Ulmus plant tissue culture medium and application thereof

Abstract

The invention discloses an ulmus plant tissue culture medium and application thereof, and belongs to the technical field of tissue culture. The invention provides an ulmus plant tissue culture medium in order to solve the technical problems of low survival rate and poor seedling stress resistance of a general culture medium for woody plants in ulmus plant culture in the prior art. The culture medium is divided into a callus first-stage culture medium, a callus second-stage culture medium, a callus cell tillering culture medium, a bud and leaf growth culture medium and a root growth culture medium. According to the method, a synergistic scheme of berberine culture medium regulation and colchicine polyploidy induction is adopted, and the stress resistance of the ulmus tissue culture seedlings is remarkably improved through a dual mechanism of activating a stress resistance signal channel and inducing chromosome doubling, so that the problems that an existing general culture medium is poor in adaptability, and the tissue culture seedlings are vitrified and yellowed are solved; the efficient and stable factory rapid propagation of the ulmus plants is realized, and the method has important application value.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, and in particular relates to a tissue culture medium for elm plants and its application. Background Technology

[0002] Elm species are important landscaping and timber trees with high ornamental and economic value. Traditional propagation methods such as cuttings and grafting are limited by season and have low propagation coefficients, making it difficult to meet the needs of large-scale production. Although plant tissue culture technology can achieve rapid propagation year-round, constructing a tissue culture system for elm, as a woody plant, is quite challenging. Currently, research on tissue culture of elm species is fragmented, mostly exploring basic culture conditions, and a highly efficient, stable, and industrially applicable general technical system has not yet been formed.

[0003] Existing general culture media for woody plants (such as MS and WPM) show obvious adaptation defects when used in the application of Ulmus species. Key parameters such as inorganic salt concentration, nitrogen form ratio and type of organic additives in conventional culture media are not well matched with the nutritional requirements of Ulmus species, which can easily cause physiological disorders such as vitrification and chlorosis, seriously affecting the propagation efficiency and seedling quality.

[0004] Berberine, also known as berberine alkaloid, is an isoquinoline alkaloid and a major component of traditional Chinese medicines such as Coptis chinensis and Scutellaria baicalensis. It has inhibitory effects on Shigella dysenteriae, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus, and Salmonella typhi. It is generally used clinically as a heat-clearing and detoxifying agent and a broad-spectrum antibacterial agent. However, researchers have recently discovered that berberine can also be used in agriculture as a pesticide antibacterial agent on crops or cash crops. Summary of the Invention

[0005] To address the technical problems of low survival rate and poor seedling resistance in the cultivation of Elm plants using general-purpose culture media for woody plants in the prior art, this invention provides a tissue culture medium for Elm plants and its application.

[0006] One objective of this invention is to provide a tissue culture medium for elm plants, the culture medium comprising a first-stage callus culture medium, a second-stage callus culture medium, a callus cell tillering culture medium, a bud and leaf growth culture medium, and a root growth culture medium.

[0007] In a preferred embodiment of the present invention, the first-stage callus culture medium consists of the following components: MS as the basal medium, 2,4-D 2.0-3.0 mg / L, 6-BA 0.2-0.5 mg / L, berberine 0.1-0.2 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.0.

[0008] In a preferred embodiment of the present invention, the second-stage callus culture medium consists of the following components: MS basal medium, 2,4-D 2.0-3.0 mg / L, 6-BA 0.2-0.5 mg / L, berberine 0.1-0.2 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.0.

[0009] In a preferred embodiment of the present invention, the callus cell tillering culture medium consists of the following components: MS basal medium, 6-BA 1.0-1.5 mg / L, KT 0.5-1.0 mg / L, berberine 0.3-0.4 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.0.

[0010] In a preferred embodiment of the present invention, the bud and leaf growth medium consists of the following components: MS basal medium, 6-BA 0.5-0.8 mg / L, IAA 0.2-0.5 mg / L, berberine 0.4-0.5 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.0.

[0011] In a preferred embodiment of the present invention, the root growth medium is composed of the following components: 1 / 2 MS basal medium, NAA 0.5-1.0 mg / L, berberine 0.3-0.4 mg / L, sucrose 15-25 g / L, agar 6.5-7.5 g / L, pH=5.8-6.0.

[0012] The second objective of this invention is to provide the application of the above-mentioned elm tissue culture medium in elm tissue culture.

[0013] In a preferred embodiment of the present invention, the elm tissue culture medium used in the application can improve the stress resistance of elm tissue culture seedlings, wherein the stress resistance includes at least one of antifungal, antibacterial, drought-resistant, salt-tolerant, and low-temperature tolerant.

[0014] The third objective of this invention is to provide a method for tissue culture of elm plants, the method comprising the following steps: S1: Collect callus tissue from elm plants and disinfect it; S2: The callus tissue obtained in S1 was inoculated onto the first-stage callus culture medium described above and cultured for 14 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the first-stage culture. The material after the first-stage culture was cut off on a sterile operating table and placed on the second-stage callus culture medium described above. It was cultured for 10 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the second-stage culture. S3: The material obtained in S2 after the second stage of culture was cut off on a sterile operating table and placed on the above-mentioned callus cell tillering culture medium. It was cultured for 16 days at a temperature of 25±2℃, a light intensity of 3000-4000lx, and a light duration of 14 h / d to obtain the material after cell tillering culture. The material after cell tillering culture was soaked in colchicine solution and washed 4 times with sterile water before use. S4: The material treated with colchicine in S3 was cut off on a sterile operating table and placed on the above-mentioned bud and leaf growth medium. It was cultured for 20 days under the conditions of temperature 25±2℃, light intensity 3000-4000lx, and light duration 14 h / d to obtain the material after bud and leaf growth culture. S5: The material obtained from the bud and leaf growth culture in S4 was cut off on a sterile operating table and placed on the above-mentioned root growth medium. It was cultured for 18 days at a temperature of 23±2℃, a light intensity of 2000-3000lx, and a light duration of 12 h / d to obtain elm tissue culture seedlings.

[0015] In a preferred embodiment of the present invention, the conditions for the colchicine solution soaking treatment in S3 are: the concentration of colchicine is 0.5%-1.0%, and the soaking time is 24-48 h.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a tissue culture medium for elm plants, wherein the culture medium is divided into a first-stage callus culture medium, a second-stage callus culture medium, a callus cell tillering culture medium, a bud and leaf growth culture medium, and a root growth culture medium.

[0017] This invention innovatively employs a synergistic technical solution of "berberine culture medium regulation + colchicine polyploid induction". In addition to achieving efficient sterilization during tissue culture, the core is to enhance seedling stress resistance through the superposition of dual mechanisms. Berberine significantly enhances plant resistance to biotic and abiotic stresses through the dual mechanisms of activating stress resistance signaling pathways and regulating gene expression. Colchicine induces polyploid formation through chromosome doubling, resulting in a superposition synergistic effect of "physiological regulation + structural optimization", which is significantly superior to single stress resistance technologies.

[0018] Berberine, as a natural alkaloid, exhibits the following resistance to biotic and abiotic stresses: (1) Inducing plant defense against fungi: Berberine activates plant “systemically acquired resistance (SAR)”, induces salicylic acid (SA) accumulation, upregulates PR gene (pathogenesis-related protein genes, such as PR-1 and PR-5) expression, and enhances plant resistance to subsequent fungal infections.

[0019] (2) Inducing plant defense against bacteria: Berberine induces plants to produce reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which activates defense enzyme systems such as peroxidase (POD) and polyphenol oxidase (PPO), enhances the degree of cell wall lignification, and prevents bacterial invasion.

[0020] (3) Drought stress resistance: Berberine enhances the antioxidant system: increases the activity of SOD (superoxide dismutase), CAT (catalase), and APX (ascorbic acid peroxidase), clears drought-induced ROS, and reduces cell membrane lipid peroxidation.

[0021] (4) Salt stress: Berberine promotes the synthesis of proline and soluble protein, and resists cell dehydration caused by salt stress.

[0022] (5) Low temperature stress: Berberine induces the expression of antifreeze genes: upregulates the expression of cold response genes (such as DREB2A, COR15a) and antifreeze protein (AFP) genes, promotes the accumulation of antifreeze proteins, and lowers the freezing point of cells.

[0023] This invention employs a colchicine soaking process in the tissue culture method of Ulmus species, which induces polyploidy and enhances seedling stress resistance. Its core mechanism is polyploidization-mediated structural remodeling, physiological metabolic optimization, and molecular network activation: colchicine inhibits spindle formation and induces chromosome doubling. Polyploid plants enhance their tolerance to stresses such as drought, salinity, low temperature, and pests and diseases through cell structure strengthening (physiological barrier), osmotic regulation and antioxidant system enhancement (physiological metabolism), and stress resistance gene network activation (molecular regulation). Detailed Implementation

[0024] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0026] Example 1: S1: Collect callus tissue from elm plants. First, soak the collected elm callus tissue in a 5% detergent solution for 20 min. After brushing the axillary buds, rinse it under running water overnight. On a sterile operating table, treat it sequentially with 75% alcohol for 30 s, rinse it with sterile water 3 times, treat it with 0.1% mercuric chloride for 10 min, rinse it with sterile water 5 times, and air dry it to obtain callus tissue. S2: The sterilized callus tissue from S1 was inoculated onto the first-stage callus culture medium (the first-stage callus culture medium consists of the following components: MS basal medium, 2,4-D 2.5 mg / L, 6-BA 0.3 mg / L, berberine 0.15 mg / L, sucrose 30 g / L, agar 7 g / L, pH 5.9), and cultured for 14 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the first-stage culture. The material after the first-stage culture was cut off on a sterile operating table and placed on the second-stage callus culture medium (the second-stage callus culture medium consists of the following components: MS basal medium, 2,4-D 1.2 mg / L, 6-BA 0.6 mg / L, berberine 0.25 mg / L, sucrose 30 g / L, agar 7 g / L, pH 5.9). The material was cultured for 10 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the second stage of culture. S3: The material obtained in S2 after the second stage of culture was cut off on a sterile operating table and placed on callus cell tillering medium (the callus cell tillering medium consists of the following components: MS basal medium, 6-BA 1.2 mg / L, KT 0.8 mg / L, berberine 0.35 mg / L, sucrose 30 g / L, agar 7 g / L, pH 5.9). It was cultured for 16 days at a temperature of 25±2℃, a light intensity of 3000-4000 lx, and a light duration of 14 h / d to obtain the material after cell tillering culture. The material was then soaked in 0.08% colchicine solution for 36 h, washed four times with sterile water, and used for later use. S4: The colchicine-treated material from S3 was cut off on a sterile operating table and placed on a bud and leaf growth medium (the bud and leaf growth medium consists of the following components: MS basal medium, 6-BA 0.6 mg / L, IAA 0.3 mg / L, berberine 0.45 mg / L, sucrose 30 g / L, agar 7 g / L, pH 5.9). It was cultured for 20 days at a temperature of 25±2℃, a light intensity of 3000-4000 lx, and a light duration of 14 h / d to obtain the material after bud and leaf growth culture. S5: The material obtained from the bud and leaf growth culture in S4 was cut off on a sterile operating table and placed on root growth medium (the root growth medium consists of the following components: 1 / 2 MS basal medium, NAA 0.8 mg / L, berberine 0.35 mg / L, sucrose 20 g / L, agar 7 g / L, pH 5.9). It was cultured for 18 days at a temperature of 23±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain elm tissue culture seedlings.

[0027] Comparative Example 1: S1: Collect callus tissue from elm plants. First, soak the collected elm callus tissue in a 5% detergent solution for 20 min. After brushing the axillary buds, rinse it under running water overnight. On a sterile operating table, treat it sequentially with 75% alcohol for 30 s, rinse it with sterile water 3 times, treat it with 0.1% mercuric chloride for 10 min, rinse it with sterile water 5 times, and air dry it to obtain callus tissue. S2: The sterilized callus tissue from S1 was inoculated onto conventional MS medium (containing 2,4-D 2.0 mg / L, 6-BA 0.5 mg / L, sucrose 30 g / L, agar 7 g / L, pH 5.8) and cultured for 24 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain callus differentiation material; S3: The callus differentiation material from S2 was inoculated onto conventional shoot differentiation medium (MS basal medium + 6-BA 1.0 mg / L + IAA 0.3 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.8) and cultured for 20 days at a temperature of 25±2℃, a light intensity of 3000-4000 lx, and a light duration of 14 h / d to obtain the shoot-differentiated material; S4: The bud-differentiated material from S3 was inoculated onto conventional rooting medium (1 / 2 MS basal medium + NAA 0.8 mg / L + sucrose 20 g / L + agar 7 g / L, pH 5.8) and cultured for 18 days at a temperature of 23±2℃, a light intensity of 2000-3000 lx, and a photoperiod of 12 h / d to obtain conventional Ulmus tissue culture seedlings.

[0028] Effect Experiment: 1. Salicylic acid content detection This invention employs high-performance liquid chromatography (HPLC) to detect the salicylic acid content in *Ulmus* tissue culture seedlings obtained in Example 1 and conventional *Ulmus* tissue culture seedlings obtained in Comparative Example 1. The instrument used is an Agilent 1260 HPLC system with a C18 column (4.6 mm × 250 mm, 5 μm), a mobile phase of methanol-water-glacial acetic acid (40:60:1, v / v / v), a flow rate of 1.0 mL / min, a detection wavelength of 308 nm, and a column temperature of 30 °C.

[0029] The results are shown in Table 1. The elm plants obtained by activation culture of the elm plant tissue culture medium provided by the present invention exhibited "systematically acquired resistance (SAR)" and significantly increased the accumulation of salicylic acid (SA). Compared with the conventional tissue culture technology in Comparative Example 1, the salicylic acid content of the obtained elm tissue culture seedlings was increased by 42%.

[0030] Table 1

[0031] 2. Detection of expression levels of disease-related protein genes (1) DNA extraction: The genomic DNA of the Ulmus tissue culture seedlings obtained in Example 1 and the conventional Ulmus tissue culture seedlings obtained in Comparative Example 1 was extracted using the TIANGEN Plant Genomic DNA Extraction Kit (product number DP305) according to the kit instructions. (2) Primer design: PR-1 Upstream primer for gene: 5'-ATGGTGCTGCTGCTGCTG-3', SEQ ID NO.1; PR-1 Downstream primer for gene: 5'-TCAGCTGCTGCTGCTGCTT-3', SEQ ID NO.2; PR-5 Upstream primer for gene: 5'-GATGGTGCTGCTGCTGCTG-3', SEQ ID NO.3; PR-5 Downstream primer for gene: 5'-CAGCTGCTGCTGCTGCTTC-3', SEQ ID NO.4; Internal reference gene ( ACTIN Upstream primer: 5'-GATGGTGATGGTGCTGATG-3', SEQ ID NO.5; Internal reference gene ( ACTIN Downstream primer: 5'-TCAGCTGCTGCTGCTGCTT-3', SEQ ID NO.6.

[0032] (3) Amplification system: 10 μL of 2×SYBR Green PCR Master Mix, 0.5 μL of upstream primer (10 μmol / L), 0.5 μL of downstream primer (10 μmol / L), 2 μL of DNA template, 7 μL of ddH2O, and a total system of 20 μL.

[0033] (4) Amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles; melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s.

[0034] The results showed that the elm tissue culture seedlings obtained in Example 1 PR-1、PR-5 The relative gene expression levels were 3.2 times and 3.7 times that of Comparative Example 1, respectively, indicating that the elm tissue culture medium provided by this invention significantly upregulated the expression levels of disease-related protein genes.

[0035] 3. Testing of antibacterial ability Using the pot inoculation method, the *Ulmus* tissue culture seedlings obtained in Example 1 and the conventional *Ulmus* tissue culture seedlings obtained in Comparative Example 1 were transplanted into flowerpots with the same substrate. After 10 days of acclimatization, they were inoculated with a suspension of *Peronobacterium* spores (1×10⁻⁶). 6 (cells / mL) and powdery mildew spore suspension (1×10) 5 (Inoculation rate / mL), after inoculation, the cells were placed in an environment with a temperature of 22±2℃, humidity of 85%, and light intensity of 12 h / d for 7 days, and the leaf infection rate was counted.

[0036] The results are shown in Table 2. The antibacterial ability of the elm tissue culture seedlings obtained by culturing with the elm tissue culture medium provided by the present invention is significantly better than that of the elm tissue culture seedlings obtained by conventional tissue culture techniques.

[0037] Table 2

[0038] 4. Testing of drought stress resistance Superoxide dismutase activity was detected in the tissue culture seedlings of *Ulmus* obtained in Example 1 and the conventional tissue culture seedlings of *Ulmus* obtained in Comparative Example 1 using the nitroblue tetrazolium method, catalase activity was detected using ultraviolet spectrophotometry, and lipid peroxide content was detected using the thiobarbituric acid method. All reagent kits used were purchased from Nanjing Jiancheng Biotechnology Institute.

[0039] Soil moisture content was tested by weighing the elm tissue culture seedlings obtained in Example 1 and the conventional elm tissue culture seedlings obtained in Comparative Example 1. The rhizosphere soil of the seedlings was taken, the fresh weight was weighed, and then dried at 105°C to constant weight. The soil moisture content was then calculated.

[0040] After the seedlings recovered, the elm tissue culture seedlings obtained in Example 1 and the conventional elm tissue culture seedlings obtained in Comparative Example 1 were placed in an environment where the soil moisture content was gradually reduced to 15% and cultured for 15 days. The survival rate was then calculated.

[0041] The results are shown in Table 3. Compared with the conventional tissue culture technique in Comparative Example 1, the superoxide dismutase activity of the elm tissue culture seedlings obtained by culture in the elm tissue culture medium provided by this invention increased by 38%, the catalase activity increased by 32%, the lipid peroxide content decreased by 56%, and the survival rate increased by 63% when the soil moisture content was 15%, indicating that the drought resistance was significantly enhanced.

[0042] Table 3

[0043] 5. Testing of salt stress tolerance Proline content was determined using the acidic ninhydrin colorimetric method, soluble protein content was determined using the Coomassie brilliant blue method (Reference: Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding[J]. Analytical Biochemistry, 1976, 72(1-2):248-254.), and cell osmolarity was determined using the freezing point osmoregometer method; Salt stress treatment: The elm tissue culture seedlings obtained in Example 1 and the conventional elm tissue culture seedlings obtained in Comparative Example 1 after the seedlings had recovered were placed in Hogrange nutrient solution containing 0.9% NaCl and cultured for 10 days. The growth status was observed and relevant indicators were detected.

[0044] The results are shown in Table 4. Compared with the conventional tissue culture technique in Comparative Example 1, the proline synthesis of elm tissue culture seedlings obtained by culturing with the elm tissue culture medium provided by this invention increased by 42%, the soluble protein content increased by 30%, the cell osmotic pressure was significantly increased, and they could grow normally at a concentration of 0.9% NaCl, with a salt tolerance concentration increased by 0.4% compared with the conventional technique.

[0045] Table 4

[0046] 6. Testing of resistance to low-temperature stress (1) Low temperature stress treatment: The elm tissue culture seedlings obtained in Example 1 and the conventional elm tissue culture seedlings obtained in Comparative Example 1 after the seedlings were recovered were placed in a -6℃ low temperature incubator for 24 h. After restoring to normal temperature for 7 days, the survival rate was counted.

[0047] (2) The degree of cell wall lignification was detected by phloroglucinol-hydrochloric acid staining: leaf cross sections were prepared, stained and observed under a microscope, and the proportion of the lignified area to the total cell wall area was measured.

[0048] (3) Seedling robustness rate test: Based on indicators such as seedling height, stem diameter, and root fresh weight, the proportion of seedlings that meet the robustness standard (seedling height ≥8cm, stem diameter ≥0.3cm, root fresh weight ≥0.5g) to the total number of seedlings.

[0049] (4) Gene expression detection DNA extraction: Genomic DNA was extracted from the Ulmus tissue culture seedlings obtained in Example 1 and the conventional Ulmus tissue culture seedlings obtained in Comparative Example 1 using the TIANGEN Plant Genomic DNA Extraction Kit (catalog number DP305) according to the kit instructions.

[0050] Primer design: DREB2A Upstream primer for gene: 5'-GATGGTGCTGCTGCTGCTA-3', SEQ ID NO.7; DREB2A Downstream primer for gene: 5'-TCAGCTGCTGCTGCTGCTG-3', SEQ ID NO.8; COR15a Upstream primer for gene: 5'-ATGGTGCTGCTGCTGCTGC-3', SEQ ID NO.9; COR15a Downstream primer for gene: 5'-CAGCTGCTGCTGCTGCTTG-3', SEQ ID NO.10; AFP Upstream primer for gene: 5'-GATGGTGCTGCTGCTGCTC-3', SEQ ID NO.11; AFP Downstream primer for gene: 5'-TCAGCTGCTGCTGCTGCTA-3', SEQ ID NO.12; Internal reference gene ( ACTIN Upstream primer: 5'-GATGGTGATGGTGCTGATG-3', SEQ ID NO.5; Internal reference gene ( ACTIN Downstream primer: 5'-TCAGCTGCTGCTGCTGCTT-3', SEQ ID NO.6.

[0051] Amplification system: 10 μL 2×SYBR Green PCR Master Mix, 0.5 μL upstream primer (10 μmol / L), 0.5 μL downstream primer (10 μmol / L), 2 μL DNA template, 7 μL ddH2O, total system 20 μL.

[0052] Amplification program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles; melting curve analysis: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s.

[0053] The results are shown in Table 5. Compared with the conventional tissue culture technique in Comparative Example 1, the relative expression levels of cold response genes and antifreeze protein genes in the Ulmus tissue culture seedlings obtained by culture in the Ulmus tissue culture medium provided by this invention increased by 3-4.5 times, the accumulation of antifreeze protein increased by 45%, the cell freezing point decreased by 3℃, the survival rate after treatment at -6℃ increased from 32% to 79%, the degree of cell wall lignification increased by 38%, and the seedling vigor rate reached 95%.

[0054] Table 5

[0055] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A tissue culture medium for elm plants, characterized in that, The culture media include callus first-stage culture medium, callus second-stage culture medium, callus cell tillering culture medium, bud and leaf growth culture medium, and root growth culture medium.

2. The elm tissue culture medium according to claim 1, characterized in that, The first-stage culture medium for the callus consisted of the following components: MS basal medium, 2,4-D 2.0-3.0 mg / L, 6-BA 0.2-0.5 mg / L, berberine 0.1-0.2 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.

0.

3. The elm tissue culture medium according to claim 1, characterized in that, The second-stage callus culture medium consisted of the following components: MS basal medium, 2,4-D 2.0-3.0 mg / L, 6-BA 0.2-0.5 mg / L, berberine 0.1-0.2 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.

0.

4. The elm tissue culture medium according to claim 1, characterized in that, The callus cell tillering culture medium consists of the following components: MS basal medium, 6-BA 1.0-1.5 mg / L, KT 0.5-1.0 mg / L, berberine 0.3-0.4 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.

0.

5. The elm tissue culture medium according to claim 1, characterized in that, The bud and leaf growth medium consists of the following components: MS basal medium, 6-BA 0.5-0.8 mg / L, IAA 0.2-0.5 mg / L, berberine 0.4-0.5 mg / L, sucrose 25-35 g / L, agar 6.5-7.5 g / L, pH=5.8-6.

0.

6. The elm tissue culture medium according to claim 1, characterized in that, The root growth medium consists of the following components: 1 / 2 MS basal medium, NAA 0.5-1.0 mg / L, berberine 0.3-0.4 mg / L, sucrose 15-25 g / L, agar 6.5-7.5 g / L, pH=5.8-6.

0.

7. The use of the elm plant tissue culture medium according to any one of claims 1 to 5 in the tissue culture of elm plants.

8. The application according to claim 7, characterized in that, The application of the elm tissue culture medium can improve the stress resistance of elm tissue culture seedlings, and the stress resistance includes at least one of antifungal, antibacterial, drought resistance, salt tolerance and low temperature tolerance.

9. A method for tissue culture of Ulmus plants, characterized in that, The method includes the following steps: S1: Collect callus tissue from elm plants and disinfect it; S2: The callus obtained in S1 is inoculated onto the first-stage callus culture medium of any one of claims 1 to 5 and cultured for 14 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the first-stage culture; the material after the first-stage culture is cut off on a sterile operating table and placed on the second-stage callus culture medium of any one of claims 1 to 5 and cultured for 10 days at a temperature of 25±2℃, a light intensity of 2000-3000 lx, and a light duration of 12 h / d to obtain the material after the second-stage culture; S3: The material obtained in S2 after the second stage of culture is cut off on a sterile operating table and placed on the callus cell tillering culture medium as described in any one of claims 1 to 5. It is cultured for 16 days at a temperature of 25±2℃, a light intensity of 3000-4000lx, and a light duration of 14 h / d to obtain the material after cell tillering culture. The material after cell tillering culture is soaked in colchicine solution and washed 4 times with sterile water before use. S4: The material treated with colchicine in S3 is cut off on a sterile operating table and placed on the bud and leaf growth culture medium described in any one of claims 1 to 5. It is cultured for 20 days under the conditions of temperature 25±2℃, light intensity 3000-4000lx, and light time 14 h / d to obtain the material after bud and leaf growth culture. S5: The material obtained from the bud and leaf growth culture in S4 is cut off on a sterile operating table and placed on the root growth culture medium described in any one of claims 1 to 5. It is cultured for 18 days under the conditions of temperature 23±2℃, light intensity 2000-3000lx, and light duration 12 h / d to obtain elm tissue culture seedlings.

10. The method according to claim 9, characterized in that, The conditions for the colchicine solution soaking treatment described in S3 are: the concentration of colchicine is 0.5%-1.0%, and the soaking time is 24-48 h.

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