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

Abstract

The application provides a tissue culture and rapid propagation culture medium for an anthurium and a preparation method thereof, the culture medium contains a large amount of elements, trace elements, iron salts, organic components and plant growth regulators in specific weights per liter, and the culture medium is additionally added with an osmotic regulator and an anti-reverse enhancer and the like to improve the effect of tissue culture and rapid propagation of the anthurium. The elderberry extract is obtained by a supercritical extraction method, and the purity and quality of the elderberry extract are ensured, the preparation method covers multiple steps, and includes operations such as sequential dissolution of components, stirring, pH value adjustment, sub-packaging and sterilization and the like, and the key parameters of each step are clear, such as sterilization conditions, a mass-volume ratio of a plant growth regulator to anhydrous ethanol and the like. The scheme provides an optimized culture medium and a matching preparation method for tissue culture and rapid propagation of the anthurium, and is helpful to improve the culture effect of the anthurium.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a cultivar of anaphalis contorta and a preparation method thereof. BACKGROUND

[0002] As a plant with high ornamental value, the market demand for anaphalis contorta is increasing. In order to meet this demand, the rapid propagation technology of anaphalis contorta 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 demand of large-scale production. Therefore, tissue culture technology as a rapid and efficient plant propagation method is widely used in the propagation of anaphalis contorta. However, the existing anaphalis contorta tissue culture technology and the corresponding medium formula still have many deficiencies.

[0003] In terms of medium composition, although the traditional anaphalis contorta tissue culture medium covers a large number of common elements, trace elements and organic components, the ratio of each component is often not optimized, which leads to the fact that the dynamic needs of anaphalis contorta at different growth stages for nutrients cannot be fully met in actual application, so that the growth rate of anaphalis contorta tissue culture seedlings is limited, the proliferation coefficient is low, and large-scale rapid propagation cannot be efficiently realized.

[0004] For the use of plant growth regulators, the existing technology often has the problem of not fine enough control of concentration, which can easily cause abnormal growth of tissue culture seedlings, such as excessive growth and malformation, and the synergistic effect is not good when regulating the key growth links such as rooting and bud differentiation of anaphalis contorta, which affects the overall quality of tissue culture.

[0005] Anaphalis contorta tissue culture seedlings are easily affected by various biological and non-biological stresses such as high temperature and drought during the culture process. The traditional medium formula does not add a targeted combination of ingredients that can comprehensively improve the stress resistance of anaphalis contorta. Some studies have tried to add individual stress resistance substances, but the effect is not significant or has limitations.

[0006] In terms of osmotic regulation, the existing medium mostly relies on a single or a few common osmotic regulation substances such as sucrose. Although these traditional osmotic regulation substances can regulate the osmotic pressure of the medium to a certain extent, their regulation ability is relatively limited when facing complex changes in the culture environment. In the process of anaphalis contorta tissue culture, when facing temperature fluctuations, humidity changes and drought stress factors in the external environment, a single osmotic regulation substance is difficult to maintain a stable osmotic pressure environment in the cell, which can easily lead to excessive water loss or water absorption of the cell, and then affect the normal growth, division and differentiation of the cell, and reduce the survival rate and quality of the tissue culture seedlings.

[0007] In summary, the existing anthurium andraeanum tissue culture technology has deficiencies in the medium formula and preparation method, which limits the efficiency and quality of the anthurium andraeanum tissue culture and rapid propagation, and an optimized medium and a scientific preparation method are urgently needed to improve the current situation. SUMMARY

[0008] In view of this, the present application provides an anthurium andraeanum tissue culture and rapid propagation medium and a preparation method thereof, which solves the above problems.

[0009] The technical scheme of the present application is as follows: an anthurium andraeanum tissue culture and rapid propagation medium: per liter of 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, microelements: 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: iron disodium ethylenediaminetetraacetate 36.7-40 mg, organic components: inositol 100-120 mg, nicotinic acid 0.5-0.8 mg, pyridoxal 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, naphthalene acetic acid 0.1-0.3 mg; and agar 6-8 g, sucrose 30-35 g, osmotic regulator 8-10 mg, stress-enhancing agent 12-25 mg, the balance being water, pH adjusted to 5.8-6.0.

[0010] Further, per liter of medium includes the following raw materials by weight: macroelements: ammonium nitrate 1700 mg, potassium nitrate 2000 mg, potassium dihydrogen phosphate 180 mg, magnesium sulfate 380 mg, microelements: potassium iodide 1.0 mg, boric acid 6.5 mg, manganese sulfate 24 mg, zinc sulfate 9.5 mg, sodium molybdate 0.30 mg, copper sulfate 0.030 mg, cobalt chloride 0.030 mg, iron salt: iron disodium ethylenediaminetetraacetate 38.5 mg, organic components: inositol 110 mg, nicotinic acid 0.7 mg, pyridoxal hydrochloride 0.7 mg, thiamine hydrochloride 0.2 mg, glycine 2.5 mg, plant growth regulator: 6-benzylaminoadenine 1.5 mg, naphthalene acetic acid 0.2 mg; and agar 6-8 g, sucrose 33 g, osmotic regulator 9 mg, stress-enhancing agent 20 mg, the balance being water.

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

[0012] Further, the plant polysaccharide is selected from the group consisting of Ulmus pumila polysaccharide and ginseng polysaccharide, and the molecular weight is 1000-3000 Da.

[0013] Further, the stress resistance enhancer comprises a mass ratio of 3.2-5.5:8.8-10.2:5.3-7.5:15.6-22.3 of the Camellia sinensis polypeptide solution, salicylic acid, elderberry extract and methyl jasmonic acid.

[0014] Further, the Camellia sinensis polypeptide solution is a small-molecule water-soluble peptide obtained by alkaline complex enzyme hydrolysis, and the molecular weight is ≤1000 Da.

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

[0016] Further, the elderberry extract is obtained by the following steps: selecting fresh mature elderberry fruits, washing and crushing, placing the fruits into an extraction kettle of a supercritical extraction device, setting the extraction pressure to 10-30 MPa, the extraction temperature to 35-55 DEG C, and the extraction time to 1-3 hours, separating CO2 from the supercritical state into a gaseous state by reducing pressure and increasing temperature, thereby separating the CO2 from the extract, and concentrating and purifying the concentrate to obtain the elderberry extract.

[0017] Further, a preparation method of a red palm tissue culture and rapid propagation culture medium comprises the following steps:

[0018] S1, dissolving the above-mentioned macroelements, microelements, iron salts, organic components and corresponding concentrations in 2.5-5 times the volume of water, fully stirring to form a mixed solution A;

[0019] S2, dissolving the plant growth regulator in anhydrous ethanol, adding it to the mixed solution A, and continuously stirring to form a mixed solution B;

[0020] S3, adding the osmotic regulator and stress resistance enhancer to the mixed solution B, stirring uniformly, adding agar, heating and continuously stirring until the agar is completely dissolved, then adding sucrose and stirring uniformly, adding water to 1L, and finally adjusting the pH value to 5.8-6.0 with an acid-base regulator, and hotly dispensing into a culture container to obtain the red palm tissue culture and rapid propagation culture medium.

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

[0022] Compared with the prior art, the present application has the following advantages:

[0023] (1) Optimized nutrient component proportioning, guaranteeing the growth demand of anthurium: the anthurium tissue culture and rapid propagation medium of the application provides comprehensive and balanced nutritional support for anthurium tissue culture seedlings in different growth stages by precisely setting the content range of macroelements (such as ammonium nitrate, potassium nitrate, potassium dihydrogen phosphate, magnesium sulfate, etc.), microelements (potassium iodide, boric acid, manganese sulfate, etc.) and organic components (inositol, various vitamins and glycine, etc.), and clearly optimizing the specific content. The mutual coordination of various nutrient components can effectively meet the dynamic demand of anthurium for various nutrients in the whole growth and development process from explant induction, bud differentiation, proliferation to rooting, which is helpful to the healthy growth of anthurium tissue culture seedlings and improves their survival rate and quality, laying a good foundation for subsequent transplanting and large-scale planting.

[0024] (2) Reasonable addition of plant growth regulators, promoting growth and differentiation: the addition of two plant growth regulators, 6-benzylaminopurine and naphthalene acetic acid, in a specific concentration range in the medium, and the fine adjustment of the use amount of the two, can precisely control the growth and differentiation process of anthurium tissue culture seedlings. At a suitable concentration, they can effectively stimulate the rapid induction of high-quality callus from explants, significantly improve the bud differentiation rate and proliferation coefficient, so that anthurium can efficiently produce a large number of high-quality buds in the tissue culture stage, and also help the timely and healthy growth of the root system, ensuring the overall growth of the plant to be in good condition, avoiding the occurrence of abnormal growth (such as overgrowth, deformity, etc.) due to improper concentration of growth regulators, and guaranteeing the efficiency and stability of anthurium tissue culture and rapid propagation.

[0025] (3) By adding specific proportions of Euonymus alatus polysaccharide, erythritol and galactinol as osmotic regulators, synergistic effect, effectively cope with various environmental changes, precisely regulate the osmotic pressure of the medium, promote the stable absorption of nutrients by cells, create an ideal growth microenvironment for anthurium tissue culture cells, and improve cell survival rate and vitality. The synergistic effect of the stress resistance enhancer composed of Euonymus alatus polysaccharide, salicylic acid, Sambucus williamsii extract and methyl jasmonic acid activates the anthurium cell stress resistance signal pathway, induces systemic resistance, resists biological and non-biological stress through antibacterial, antioxidant and other properties, enhances its ability to adapt to complex environments, reduces mortality and deformity rate, and guarantees the quality and robustness of tissue culture seedlings. The dual advantages of osmotic regulation and stress resistance enhancement work together to significantly promote the growth and development of anthurium tissue culture seedlings at each stage, improve the adaptation and differentiation speed of primary culture explants, the proliferation coefficient of subculture, and the rooting rate and root activity of rooting culture, not only greatly improve the propagation efficiency and shorten the production cycle, but also ensure the excellent quality of seedlings, including healthy plants, fresh and thick leaves, thick stems, developed roots and high genetic stability, which can quickly adapt to the external environment after transplanting and has good growth consistency, providing high-quality seedling source for anthurium high-quality flower production, greatly enhancing its competitiveness in the market, and effectively promoting the development of anthurium tissue culture industry.

[0026] In addition, the Sambucus williamsii Hance extract obtained by using the specific supercritical extraction method is rich in various bioactive substances. After the bioactive substances are added to the culture medium, on one hand, the bioactive substances can play a role similar to that of plant growth regulators to further promote cell division, differentiation, and growth and development of roots and stems and leaves of the anthurium cutting seedlings in cooperation with other plant growth regulators. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0028] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0029] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0030] Embodiment 1

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

[0032] The osmotic regulator includes 8:2:6 of Sanguisorba officinalis polysaccharide, erythritol and galactinol by mass fraction; the Sanguisorba officinalis polysaccharide has a molecular weight of 1000 Da;

[0033] The stress resistance enhancer includes 3.2:8.8:5.3:15.6 of Camellia pitardii polypeptide solution, salicylic acid, Sambucus williamsii Hance extract and methyl jasmonic acid by mass fraction;

[0034] The Camellia pitardii polypeptide solution is a small molecular water-soluble peptide obtained by enzymolysis of an alkaline complex enzyme, and the complex enzyme has an addition amount of 8.5% of the mass of the Camellia pitardii;

[0035] The sambucus williamsii extract is specifically obtained by selecting fresh mature sambucus williamsii fruits, washing and crushing the fruits, placing the fruits into an extraction kettle of a supercritical extraction device, setting the extraction pressure to 10 MPa, setting the extraction temperature to 35 DEG C, and setting the extraction time to 1 hour, and then converting CO2 from a supercritical state into a gaseous state through pressure reduction and temperature increase, so as to separate the CO2 from the extract, and concentrating and purifying the concentrate to obtain the sambucus williamsii extract.

[0036] Example 2

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

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

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

[0040] The small peony polypeptide liquid is a small-molecule water-soluble peptide obtained by enzymolysis of an alkaline compound enzyme, and has a molecular weight of ≤1000 Da; the compound enzyme is added in an amount of 10.2% of the mass of the small peony.

[0041] The sambucus williamsii extract is specifically obtained by selecting fresh mature sambucus williamsii fruits, washing and crushing the fruits, placing the fruits into an extraction kettle of a supercritical extraction device, setting the extraction pressure to 30 MPa, setting the extraction temperature to 55 DEG C, and setting the extraction time to 3 hours, and then converting CO2 from a supercritical state into a gaseous state through pressure reduction and temperature increase, so as to separate the CO2 from the extract, and concentrating and purifying the concentrate to obtain the sambucus williamsii extract.

[0042] Example 3

[0043] A tissue culture medium for Anthurium andraeanum, each liter of the medium comprising the following raw materials by weight: macroelements: ammonium nitrate 1700 mg, potassium nitrate 2000 mg, potassium dihydrogen phosphate 180 mg, magnesium sulfate 380 mg, microelements: potassium iodide 1.0 mg, boric acid 6.5 mg, manganese sulfate 24 mg, zinc sulfate 9.5 mg, sodium molybdate 0.30 mg, copper sulfate 0.030 mg, cobalt chloride 0.030 mg, iron salt: iron disodium ethylenediaminetetraacetate 38.5 mg, organic components: myo-inositol 110 mg, nicotinic acid 0.7 mg, pyridoxine hydrochloride 0.7 mg, thiamine hydrochloride 0.2 mg, glycine 2.5 mg, plant growth regulators: 6-benzylaminopurine 1.5 mg, naphthalene acetic acid 0.2 mg, agar 6-8 g, sucrose 33 g, osmotic regulator 9 mg, stress resistance enhancer 20 mg, the rest being water, and the pH being adjusted to 6.0;

[0044] The osmotic regulator comprises a mass ratio of 15:4:10 of Sibirian elm polysaccharide, erythritol and myo-inositol galactoside; the Sibirian elm polysaccharide has a molecular weight of 2000 Da;

[0045] The stress resistance enhancer comprises a mass ratio of 4.5:9.5:6.2:20 of Camellia pitardii polypeptide solution, salicylic acid, elderberry extract and methyl jasmonic acid;

[0046] The Camellia pitardii polypeptide solution is a small-molecule water-soluble peptide obtained by enzymolysis of the Camellia pitardii with an alkaline complex enzyme, and has a molecular weight of ≤1000 Da; the complex enzyme is added in an amount of 9.8% of the mass of the Camellia pitardii;

[0047] The elderberry extract is obtained by the following steps: selecting fresh mature elderberry fruits, washing and crushing, placing the fruits into an extraction kettle of a supercritical extraction device, setting the extraction pressure to 20 MPa, the extraction temperature to 45℃ and the extraction time to 2 hours, separating CO2 from the supercritical state into a gaseous state by reducing the pressure and increasing the temperature, thereby separating the CO2 from the extract, and concentrating and purifying the concentrate to obtain the elderberry extract;

[0048] The above examples 1-3 are prepared by the following method:

[0049] S1, dissolving the macroelements, microelements, iron salt, organic components and corresponding concentrations in 3.5 times the volume of water, and fully stirring to obtain a mixed solution A;

[0050] S2, dissolving the plant growth regulators in anhydrous ethanol, and adding the solution to the mixed solution A, and fully stirring to obtain a mixed solution B;

[0051] S3, add the osmotic regulator and the stress resistance enhancer to the mixed solution B, stir until uniform, then add agar, heat and continuously stir until the agar is completely dissolved, then add sucrose, stir until uniform, add water to 1 L, finally adjust the pH value to 6.0 with the acid-base regulator, and then pour into culture containers while hot, and the culture medium for tissue culture and rapid propagation of Caladium bicolor cv. Splendens is obtained after sterilization.

[0052] Comparative Example 1

[0053] The difference between the present comparative example and Example 3 is that the culture medium for tissue culture and rapid propagation of Caladium bicolor cv. Splendens comprises the following raw materials per liter: macronutrients: ammonium nitrate 1200 mg, potassium nitrate 1000 mg, potassium dihydrogen phosphate 250 mg, magnesium sulfate 350 mg, micronutrients: potassium iodide 1.5 mg, boric acid 8.0 mg, manganese sulfate 20 mg, zinc sulfate 7.5 mg, sodium molybdate 0.20 mg, copper sulfate 0.055 mg, cobalt chloride 0.012 mg, iron salt: iron disodium ethylenediaminetetraacetate 48 mg, organic components: myo-inositol 58 mg, nicotinic acid 1.5 mg, pyridoxine hydrochloride 0.1 mg, thiamine hydrochloride 0.8 mg, glycine 3.5 mg, plant growth regulator: 6-benzylaminopurine 2.8 mg, naphthalene acetic acid 1.2 mg; and agar 5 g, sucrose 22 g, and the balance is water.

[0054] Comparative Example 2

[0055] The difference between the present comparative example and Example 3 is that the culture medium for tissue culture and rapid propagation of Caladium bicolor cv. Splendens does not contain an osmotic regulator.

[0056] Comparative Example 3

[0057] The difference between the present comparative example and Example 3 is that the culture medium for tissue culture and rapid propagation of Caladium bicolor cv. Splendens does not contain a stress resistance enhancer.

[0058] I. Test of plant growth and development effect

[0059] 1. Material preparation

[0060] Plant material: select healthy Caladium bicolor cv. Splendens explants (young leaves) with consistent growth state, and no less than 30 explants per treatment group to ensure that the samples are representative.

[0061] Culture medium: prepare the culture medium for tissue culture and rapid propagation of Caladium bicolor cv. Splendens prepared in Examples 1-3 and Comparative Examples 1-3 above and qualified by sterile detection, and then divide and label according to different treatments.

[0062] Culture container: select appropriate tissue culture bottles or culture dishes, etc., to ensure that they are sterile and uniform in size, which is convenient for subsequent observation and operation.

[0063] 2. Inoculation and culture

[0064] In the clean bench, strictly in accordance with the aseptic operation specification, the red palm explant is inoculated into the corresponding tissue culture medium, and 5 repeats are set for each treatment to reduce errors.

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

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

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

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

[0069] (1) Callus induction

[0070] Induction time record: from the inoculation of explants, observe the explants every day, record the time when visible callus appears on each explant, take the earliest time as the standard, and calculate the average induction time of different treatment groups to compare the effects of different medium formulations on callus induction speed.

[0071] Callus feature observation: after the callus is formed, observe its texture (loose or dense), color (such as light yellow, green, etc.), growth state (whether it grows vigorously or not, whether it is browned or not, etc.) regularly (every 7 days), and record in detail through written description combined with photographic record, and 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 buds differentiated from each explant, and calculate the bud differentiation rate.

[0074] Bud differentiation rate = (number of differentiated explants / total number of inoculated explants) x 100%, compare the bud differentiation rates of different treatment groups, and evaluate the promoting ability of the medium for bud differentiation.

[0075] Bud proliferation coefficient determination: after 2-3 weeks of continuous culture, count the average number of buds produced by each explant, i.e. bud proliferation coefficient = total bud number / total number of explants, to measure the effect of the medium on bud proliferation and determine the most favorable medium conditions for large-scale bud proliferation.

[0076] (3) Rooting

[0077] Rooting time recording: Observe the time when the roots of the tissue culture seedlings begin to appear. The average rooting time of different treatment groups is calculated to understand the effect of the medium on the initiation of rooting.

[0078] Root morphology index measurement: After 4-6 weeks of rooting culture, randomly select 10 tissue culture seedlings and measure the length and diameter of each seedling's root using an electronic vernier caliper. Calculate the average root length and diameter, and count the number of roots to comprehensively evaluate the growth and development of the root system and compare the effects of different media on the quality of rooting.

[0079] (4) Morphology 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 and record the changes in plant height at different culture stages to draw growth curves and intuitively compare the growth rates of tissue culture seedlings in different treatment groups.

[0081] Leaf number and area measurement: Regularly count the number of leaves in each group of tissue culture seedlings and measure the leaf area using a leaf area meter to analyze the effects of different treatments on leaf growth. An increase in leaf number and expansion of leaf area generally indicates good plant growth conditions.

[0082] Chlorophyll content determination: Select leaves from the same part and use a spectrophotometer to determine the chlorophyll a, chlorophyll b, and total chlorophyll content according to the standard operating procedures for chlorophyll extraction and determination. The chlorophyll content reflects the photosynthetic capacity of tissue culture seedlings and indirectly represents their growth vigor. Comparing the differences in chlorophyll content between different treatment groups can help determine the effects of the medium on the photosynthetic capacity and growth of tissue culture seedlings.

[0083] 4. Test indicators and results data table

[0084] Table 1: Induction of Anthurium andraeanum callus

[0085]

[0086] Table 2: Bud differentiation and proliferation of Anthurium andraeanum

[0087] treatment group rate of shoot differentiation (%) shoot multiplication factor 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 of Anthurium andraeanum

[0089]

[0090] Table 4: Morphology and physiological indicators of Anthurium andraeanum tissue culture seedlings (after 6 weeks of culture)

[0091]

[0092] From the analysis of the above growth and development indicators, it can be seen that the optimized anthurium tissue culture and rapid propagation medium formula represented by examples 1-3 has shown significant advantages in the process of anthurium tissue culture. Whether it is callus induction, bud differentiation and proliferation, rooting, or the overall morphology and physiological function of the tissue culture seedlings, it is significantly better than the medium used by comparative examples 1-3 as a control. It effectively improves the tolerance to high temperature and drought environment. This highlights the importance of reasonable deployment of medium components, including precise control of plant growth regulator concentration, optimization of nutrient element matching, etc., for improving anthurium tissue culture and rapid propagation effect and promoting healthy growth and development of anthurium plants.

[0093] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A rapid propagation culture medium for Anthurium tissue culture, characterized in that: Each liter of culture medium consists of the following ingredients by weight: Macroelements: Ammonium nitrate 1650-1800 mg, Potassium nitrate 1900-2100 mg, Potassium dihydrogen phosphate 170-200 mg, Magnesium sulfate 370-400 mg; Microelements: 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 36.7-40 mg of disodium ferric tetraacetate; organic components: 100-120 mg of inositol, 0.5-0.8 mg of nicotinic acid, 0.5-0.8 mg of pyridoxine hydrochloride, 0.1-0.3 mg of thiamine hydrochloride, and 2.0-3.0 mg of glycine; plant growth regulators: 1.0-2.0 mg of 6-benzylaminopurine and 0.1-0.3 mg of naphthaleneacetic acid; as well as 6-8 g of agar, 30-35 g of sucrose, 8-10 mg of osmotic regulator, 12-25 mg of stress resistance enhancer, with the remainder being water, and the pH adjusted to 5.8-6.

0. The osmotic regulator is composed of plant polysaccharides, erythritol and inositol galactoside in a mass ratio of (8-23):(2-5):(6-17); The plant polysaccharides are selected from Sanguisorba officinalis polysaccharide or ginseng polysaccharide, with a molecular weight of 1000-3000 Da; The stress-resistance enhancer is composed of 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). The partridge tea polypeptide liquid is a small-molecule water-soluble peptide obtained by alkaline complex enzyme hydrolysis, with a molecular weight ≤1000 Da. The amount of the complex enzyme added is 8.5-10.2% of the mass of the partridge tea. The alkaline complex enzyme is alkaline pectinase and high alkaline cellulase in a mass ratio of 1:1-2. The elderberry extract is specifically prepared by selecting fresh, ripe elderberry fruits, washing and crushing them, and placing them in the extraction vessel of a supercritical extraction device. The extraction pressure is 10-30 MPa, the extraction temperature is 35-55℃, and the extraction time is 1-3 hours. The extracted mixture is then subjected to depressurization and heating to convert CO2 from a supercritical state to a gaseous state, thereby separating it from the extract. The concentrate is then concentrated and purified to obtain the elderberry extract.

2. The anthurium tissue culture rapid propagation medium as described in claim 1, characterized in that: Each liter of culture medium consists of the following ingredients by weight: macroelements: ammonium nitrate 1700 mg, potassium nitrate 2000 mg, potassium dihydrogen phosphate 180 mg, magnesium sulfate 380 mg; microelements: potassium iodide 1.0 mg, boric acid 6.5 mg, manganese sulfate 24 mg, zinc sulfate 9.5 mg, sodium molybdate 0.30 mg, copper sulfate 0.030 mg, cobalt chloride 0.030 mg; iron salt: disodium ferric ethylenediaminetetraacetate 38.5 mg; organic components: inositol 110 mg, nicotinic acid 0.7 mg, pyridoxine hydrochloride 0.7 mg, thiamine hydrochloride 0.2 mg, glycine 2.5 mg; plant growth regulators: 6-benzylaminopurine 1.5 mg, naphthaleneacetic acid 0.2 mg; as well as agar 7 g, sucrose 33 g, osmotic regulator 9 mg, stress resistance enhancer 20 mg, and the remainder is water.

3. The method for preparing a rapid propagation culture medium for Anthurium tissue culture as described in claim 1 or 2, characterized in that: The process includes the following steps: S1. Dissolve the above macro-elements, micro-elements, iron salts, organic components and their corresponding concentrations in 2.5-5 times their volume of water, stir thoroughly to form mixed solution A; S2. Dissolve the plant growth regulator in anhydrous ethanol and add it to mixed solution A. Continue stirring until homogeneous to form mixed solution B. S3. Add the osmotic regulator and stress-resistance 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 up to 1L, finally adjust the pH value to 5.8-6.0 with acid-base regulator, and dispense into culture containers while hot to obtain the Anthurium tissue culture rapid propagation medium.

4. The method for preparing a rapid propagation culture medium for Anthurium tissue culture as described in claim 3, characterized in that: The mass-to-volume ratio (mg / mL) of the plant growth regulator and anhydrous ethanol in S2 is 1:0.2-1.

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