25° C., the illumination time is 15 h / d, and the culture time is 30 days.

[0019] Furthermore, the culture temperature of the rooting culture is 25° C., the light exposure time is 15 h / d, and the culture time is 35 days.

[0020] Furthermore, the culture temperature of the seedling culture is 25°C, the light exposure time is 15h / d, and the culture time is 30d.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses honey locust stem segments as explants and screens out the optimal combination (culture medium + agar + sucrose + hormones) for tissue culture of honey locust stem segments through experiments with different culture media, different hormone types and combinations, and different concentrations of plant growth regulators. This solves the browning problem in tissue culture of honey locust stem segments. The method also has advantages such as a high bud induction rate, rooting rate, and seedling survival rate.

[0023] The invention has important significance for promoting and accelerating the popularization and application of excellent new varieties of honey locust, carrying out the protection, development and utilization of germplasm resources of honey locust tree species and enriching the gardening tree species in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 Schematic diagram of the cultivation process of Example 2; wherein A is a diagram of the state of the stem segment of the soap locust tree after disinfection and inoculation into the primary culture medium; B is a schematic diagram of the germination of the stem segment; C is a schematic diagram of the growth of callus tissue; D is a schematic diagram of the proliferation of callus tissue; E is a schematic diagram of the seedlings in the rooting culture stage; F is a schematic diagram of the seedlings in the seedling cultivation stage. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Terminology Notes:

[0032] IBA refers to 3-indolebutyric acid;

[0033] 6-BA refers to 6-benzylaminopurine;

[0034] NAA refers to naphthaleneacetic acid;

[0035] GA3 refers to gibberellin;

[0036] KT refers to kinetin;

[0037] AC refers to adenylate cyclase;

[0038] 2,4-D refers to 2,4-dichlorophenoxyacetic acid.

[0039] The MS, 1 / 2MS and WPM culture media used in the following examples were purchased from Beijing Solebow Technology Co., Ltd.

[0040] Example 1

[0041] 1. Materials and Methods

[0042] 1. Plant Materials

[0043] The stem segment material used in the present invention is cut from a honey locust branch that is in good growth condition and free of pests and diseases. The dust attached to the surface of the honey locust branch is first gently removed with a brush, and then rinsed with running water for 1 hour, and continuously shaken and washed. After that, the leaves and petioles on the branch are first cut off, and then the branch is cut into 2-3 cm long stem segments (i.e., explants), each stem segment retaining 1-2 axillary buds, for research on a tissue culture in vitro rapid propagation system.

[0044] 2. Tissue culture method

[0045] 2.1 Screening of disinfection method

[0046] After sterilization for 30 min by ultraviolet lamp of the super-clean workbench, the stem segments of Gleditsia sinensis L. were placed in it, and then were sterilized for 30 s by 75% alcohol, washed for 1 min by shaking with sterile water, and sterilized by 0.1% mercury or 2% sodium hypochlorite solution. The sterilization time of 0.1% mercury was set to be 8, 10, 12 and 15 min respectively, and the sterilization time of sodium hypochlorite was set to be 10, 15, 20 and 25 min respectively. After treatment by 0.1% mercury or sodium hypochlorite, the stem segments were washed for 3 times by sterile water, and the whole process was shaken by hand to make the stem segments fully washed. Then, the stem segments were placed on sterile filter paper to absorb surface moisture, and were inoculated into blank MS medium. After sterilization treatment of the stem segments of Gleditsia sinensis L., 12 bottles were inoculated for each treatment, and the whole process was repeated for 3 times. After inoculation and culture of the stem segments for 30 d, the contamination rate and the germination rate were counted, so as to screen out the best sterilization time and determine the best disinfection method.

[0047] 2.2 Screening of basic medium

[0048] After sterilization of the stem segments of Gleditsia sinensis L., the stem segments with good growth state were selected and inoculated into three kinds of culture media (named as medium A, B and C) for test. 30 explants were inoculated for each medium, and the whole process was repeated for 3 times. The growth state of the buds was recorded every 7 d, and the induction rate was counted after culture for 30 d, so as to screen out the most suitable basic medium for axillary bud induction.

[0049] Medium A: MS + sucrose 30 g / L + agar 8.0 g / L + IBA 0.20 mg / L + 6-BA 0.5 mg / L;

[0050] Medium B: 1 / 2MS + sucrose 30 g / L + agar 8.0 g / L + IBA 0.20 mg / L + 6-BA 0.5 mg / L;

[0051] Medium C: WPM + sucrose 30 g / L + agar 8.0 g / L + IBA 0.20 mg / L + 6-BA 0.5 mg / L.

[0052] 2.3 Primary culture of explants

[0053] Based on the selected basal medium, a three-factor, three-level, completely randomized experiment was conducted using 6-BA, NAA, and IBA. 6-BA concentrations were set at 0.5, 1.0, and 1.5 mg / L, NAA at 0.05, 0.1, and 0.2 mg / L, and IBA at 0.1, 0.3, and 0.5 mg / L. Each basal medium was supplemented with 8 g / L agar and 30 g / L sucrose, and the pH of the medium was adjusted to 5.8. Twenty explants were treated with each concentration, and each experiment was replicated three times. Growth was observed and recorded after 30 days, and the induction rate of clustered shoots was calculated to determine the optimal hormone concentration combination for primary culture.

[0054] The culture conditions for primary culture were as follows: culture in a tissue culture room at a temperature of 25°C and a light intensity of 15h / d.

[0055] 2.4 Subculture proliferation

[0056] The uniformly growing and robust clustered shoots induced in the induction medium were cut into single shoots and inoculated into the subculture proliferation medium for cultivation. The subculture proliferation culture used MS as the basal medium, and different mass concentrations of 6-BA (0.5, 1.0, 1.5 mg / L), NAA (0.1, 0.3, 0.6 mg / L) and GA3 (1.0, 1.5, 2 mg / L) were added, and the pH of the culture medium was adjusted to 5.8. According to the three-factor three-level orthogonal design experiment, the elongation growth of the young shoots and the number of adventitious shoots produced were observed with a subculture cycle of 30 days. The optimal hormone concentration combination was screened, the average length of the new shoots was counted, and the proliferation coefficient was calculated.

[0057] Subculture proliferation culture conditions: culture in a tissue culture room at a temperature of 25℃ and a light intensity of 15h / d.

[0058] 2.5 Rooting culture

[0059] When adventitious buds grew to over 2 cm, selected well-growing buds were inoculated into rooting medium for rooting culture. The rooting medium consisted of MS, 1 / 2MS, and WPM as the basic medium, supplemented with varying concentrations of IBA (0.8 mg / L, 1.2 mg / L, and 2.0 mg / L) and zeatin (0.1 mg / L, 0.15 mg / L, and 0.2 mg / L), and the pH of the medium was adjusted to 5.8. The control group consisted of MS, 1 / 2MS, and WPM culture medium without hormones. After 35 days of rooting culture, the number of roots and root length of the plants were counted, and the rooting rate and average root number were calculated.

[0060] Rooting culture conditions: Culture in a tissue culture room at a temperature of 25°C and a light intensity of 15h / d.

[0061] 2.6 Seedling cultivation

[0062] The seedlings after 30 days of rooting culture were inoculated into the seedling culture medium for seedling culture. The seedling culture medium was based on MS, and 6-BA and GA3 were added to adjust the pH of the culture medium to 5.8. The experimental design was as follows: 6-BA (1 mg / L,

[0063] 1.5 mg / L, 2 mg / L) and IBA (0.1 mg / L, 0.2 mg / L) were randomly combined, for a total of 6 combinations, 12 explants were inoculated in each treatment, repeated 3 times, and the seedling growth was observed after 30 days of culture and the seedling height was counted.

[0064] Conditions for strong seedling culture: culture in a tissue culture room with a temperature of 25℃ and a light intensity of 15h / d.

[0065] 2. Results

[0066] 2.1 Determination of disinfection method

[0067] The statistical results of the contamination rate and survival rate of the stem segments of honey locust after different disinfection treatments are shown in Table 1. Based on the statistical results of contamination rate and survival rate, the disinfection treatment method was selected as follows: 75% alcohol disinfection for 30 seconds + 0.1% mercuric chloride disinfection for 10 minutes.

[0068] Table 1 Contamination rate and survival rate of honey locust stem segments after different disinfection treatments

[0069]

[0070] 2.2 Effect of basic culture medium on axillary bud induction rate

[0071] After using different basic culture media to inoculate the stem segments of honey locust for culture, the cluster bud induction rates were calculated. The results are shown in Table 2.

[0072] Table 2 Cluster bud induction rate of honey locust stem segments cultured on different basic media

[0073] Basic culture medium Cluster bud induction rate MS 88.14 1 / 2MS 75.18 WPM 52.58

[0074] 2.3 Effects of different hormone combinations on primary culture

[0075] Based on the selected basic culture medium, a three-factor, three-level, completely randomized experiment with 6-BA, NAA, and IBA was conducted to determine the optimal hormone concentration combination for primary culture. The results are shown in Table 3. The results showed that the optimal cluster bud induction rate was achieved when the concentrations of 6-BA, NAA, and IBA were 1.0 mg / L, 0.1 mg / L, and 0.5 mg / L, respectively.

[0076] Table 36 - Results of a 3-factor 3-level completely randomized trial of BA, NAA, and IBA

[0077]

[0078] 2.4 Effects of different hormone combinations on subculture proliferation

[0079] Subculture proliferation was performed using MS as the basal medium, and a three-factor, three-level orthogonal experiment was conducted with 6-BA, NAA, and GA3 to determine the optimal hormone concentration combination for subculture proliferation. The results are shown in Table 4. The results showed that the optimal number of adventitious buds was obtained when the concentrations of 6-BA, NAA, and GA3 were 1.0 mg / L, 0.1 mg / L, and 0.8 mg / L, respectively.

[0080] Table 46 - Results of 3-factor 3-level orthogonal test of BA, NAA and GA3

[0081]

[0082] 2.5 Effects of different rooting media on rooting culture

[0083] Rooting cultures were performed using MS, 1 / 2MS, and WPM as basal media, supplemented with varying concentrations of IBA and zeatin. The optimal medium for rooting was determined, as shown in Table 5. Based on the statistical results of rooting rate and root length, the rooting medium selected was: 1 / 2MS + 1.2 mg / L IBA + 0.15 mg / L zeatin.

[0084] Table 5 Rooting rate and root length of different rooting medium treatment groups

[0085]

[0086]

[0087] 2.6 Effects of different hormone combinations on seedling cultivation

[0088] Using MS as the basic culture medium, the effects of different hormone combinations on seedling cultivation were investigated. The results are shown in Table 6. The results showed that when the concentrations of 6-BA and IBA were 1.5 mg / L and 0.1 mg / L, respectively, the seedling cultivation effect was the best.

[0089] Table 6 Seedling height after treatment with different hormone combinations

[0090] deal with Culture medium type 6-BA (mg / L) IBA (mg / L) Seedling height (cm) D1 MS 1 0.1 3.59 D2 MS 1 0.2 4.04 D3 MS 1.5 0.1 5.64 D4 MS 1.5 0.2 5.15 D5 MS 2 0.1 4.38 D6 MS 2 0.2 4.69

[0091] Example 2

[0092] 1. Culture medium

[0093] Primary culture medium: MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+IBA 0.5 mg / L;

[0094] Subculture proliferation medium: MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+GA3 0.8 mg / L;

[0095] Rooting medium: 1 / 2MS + IBA 1.2 mg / L + zeatin 0.15 mg / L;

[0096] Seedling growth medium: MS+6-BA 1.5mg / L+IBA0.1mg / L.

[0097] 2. Culture Methods

[0098] (1) After sterilization under ultraviolet light for 30 min, the stem segments of the honey locust collected and cut in Example 1 were placed in the ultraclean bench, disinfected with 75% alcohol for 30 s, rinsed with sterile water three times, and then disinfected with 0.1% mercuric chloride solution for 10 min, and rinsed with sterile water three times to obtain sterilized stem segments, which were used as explants for subsequent culture.

[0099] (2) Cluster bud induction culture: The sterilized explants were inoculated into the primary culture medium for cluster bud induction culture. The culture temperature was 25°C, the light duration was 15 h / d, and the culture time was 30 days.

[0100] (3) Subculture proliferation culture: The cluster buds obtained by the cluster bud induction culture in step (2) are inoculated into the subculture proliferation culture medium for subculture proliferation culture. The culture temperature is 25°C, the light duration is 15h / d, and the culture time is 30d.

[0101] (4) Rooting culture: The adventitious buds that grew to more than 2 cm obtained in the subculture proliferation culture in step (3) were inoculated into the rooting medium for rooting culture. The culture temperature was 25°C, the light duration was 15 h / d, and the culture time was 35 days.

[0102] (5) Seedling cultivation: The seedlings obtained after rooting culture were inoculated into the seedling cultivation medium for seedling cultivation. The cultivation temperature was 25°C, the light duration was 15 h / d, and the cultivation time was 30 days.

[0103] (6) Hardening and transplanting: The seedlings obtained from the seedling hardening culture in step (5) were transplanted into the field soil in a plastic greenhouse, and the survival rate was calculated 40 days after planting.

[0104] The schematic diagram of the cultivation process of this embodiment is shown in Figure 1 .

[0105] Comparative Example 1

[0106] 1. Culture medium

[0107] Primary culture medium: MS + NAA 0.1 mg / L + 6-BA 4.0 mg / L + KT 1.4 mg / L + AC 0.6 g / L + sucrose 24 g / L + agar 4.8 g / L, pH 5.6.

[0108] Subculture proliferation medium: MS + 2,4-D 0.5 mg / L + 6-BA 3.9 mg / L + NAA 0.4 mg / L + AC 0.8 g / L + sucrose 24 g / L + agar 4.9 g / L, pH 5.6.

[0109] The rooting medium was: MS + NAA 0.2 mg / L + IBA 1.2 mg / L + sucrose 16 g / L + agar 3.8 g / L, pH 5.6.

[0110] Seedling growth medium: MS+6-BA 1.5mg / L+IBA0.1mg / L.

[0111] 2. Culture Methods

[0112] (1) After sterilization under ultraviolet light for 30 minutes, the stem segments of honey locust collected and cut in Example 1 were placed in the ultraclean bench, soaked in 75% ethanol solution for 18 seconds, rinsed with sterile water 4 times, and then disinfected with 0.3% mercuric chloride solution for 4 minutes. After rinsing with sterile water, the sterilized stem segments were obtained and used as explants for subsequent culture.

[0113] (2) Cluster bud induction culture: The sterilized explants were inoculated into primary culture medium for cluster bud induction culture. After inoculation, the culture was placed under conditions of 15 hours of light per day, 1900 lx, a culture temperature of 23°C, and a relative humidity of 75% for 28 days.

[0114] (3) Subculture: The buds obtained by induction culture in step (2) are inoculated into a subculture proliferation medium for subculture proliferation. After inoculation, the culture medium is placed under conditions of 10 hours of light per day, a light intensity of 1200 lx, a culture temperature of 23°C, and a relative humidity of 75% for 38 days.

[0115] (4) Rooting culture: The adventitious buds that grew to more than 2 cm obtained from the subculture proliferation culture in step (3) were inoculated into a rooting medium for rooting culture. After inoculation, the culture medium was placed under conditions of 12 hours of light per day, 1800 lx of light intensity, a culture temperature of 23°C, and a relative humidity of 75% for 32 days.

[0116] (5) Seedling cultivation: The seedlings obtained after rooting culture were inoculated into the seedling cultivation medium for seedling cultivation. The cultivation temperature was 25°C, the light duration was 15 h / d, and the cultivation time was 30 days.

[0117] (6) Hardening and transplanting: The seedlings obtained from the seedling hardening culture in step (5) were transplanted into the field soil in a plastic greenhouse, and the survival rate was calculated 40 days after planting.

[0118] The cluster bud induction rate, the browning rate in the subculture proliferation culture stage, the rooting rate in the rooting culture stage and the final seedling survival rate of Example 2 and Comparative Example 1 were statistically analyzed. The results are shown in Table 7.

[0119] Cluster bud induction rate = number of explants induced to produce cluster buds / total number of explants inoculated for cluster bud induction culture × 100%;

[0120] Browning rate = number of browning buds / total number of inoculated buds × 100%;

[0121] Rooting rate = number of induced rooting adventitious buds / total number of inoculated adventitious buds × 100%;

[0122] Survival rate = number of surviving seedlings / total number of transplanted seedlings × 100%.

[0123] Table 7 Cluster bud induction rate, browning rate, rooting rate and seedling survival rate of Example 2 and Comparative Example 1

[0124] Example / Comparative Example Cluster bud induction rate Browning rate Rooting rate Seedling survival rate Example 2 98％ 15％ 98％ 96.5％ Comparative Example 1 98％ 65％ 95％ 90％

[0125] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for rapid propagation of honey locust stem segments through tissue culture, characterized in that: The following steps are involved: After sterilizing the stem segments of honey locust, sterilized explants are obtained; Inoculating the sterilized explants into a primary culture medium for cluster bud induction culture; Inoculating the clustered buds obtained by the clustered bud induction culture into a subculture proliferation culture medium for subculture proliferation culture; inoculating the adventitious buds obtained from the subculture proliferation culture into a rooting medium for rooting culture; Inoculating the seedlings obtained through the rooting culture into a seedling-strengthening culture medium for seedling-strengthening culture to obtain Gleditsia sinensis tissue culture seedlings; The primary culture medium is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+IBA 0.5 mg / L; The secondary proliferation medium is MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+GA3 0.8 mg / L; The rooting medium is 1 / 2MS+IBA 1.2mg / L+zeatin 0.15mg / L; The seedling-strengthening culture medium is MS+6-BA 1.5mg / L+IBA 0.1mg / L; The honey locust stem segment is a honey locust stem segment with axillary buds; The disinfection method comprises the steps of disinfecting with 0.1% mercuric chloride solution for 10 minutes; The bud induction culture temperature is 25°C, the light intensity is 15h / d, and the culture time is 30d. The culture temperature of the subculture proliferation culture is 25°C, the light exposure time is 15h / d, and the culture time is 30d; The rooting culture temperature is 25°C, the light intensity is 15h / d, and the culture time is 35d; The culture temperature of the seedling culture is 25° C., the light intensity is 15 h / d, and the culture time is 30 d.

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