Anthurium andraeanum tissue culture and rapid propagation culture medium and preparation method thereof

Abstract

The invention provides an anthurium andraeanum tissue culture and rapid propagation culture medium and a preparation method thereof, each liter of the culture medium contains major elements, trace elements, ferric salt, organic components and a plant growth regulator with specific weight, and components such as an osmotic regulator and a stress-resistant enhancer are also added in the culture medium, so that the anthurium andraeanum tissue culture and rapid propagation effect is improved. The sambucus williamsii extract is obtained through a supercritical extraction method, the purity and quality of the sambucus williamsii extract are ensured, the preparation method of the sambucus williamsii extract comprises multiple steps including sequential dissolution of all components, stirring, pH value adjustment, subpackaging, sterilization and other operations, and key parameters of all the steps such as sterilization conditions, the mass-volume ratio of a plant growth regulator to absolute ethyl alcohol and the like are defined. According to the scheme, an optimized culture medium and a matched preparation method are provided for tissue culture and rapid propagation of anthurium andraeanum, and the anthurium andraeanum culture effect can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation medium for tissue culture of anthurium andraeanum and a preparation method thereof. Background Art

[0002] As a plant with extremely high ornamental value, the market demand for anthurium is increasing day by day. In order to meet this demand, the rapid propagation technology of anthurium is particularly important. Traditional propagation methods such as seed propagation and division propagation have problems such as long propagation cycle and low propagation efficiency, which are difficult to meet the needs of large-scale production. Therefore, tissue culture technology, as a fast and efficient plant propagation method, is widely used in the propagation of anthurium. However, the existing anthurium tissue culture technology and corresponding culture medium formula still have many shortcomings.

[0003] In terms of culture medium components, although traditional anthurium tissue culture medium covers common macroelements, trace elements and organic components, the ratio of each component is often not accurately optimized, resulting in the inability to fully meet the dynamic needs of anthurium for nutrients at different growth stages in actual applications. This limits the growth rate of anthurium tissue culture seedlings and reduces the proliferation coefficient, making it difficult to efficiently achieve large-scale rapid propagation.

[0004] Regarding the use of plant growth regulators, the existing technology often has the problem of insufficient concentration control, which can easily lead to abnormal growth of tissue culture seedlings, such as excessive growth and deformity. In addition, the synergistic effect is poor when regulating key growth links such as rooting and bud differentiation of anthurium, affecting the overall tissue culture quality.

[0005] During the culture process, the tissue culture seedlings of Anthurium are easily affected by various biological and abiotic stresses, such as high temperature and drought. The traditional culture medium formula does not specifically add a combination of ingredients that can comprehensively improve the stress resistance of Anthurium. Although some studies have tried to add individual stress resistance substances, the effects are often not significant enough or have limitations.

[0006] In terms of osmotic regulation, most existing culture media rely on a single or a few common osmotic regulating substances, such as sucrose. Although these traditional osmotic regulating substances can regulate the osmotic pressure of the culture medium to a certain extent, their regulatory ability is relatively limited when dealing with complex changes in the tissue culture environment. In the process of anthurium tissue culture, when faced with stress factors such as temperature fluctuations, humidity changes, and drought in the external environment, a single osmotic regulating substance is difficult to maintain a stable osmotic pressure environment in the cell, which can easily lead to cell dehydration or excessive water absorption, thereby affecting the normal growth, division and differentiation of the cells, and reducing the survival rate and quality of the tissue culture seedlings.

[0007] In summary, the shortcomings of the existing anthurium tissue culture technology in terms of culture medium formula and preparation method limit the efficiency and quality of anthurium tissue culture rapid propagation. An optimized culture medium and scientific preparation method are urgently needed to improve this situation. Summary of the invention

[0008] In view of this, the present invention provides a tissue culture rapid propagation medium for Anthurium andraeanum and a preparation method thereof to solve the above problems.

[0009] The technical solution of the present invention is achieved as follows: a culture medium for rapid propagation of anthurium tissue culture: each liter of culture medium includes the following raw materials by weight: macroelements: 1650-1800 mg of ammonium nitrate, 1900-2100 mg of potassium nitrate, 170-200 mg of potassium dihydrogen phosphate, 370-400 mg of magnesium sulfate, trace elements: 0.8-1.2 mg of potassium iodide, 6.2-7.0 mg of boric acid, 22.3-25 mg of manganese sulfate, 8.6-10 mg of zinc sulfate, 0.25-0.35 mg of sodium molybdate, 0.025-0.035 mg of copper sulfate, 0.025-0.035 mg of cobalt chloride. .035mg, iron salt: disodium ferric ethylenediaminetetraacetate 36.7-40mg, organic ingredients: inositol 100-120mg, niacin 0.5-0.8mg, pyridoxine hydrochloride 0.5-0.8mg, thiamine hydrochloride 0.1-0.3mg, glycine 2.0-3.0mg, plant growth regulator: 6-benzylaminoadenine 1.0-2.0mg, naphthaleneacetic acid 0.1-0.3mg; and agar 6-8g, sucrose 30-35g, osmotic regulator 8-10mg, stress resistance enhancer 12-25mg, the balance is water, and the pH is adjusted to 5.8-6.0.

[0010] Furthermore, each liter of culture medium includes the following raw materials by weight: macroelements: 1700 mg ammonium nitrate, 2000 mg potassium nitrate, 180 mg potassium dihydrogen phosphate, 380 mg magnesium sulfate, trace elements: 1.0 mg potassium iodide, 6.5 mg boric acid, 24 mg manganese sulfate, 9.5 mg zinc sulfate, 0.30 mg sodium molybdate, 0.030 mg copper sulfate, 0.030 mg cobalt chloride, iron salt: 38.5 mg disodium ferric ethylenediaminetetraacetate, organic ingredients: 110 mg inositol, 0.7 mg niacin, 0.7 mg pyridoxine hydrochloride, 0.2 mg thiamine hydrochloride, 2.5 mg glycine, plant growth regulator: 1.5 mg 6-benzylaminoadenine, 0.2 mg naphthaleneacetic acid; as well as 6-8 g agar, 33 g sucrose, 9 mg osmotic regulator, 20 mg stress resistance enhancer, and the balance is water.

[0011] Furthermore, the osmotic regulator comprises plant polysaccharides, erythritol and inositol galactoside in a mass ratio of (8-23):(2-5):(6-17).

[0012] Furthermore, the plant polysaccharide is selected from Sanguisorba officinalis polysaccharide or Panax ginseng polysaccharide, and its molecular weight is 1000-3000Da.

[0013] Furthermore, the stress resistance enhancer comprises partridge tea polypeptide liquid, salicylic acid, elderberry extract, and methyl jasmonic acid in a mass ratio of (3.2-5.5):(8.8-10.2):(5.3-7.5):(15.6-22.3).

[0014] Furthermore, the partridge tea polypeptide liquid is a small molecule water-soluble peptide obtained after enzymatic hydrolysis with an alkaline complex enzyme, and its molecular weight is ≤1000Da. The addition amount of the complex enzyme is 8.5-10.2% of the mass of the partridge tea.

[0015] Furthermore, the alkaline complex enzyme is alkaline pectinase and high alkaline cellulase in a mass ratio of 1:1-2.

[0016] Furthermore, the elderberry extract is prepared by washing and crushing fresh and ripe elderberry fruits, placing them in an extraction kettle of a supercritical extraction device, with an extraction pressure of 10-30 MPa, an extraction temperature of 35-55° C., and an extraction time of 1-3 hours. The extracted mixture is decompressed and heated to convert CO2 from a supercritical state to a gaseous state, thereby separating CO2 from the extract, and the concentrate is concentrated and purified to obtain the elderberry extract.

[0017] Further, a method for preparing a culture medium for rapid propagation of anthurium andraeanum tissue culture comprises the following steps:

[0018] S1. Dissolve the above macroelements, trace elements, iron salts, organic components and their corresponding concentrations in 2.5-5 times the volume of water, stir well and evenly to form a mixed solution A;

[0019] S2, dissolving the plant growth regulator in anhydrous ethanol, adding the mixture to the mixed solution A, and continuing to stir evenly to form a mixed solution B;

[0020] S3. Add the osmotic regulator and stress enhancer to the mixed solution B, stir evenly, then add agar, heat and stir continuously until the agar is completely dissolved, then add sucrose, stir evenly, add water to make the volume to 1 L, and finally adjust the pH value to 5.8-6.0 with an acid-base regulator, and dispense into culture containers while hot to obtain the anthurium andraeanum tissue culture rapid propagation medium.

[0021] Furthermore, the mass volume ratio of the plant growth regulator and anhydrous ethanol in S2 is 1:0.2-1 in mg / mL.

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

[0023] (1) Optimized nutritional composition ratio to ensure the growth requirements of anthurium: The anthurium tissue culture rapid propagation medium of the present invention accurately sets the content range of macroelements (such as ammonium nitrate, potassium nitrate, potassium dihydrogen phosphate, magnesium sulfate, etc.), trace elements (potassium iodide, boric acid, manganese sulfate, etc.) and organic components (inositol, various vitamins and glycine, etc.), and clearly defines the preferred specific content, providing comprehensive and balanced nutritional support for anthurium tissue culture seedlings at different growth stages. The various nutritional components work together to effectively meet the dynamic requirements of anthurium for various nutrients in the entire growth and development process from explant induction, bud differentiation, proliferation to rooting, etc., which helps the anthurium tissue culture seedlings grow robustly, improves their survival rate and quality, and lays a good foundation for subsequent transplanting and large-scale planting.

[0024] (2) Reasonable addition of plant growth regulators to promote growth and differentiation: Adding 6-benzylaminoadenine and naphthylacetic acid, two plant growth regulators, in a specific concentration range to the culture medium and finely adjusting the dosage of the two can accurately regulate the growth and differentiation process of the anthurium tissue culture seedlings. At appropriate concentrations, they can effectively stimulate the explants to quickly induce high-quality callus tissue, significantly improve the differentiation rate and proliferation coefficient of buds, and enable anthurium to efficiently produce a large number of high-quality buds during the tissue culture stage. At the same time, they also help the timely and healthy growth of the root system, ensure the overall growth of the plant is good, avoid abnormal growth (such as elongation, deformity, etc.) caused by improper growth regulator concentrations, and ensure the efficiency and stability of anthurium tissue culture rapid propagation.

[0025] (3) By adding a specific ratio of Sanguisorba officinalis polysaccharide, erythritol and inositol galactoside as osmotic regulators, they work synergistically to effectively respond to various environmental changes, accurately adjust the osmotic pressure of the culture medium, promote the stable absorption of nutrients by cells, create an ideal growth microenvironment for the tissue culture cells of Anthurium andraeanum, and improve cell survival rate and vitality. The stress-resistant enhancer composed of partridge tea polypeptide liquid, salicylic acid, elderberry extract and methyl jasmonic acid in a specific mass ratio exerts a synergistic effect, fully activates the stress-resistant signaling pathway of Anthurium andraeanum cells, induces systemic resistance, and resists biological and non-biological stresses with its antibacterial and antioxidant properties, enhances its ability to adapt to complex environments, reduces mortality and deformity rates, and ensures the quality and robustness of tissue culture seedlings. The above-mentioned dual advantages of osmotic regulation and enhanced stress resistance work together to significantly promote the growth and development of anthurium tissue culture seedlings at all stages, improve the adaptation and differentiation rate of primary culture explants, the proliferation coefficient of subculture, and the rooting rate and root vitality of root culture. It not only greatly improves the reproduction efficiency and shortens the production cycle, but also ensures the excellent quality of seedlings, including strong plants, bright and thick leaves, strong stems, developed root systems and high genetic stability. After transplanting, they can quickly adapt to the external environment and have good growth consistency, providing a high-quality seedling source for the high-quality flower production of anthurium, greatly enhancing its competitiveness in the market, and effectively promoting the development of the anthurium tissue culture industry.

[0026] In addition, elderberry extract obtained by a specific supercritical extraction method is rich in a variety of bioactive substances. After these substances are added to the culture medium, they can play a role similar to plant growth regulators, and cooperate with other plant growth regulators to further promote the cell division, differentiation, and growth and development of roots, stems and leaves of anthurium tissue culture seedlings. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0029] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0030] Example 1

[0031] A culture medium for rapid propagation of anthurium tissue culture, comprising the following raw materials by weight per liter of culture medium: macroelements: 1650 mg of ammonium nitrate, 1900 mg of potassium nitrate, 170 mg of potassium dihydrogen phosphate, 370 mg of magnesium sulfate, trace elements: 0.8 mg of potassium iodide, 6.2 mg of boric acid, 22.3 mg of manganese sulfate, 8.6 mg of zinc sulfate, 0.25 mg of sodium molybdate, 0.025 mg of copper sulfate, 0.025 mg of cobalt chloride, iron salt: 36.7 mg of disodium ferric ethylenediaminetetraacetic acid, organic components: 100 mg of inositol, 0.5 mg of nicotinic acid, 0.5 mg of pyridoxine hydrochloride, 0.1 mg of thiamine hydrochloride, 2.0 mg of glycine, plant growth regulator: 1.0 mg of 6-benzylaminoadenine, 0.1 mg of naphthaleneacetic acid, 6 g of agar, 30 g of sucrose, 8 mg of osmotic regulator, 12 mg of stress resistance enhancer, and the balance is water, and the pH value is adjusted to 5.8;

[0032] The osmotic regulator comprises Sanguisorba officinalis polysaccharide, erythritol and inositol galactoside in a mass ratio of 8:2:6; the molecular weight of Sanguisorba officinalis polysaccharide is 1000Da;

[0033] The stress resistance enhancer comprises partridge tea polypeptide liquid, salicylic acid, elderberry extract and methyl jasmonic acid in a mass ratio of 3.2:8.8:5.3:15.6;

[0034] The partridge tea polypeptide liquid is a small molecule water-soluble peptide obtained after being hydrolyzed by alkaline complex enzyme, and its molecular weight is ≤1000Da, and the addition amount of the complex enzyme is 8.5% of the mass of the partridge tea;

[0035] The elderberry extract is prepared by washing and crushing fresh and ripe elderberry fruits, placing them in an extraction kettle of a supercritical extraction device, with an extraction pressure of 10 MPa, an extraction temperature of 35° C., and an extraction time of 1 hour. The extracted mixture is subjected to pressure reduction and temperature increase to convert CO2 from a supercritical state to a gaseous state, thereby separating CO2 from the extract. The concentrate is concentrated and purified to obtain the elderberry extract.

[0036] Example 2

[0037] A rapid propagation medium for tissue culture of anthurium, comprising the following raw materials by weight per liter of the medium: macroelements: 1800 mg of ammonium nitrate, 2100 mg of potassium nitrate, 200 mg of potassium dihydrogen phosphate, 400 mg of magnesium sulfate, trace elements: 1.2 mg of potassium iodide, 7.0 mg of boric acid, 25 mg of manganese sulfate, 10 mg of zinc sulfate, 0.35 mg of sodium molybdate, 0.035 mg of copper sulfate, 0.035 mg of cobalt chloride, iron salt: 40 mg of disodium ferric ethylenediaminetetraacetic acid, organic components: 120 mg of inositol, 0.8 mg of nicotinic acid, 0.8 mg of pyridoxine hydrochloride, 0.3 mg of thiamine hydrochloride, 3.0 mg of glycine, plant growth regulator: 2.0 mg of 6-benzylaminoadenine, 0.3 mg of naphthaleneacetic acid, 8 g of agar, 35 g of sucrose, 10 mg of an osmotic regulator, 25 mg of a stress resistance enhancer, and the balance is water, and the pH value is adjusted to 6.0;

[0038] The osmotic regulator comprises ginseng polysaccharide, erythritol and inositol galactoside in a mass ratio of 23:5:17; the molecular weight of ginseng polysaccharide is 3000Da;

[0039] The stress resistance enhancer comprises partridge tea polypeptide liquid, salicylic acid, elderberry extract and methyl jasmonic acid in a mass ratio of 5.5:10.2:7.5:22.3;

[0040] The partridge tea polypeptide liquid is a small molecule water-soluble peptide obtained after being hydrolyzed by alkaline complex enzyme, and its molecular weight is ≤1000Da, and the addition amount of the complex enzyme is 10.2% of the mass of the partridge tea;

[0041] The elderberry extract is specifically prepared by selecting fresh and ripe elderberry fruits, washing and crushing them, and putting them into an extraction kettle of a supercritical extraction device, with an extraction pressure of 30 MPa, an extraction temperature of 55° C., and an extraction time of 3 hours. The extracted mixture is subjected to a method of reducing pressure and heating to convert CO2 from a supercritical state to a gaseous state, thereby separating CO2 from the extract, and concentrating and purifying the concentrate to obtain the elderberry extract.

[0042] Example 3

[0043] A rapid propagation medium for anthurium tissue culture, comprising the following raw materials by weight per liter of the medium: macroelements: 1700 mg of ammonium nitrate, 2000 mg of potassium nitrate, 180 mg of potassium dihydrogen phosphate, 380 mg of magnesium sulfate, trace elements: 1.0 mg of potassium iodide, 6.5 mg of boric acid, 24 mg of manganese sulfate, 9.5 mg of zinc sulfate, 0.30 mg of sodium molybdate, 0.030 mg of copper sulfate, 0.030 mg of cobalt chloride, iron salt: 38.5 mg of disodium ferric ethylenediaminetetraacetic acid, organic components: 110 mg of inositol, 0.7 mg of nicotinic acid, 0.7 mg of pyridoxine hydrochloride, 0.2 mg of thiamine hydrochloride, 2.5 mg of glycine, plant growth regulators: 1.5 mg of 6-benzylaminoadenine, 0.2 mg of naphthaleneacetic acid, 6-8 g of agar, 33 g of sucrose, 9 mg of an osmotic regulator, 20 mg of a stress resistance enhancer, and the balance is water, and the pH value is adjusted to 6.0;

[0044] The osmotic regulator comprises Sanguisorba officinalis polysaccharide, erythritol and inositol galactoside in a mass ratio of 15:4:10; the molecular weight of Sanguisorba officinalis polysaccharide is 2000Da;

[0045] The stress resistance enhancer comprises partridge tea polypeptide liquid, salicylic acid, elderberry extract, and methyl jasmonic acid in a mass ratio of 4.5:9.5:6.2:20;

[0046] The partridge tea polypeptide liquid is a small molecule water-soluble peptide obtained after being hydrolyzed by alkaline complex enzyme, and its molecular weight is ≤1000Da. The addition amount of the complex enzyme is 9.8% of the mass of the partridge tea;

[0047] The elderberry extract is specifically prepared by selecting fresh and ripe elderberry fruits, washing and crushing them, and putting them into an extraction kettle of a supercritical extraction device, with an extraction pressure of 20 MPa, an extraction temperature of 45° C., and an extraction time of 2 hours. The extracted mixture is decompressed and heated to change CO2 from a supercritical state to a gaseous state, thereby separating CO2 from the extract, and concentrating and purifying the concentrate to obtain the elderberry extract.

[0048] The above examples 1-3 adopt the following preparation method:

[0049] S1. Dissolve the above macroelements, trace elements, iron salts, organic components and their corresponding concentrations in 3.5 times the volume of water and stir thoroughly to form a mixed solution A;

[0050] S2, dissolving the plant growth regulator in anhydrous ethanol, adding the mixture to the mixed solution A, and continuing to stir evenly to form a mixed solution B;

[0051] S3. Add the osmotic regulator and stress enhancer to the mixed solution B, stir evenly, then add agar, heat and stir continuously until the agar is completely dissolved, then add sucrose, stir evenly, add water to make the volume to 1 L, finally adjust the pH value to 6.0 with an acid-base regulator, dispense into culture containers while hot, and obtain the anthurium andraeanum tissue culture rapid propagation medium after sterilization.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 3 is that each liter of anthurium tissue culture rapid propagation medium includes the following raw materials by weight: macroelements: 1200 mg ammonium nitrate, 1000 mg potassium nitrate, 250 mg potassium dihydrogen phosphate, 350 mg magnesium sulfate, trace elements: 1.5 mg potassium iodide, 8.0 mg boric acid, 20 mg manganese sulfate, 7.5 mg zinc sulfate, 0.20 mg sodium molybdate, 0.055 mg copper sulfate, 0.012 mg cobalt chloride, iron salt: 48 mg disodium ferric ethylenediaminetetraacetic acid, organic ingredients: 58 mg inositol, 1.5 mg nicotinic acid, 0.1 mg pyridoxine hydrochloride, 0.8 mg thiamine hydrochloride, 3.5 mg glycine, plant growth regulators: 2.8 mg 6-benzylaminoadenine, 1.2 mg naphthylacetic acid; and 5 g agar, 22 g sucrose, and the balance is water.

[0054] Comparative Example 2

[0055] The difference between this comparative example and Example 3 is that the components of the rapid propagation medium for tissue culture of Anthurium andraeanum do not contain an osmotic regulator.

[0056] Comparative Example 3

[0057] The difference between this comparative example and Example 3 is that the components of the rapid propagation medium for tissue culture of Anthurium andraeanum do not contain stress resistance enhancer.

[0058] 1. Plant growth and development effect test

[0059] 1. Material Preparation

[0060] Plant materials: Select healthy anthurium explants (young leaves) with consistent growth status. Treat no less than 30 explants in each group to ensure representative samples.

[0061] Culture medium: prepare the anthurium tissue culture rapid propagation culture medium prepared in the above Examples 1-3 and Comparative Examples 1-3 and passed the sterility test, package it according to different treatments, and mark it clearly.

[0062] Culture container: Select appropriate tissue culture bottles or culture dishes to ensure they are sterile and of uniform specifications to facilitate subsequent observation and operation.

[0063] 2. Inoculation and cultivation

[0064] In the clean bench, the anthurium explants were inoculated into the corresponding tissue culture medium in strict accordance with the aseptic operation specifications, and 5 replicates were set for each treatment to reduce errors.

[0065] After the inoculation is completed, the culture container is sealed and placed in a light incubator or tissue culture room with set environmental conditions for cultivation;

[0066] Initial induction stage: stress temperature 35-40℃, light intensity 1000-1500lx, photoperiod 12-14h / d, relative humidity 70-80%;

[0067] Differentiation and proliferation stage: temperature 22-28°C, light intensity 2000-3000lx, light duration 12-16h / d, controlled stress humidity 40-50%; regularly observe and record relevant conditions during the culture process.

[0068] 3. Growth and development index determination and methods

[0069] (1) Callus induction

[0070] Induction time record: starting from the inoculation of explants, observe the explants every day and record the time when visible callus tissue begins to appear on each explant. The earliest time of appearance is taken as the basis. The average induction time of different treatment groups is calculated to compare the effects of different culture medium formulas on the callus induction speed.

[0071] Observation of callus characteristics: After callus formation, regularly (every 7 days) observe its texture (loose or dense), color (such as light yellow, green, etc.), and growth status (whether it is growing vigorously, whether there is browning, etc.), and make detailed records through text description and photographic records to analyze the effects of different treatments on callus quality.

[0072] (2) Bud differentiation and proliferation

[0073] Bud differentiation rate statistics: After 3-4 weeks of culture, observe and count the number of differentiated buds on each explant and calculate the bud differentiation rate.

[0074] Bud differentiation rate = (number of explants that differentiated into buds / total number of inoculated explants) × 100%. The bud differentiation rates of different treatment groups were compared to evaluate the ability of the culture medium to promote bud differentiation.

[0075] Determination of bud proliferation coefficient: After continuing to culture for 2-3 weeks, the average number of buds produced by each explant is counted, that is, bud proliferation coefficient = total number of buds / total number of explants, to measure the effect of the culture medium on bud proliferation and determine the culture medium conditions that are most conducive to large-scale bud proliferation.

[0076] (3) Rooting

[0077] Rooting time record: observe the time when the roots of the tissue culture seedlings begin to appear. The time when the earliest visible roots appear is also used as the standard. The average rooting time of different treatment groups is calculated to understand the effect of the culture medium on the initiation of rooting.

[0078] Measurement of root morphological indicators: After 4-6 weeks of rooting culture, 10 tissue culture seedlings were randomly selected, and the length and diameter of the roots of each seedling were measured with an electronic vernier caliper. The average root length and average root diameter were calculated, and the number of roots was counted. These indicators were used to comprehensively evaluate the growth and development of the root system and compare the effects of different culture media on rooting quality.

[0079] (4) Morphological and physiological indicators of tissue culture seedlings

[0080] Plant height measurement: Use an electronic vernier caliper or ruler to measure the height from the base to the top of the tissue culture seedlings. Measure once a week, record the changes in plant height at different culture stages, draw a growth curve, and visually compare the growth rates of tissue culture seedlings in different treatment groups.

[0081] Determination of leaf number and leaf area: Regularly count the number of leaves of each group of tissue culture seedlings, use a leaf area meter to measure the leaf area, and analyze the effects of different treatments on leaf growth. An increase in the number of leaves and an expansion of the leaf area usually mean that the plant is in good growth condition.

[0082] Determination of chlorophyll content: leaves from the same part were selected, and the chlorophyll a, chlorophyll b and total chlorophyll contents in the leaves were determined by spectrophotometry according to the standard operating procedures for chlorophyll extraction and determination. The chlorophyll content reflects the photosynthetic capacity of the tissue culture seedlings and indirectly reflects their growth vitality. The differences in chlorophyll content between different treatment groups were compared to determine the effect of the culture medium on the photosynthetic capacity and growth of the tissue culture seedlings.

[0083] 4. Test indicators and result data table

[0084] Table 1: Callus induction of Anthurium andraeanum

[0085]

[0086] Table 2: Differentiation and proliferation of anthurium buds

[0087] Treatment Group Bud differentiation rate (%) Bud proliferation coefficient Example 1 85 5.2 Example 2 88 5.8 Example 3 82 4.8 Comparative Example 1 70 3.5 Comparative Example 2 60 3.0 Comparative Example 3 68 3.3

[0088] Table 3: Rooting situation of anthurium

[0089]

[0090] Table 4: Morphological and physiological parameters of tissue culture seedlings of Anthurium (after 6 weeks of cultivation)

[0091]

[0092] Based on the analysis of the above growth and development indicators, the optimized anthurium tissue culture rapid propagation medium formula represented by Examples 1-3 showed significant advantages in the anthurium tissue culture process, whether it was callus induction, bud differentiation and proliferation, rooting, or the overall morphology and physiological function of the tissue culture seedlings, they were significantly better than the medium used in Comparative Examples 1-3 as controls, effectively improving the tolerance to high temperature and drought environments. This highlights the importance of rationally allocating the components of the culture medium, including accurately controlling the concentration of plant growth regulators, optimizing the combination of nutrient elements, etc., for improving the effect of anthurium tissue culture rapid propagation and promoting the healthy growth and development of anthurium plants.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture medium for rapid propagation of anthurium andraeanum tissue culture, characterized in that: Each liter of culture medium includes the following raw materials by weight: macroelements: ammonium nitrate 1650-1800 mg, potassium nitrate 1900-2100 mg, potassium dihydrogen phosphate 170-200 mg, magnesium sulfate 370-400 mg, trace elements: potassium iodide 0.8-1.2 mg, boric acid 6.2-7.0 mg, manganese sulfate 22.3-25 mg, zinc sulfate 8.6-10 mg, sodium molybdate 0.25-0.35 mg, copper sulfate 0.025-0.035 mg, cobalt chloride 0.025-0.035 mg, iron salt: ethylenediamine Disodium ferric tetraacetate 36.7-40 mg, organic ingredients: inositol 100-120 mg, niacin 0.5-0.8 mg, pyridoxine hydrochloride 0.5-0.8 mg, thiamine hydrochloride 0.1-0.3 mg, glycine 2.0-3.0 mg, plant growth regulator: 6-benzylaminoadenine 1.0-2.0 mg, naphthylacetic acid 0.1-0.3 mg; and agar 6-8 g, sucrose 30-35 g, osmotic regulator 8-10 mg, stress resistance enhancer 12-25 mg, the balance is water, and the pH is adjusted to 5.8-6.

0.

2. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 1, characterized in that: Each liter of culture medium includes the following raw materials by weight: macroelements: 1700 mg ammonium nitrate, 2000 mg potassium nitrate, 180 mg potassium dihydrogen phosphate, 380 mg magnesium sulfate, trace elements: 1.0 mg potassium iodide, 6.5 mg boric acid, 24 mg manganese sulfate, 9.5 mg zinc sulfate, 0.30 mg sodium molybdate, 0.030 mg copper sulfate, 0.030 mg cobalt chloride, iron salt: 38.5 mg disodium ferric ethylenediaminetetraacetate, organic ingredients: 110 mg inositol, 0.7 mg niacin, 0.7 mg pyridoxine hydrochloride, 0.2 mg thiamine hydrochloride, 2.5 mg glycine, plant growth regulator: 1.5 mg 6-benzylaminoadenine, 0.2 mg naphthylacetic acid; as well as 7 g agar, 33 g sucrose, 9 mg osmotic regulator, 20 mg stress resistance enhancer, and the balance is water.

3. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 1 or 2, characterized in that: The osmotic regulator comprises plant polysaccharides, erythritol and inositol galactoside in a mass ratio of (8-23):(2-5):(6-17).

4. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 3, characterized in that: The plant polysaccharide is selected from Sanguisorba officinalis polysaccharide or Panax ginseng polysaccharide, and has a molecular weight of 1000-3000Da.

5. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 1 or 2, characterized in that: The stress resistance enhancer comprises partridge tea polypeptide liquid, salicylic acid, elderberry extract and methyl jasmonic acid in a mass ratio of (3.2-5.5):(8.8-10.2):(5.3-7.5):(15.6-22.3).

6. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 5, characterized in that: The partridge tea polypeptide liquid is a small molecule water-soluble peptide obtained after being hydrolyzed by alkaline complex enzyme, and its molecular weight is less than or equal to 1000Da. The addition amount of the complex enzyme is 8.5-10.2% of the mass of the partridge tea.

7. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 6, characterized in that: The alkaline complex enzyme is alkaline pectinase and high alkaline cellulase in a mass ratio of 1:1-2.

8. A rapid propagation medium for tissue culture of anthurium andraeanum as claimed in claim 5, characterized in that: The elderberry extract is specifically prepared by selecting fresh and ripe elderberry fruits, washing and crushing them, and putting them into an extraction kettle of a supercritical extraction device. The extraction pressure is 10-30MPa, the extraction temperature is 35-55°C, and the extraction time is 1-3 hours. The extracted mixture is subjected to pressure reduction and temperature increase to convert CO2 from a supercritical state to a gaseous state, thereby separating CO2 from the extract. The concentrate is concentrated and purified to obtain the elderberry extract.

9. The method for preparing a culture medium for rapid propagation of anthurium andraeanum tissue culture as claimed in claim 3, characterized in that: The steps include: S1. Dissolve the above macroelements, trace elements, iron salts, organic components and their corresponding concentrations in 2.5-5 times the volume of water, stir well and evenly to form a mixed solution A; S2, dissolving the plant growth regulator in anhydrous ethanol, adding the mixture to the mixed solution A, and continuing to stir evenly to form a mixed solution B; S3. Add the osmotic regulator and stress enhancer to the mixed solution B, stir evenly, then add agar, heat and stir continuously until the agar is completely dissolved, then add sucrose, stir evenly, add water to make the volume to 1 L, and finally adjust the pH value to 5.8-6.0 with an acid-base regulator, and dispense into culture containers while hot to obtain the anthurium andraeanum tissue culture rapid propagation medium.

10. The method for preparing a culture medium for rapid propagation of anthurium andraeanum tissue culture according to claim 9, characterized in that: The mass volume ratio of the plant growth regulator and anhydrous ethanol in S2 is 1:0.2-1 in mg / mL.

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