Formula of plateau potato tissue culture medium

Abstract

The present invention discloses a plateau potato tissue culture medium formula, and relates to the technical field of plant tissue culture, the plateau potato tissue culture medium formula comprises the following components and concentration ranges: a basic inorganic salt, the basic inorganic salt comprises a macroelement, a microelement and an iron salt, wherein the macroelements are prepared from the following components: 1800 to 2000 mg / L of potassium nitrate, 1500 to 1800 mg / L of ammonium nitrate, 150 to 200 mg / L of monopotassium phosphate, 350 to 400 mg / L of magnesium sulfate and 400 to 480 mg / L of calcium chloride; according to the plateau potato tissue culture medium formula, an MS culture medium is used as a nutritional basis, and three plant growth regulators, namely indolebutyric acid (IBA), naphthylacetic acid (NAA) and indoleacetic acid (IAA), are subjected to composite compatibility at specific concentrations, so that an optimized plant growth regulator composition is constructed; the synergistic effect of the three auxin in promoting root primordium induction, root thickening and root elongation is utilized, so that the common obstacles of slow rooting, thin and weak root systems and the like of the plateau potato virus-free test-tube plantlets are effectively overcome, and the robust development and the overall growth vigor of the root systems of the plateau potato virus-free test-tube plantlets are promoted.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a formulation for a high-altitude potato tissue culture medium. Background Technology

[0002] Plant tissue culture technology, as a key branch of modern agricultural biotechnology, occupies a core position in crop genetic improvement, rapid propagation, and virus-free seedling production. By precisely controlling the in vitro culture environment, it achieves efficient regeneration of plant cells, tissues, or organs, and provides important technical support, especially for the large-scale propagation of root and tuber crops such as potatoes. Due to the unique low temperature, low oxygen, and strong radiation environment in plateau regions, higher requirements are placed on the adaptability and propagation quality of potato tissue culture. Developing special culture media adapted to plateau conditions has become the key to improving production efficiency.

[0003] However, current practices in potato tissue culture at high altitudes often encounter technical bottlenecks during the rooting stage of in vitro seedlings, including slow root development and a tendency for vitrification. This manifests as follows: under the stress of low oxygen, low temperature, and strong ultraviolet radiation at high altitudes, plant growth regulators in conventional culture media formulations are unable to synergistically promote root primordia induction and root elongation. This leads to prolonged rooting periods (usually exceeding 4 weeks), weak roots (average diameter <0.5 mm), and insufficient lateral root differentiation. More seriously, the accumulation of endogenous ethylene caused by environmental stress further exacerbates vitrification in in vitro seedlings (occurring in 30%-50% of cases), resulting in translucent, swollen leaves, fragile stems, and waterlogged roots, ultimately leading to a transplant survival rate of less than 60%. Although existing technologies attempt to improve rooting quality by adjusting single or dual hormone combinations (such as IBA+NAA), they cannot simultaneously resolve the dual contradiction between rooting speed and root robustness, especially with limited effectiveness in addressing the complex environmental stresses of high altitudes. Therefore, improvements are needed. Summary of the Invention

[0004] The purpose of this invention is to provide a high-altitude potato tissue culture medium formula to solve the problems of slow rooting, weak root system and easy vitrification of test-tube seedlings in high-altitude environments in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a high-altitude potato tissue culture medium formulation, comprising the following components and concentration ranges:

[0006] Basic inorganic salts, wherein the basic inorganic salts are composed of macroelements, microelements, and iron salts, wherein:

[0007] The macroelements consist of the following components: potassium nitrate 1800-2000 mg / L, ammonium nitrate 1500-1800 mg / L, potassium dihydrogen phosphate 150-200 mg / L, magnesium sulfate 350-400 mg / L, and calcium chloride 400-480 mg / L.

[0008] The trace elements consist of the following components: potassium iodide 0.7-0.9 mg / L, boric acid 5.5-6.5 mg / L, manganese sulfate 20.0-25.0 mg / L, zinc sulfate 8.0-9.0 mg / L, sodium molybdate 0.20-0.30 mg / L, copper sulfate 0.02-0.03 mg / L, and cobalt chloride 0.02-0.03 mg / L.

[0009] The iron salt is sodium ferric ethylenediaminetetraacetate, which is chelated from disodium ethylenediaminetetraacetate at a concentration of 36.0-38.0 mg / L and ferrous sulfate at a concentration of 27.0-28.5 mg / L.

[0010] The basic organic components consist of the following: inositol 90-110 mg / L, glycine 1.5-2.5 mg / L, thiamine hydrochloride 0.08-0.12 mg / L, pyridoxine hydrochloride 0.4-0.6 mg / L, and nicotinic acid 0.4-0.6 mg / L;

[0011] Carbon source and curing agent: sucrose 20-30 g / L, agar 5.5-7.0 g / L;

[0012] Plant growth regulator combination: indolebutyric acid 0.5-1.5 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, indoleacetic acid 0.1-0.3 mg / L;

[0013] Functional additive: Silver nitrate 1.0-5.0 mg / L;

[0014] And water;

[0015] The pH value of the culture medium is 5.6-5.8.

[0016] Furthermore, in the plant growth regulator combination, the concentration of indolebutyric acid is 1.0 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, and the concentration of indoleacetic acid is 0.2 mg / L.

[0017] Furthermore, the concentration of silver nitrate is 2.0-3.0 mg / L.

[0018] Furthermore, the culture medium is prepared by the following steps:

[0019] S1. Dissolve the basic inorganic salt components, basic organic components, sucrose and agar in water, heat and stir until completely dissolved to obtain the basic culture medium;

[0020] S2. After the temperature of the basic culture medium drops to 50-60℃, add the plant growth regulator combination and silver nitrate to it and mix well;

[0021] S3. Adjust the pH of the mixture obtained in step S2 to 5.6-5.8;

[0022] S4. Dispense and sterilize the culture medium obtained in step S3, and then cool it to obtain the final product.

[0023] Further, in step S2, the silver nitrate is added in the form of a mother liquor that has been sterilized by filtration through a 0.22 μm filter membrane.

[0024] Furthermore, in step S4, the sterilization conditions are: high-pressure steam sterilization at 121°C for 15-20 minutes.

[0025] Furthermore, the contents of the basic inorganic salt components, basic organic components, and iron salts are the same as the contents of the corresponding components in the MS medium.

[0026] Compared with existing technologies, the present invention provides a plateau potato tissue culture medium formula that uses MS medium as the nutrient base and combines three plant growth regulators—indolebutyric acid (IBA), naphthaleneacetic acid (NAA), and indoleacetic acid (IAA)—at specific concentrations to construct an optimized combination of plant growth regulators. By utilizing the synergistic effect of the three auxins in promoting root primordia induction, root thickening, and root elongation, it effectively overcomes the obstacles commonly found in virus-free test-tube seedlings of plateau potatoes, such as slow rooting and weak roots, and promotes robust root development and overall growth vigor.

[0027] By adding a specific concentration of silver nitrate as a functional regulator to the culture medium formulation, physiological disorders caused by environmental stress and endogenous ethylene accumulation during tissue culture were effectively inhibited. This significantly reduced the risk of vitrification in high-altitude potato test-tube seedlings during subculture and rooting stages, ensuring the physiological health of the tissue culture seedlings and increasing the proportion of high-quality, robust seedlings suitable for subsequent hardening and transplanting.

[0028] By integrating plant growth regulators with silver nitrate, an anti-vitrification functional additive, and combining them with standardized MS basic nutrients, carbon sources, and curing agents, a synergistic culture medium solution specifically designed for the rooting stage of tuber culture in high-altitude potatoes is provided. This achieves the dual goals of inducing efficient rooting while simultaneously ensuring normal plant morphology and robust growth, simplifying the culture process and improving the stability and seedling quality of industrialized production of virus-free tuber seedlings from high-altitude potatoes. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0030] Figure 1 This is a schematic diagram of the formula provided for an embodiment of the present invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] As attached Figure 1 As shown:

[0033] Example 1:

[0034] This invention provides a formulation for a high-altitude potato tissue culture medium, comprising the following components and their specific concentrations:

[0035] Basic inorganic salts (consistent with MS medium formulation):

[0036] Macroelements: Potassium nitrate (KNO3) 1900 mg / L; Ammonium nitrate (NH4NO3) 1650 mg / L; Potassium dihydrogen phosphate (KH2PO4) 170 mg / L; Magnesium sulfate (MgSO4·7H2O) 370 mg / L; Calcium chloride (CaCl2·2H2O) 440 mg / L.

[0037] Trace elements: Potassium iodide (KI) 0.83 mg / L; Boric acid (H3BO3) 6.2 mg / L; Manganese sulfate (MnSO4·4H2O) 22.3 mg / L; Zinc sulfate (ZnSO4·7H2O) 8.6 mg / L; Sodium molybdate (Na2MoO4·2H2O) 0.25 mg / L; Copper sulfate (CuSO4·5H2O) 0.025 mg / L; Cobalt chloride (CoCl2·6H2O) 0.025 mg / L.

[0038] Iron salts: Disodium ethylenediaminetetraacetate (Na2EDTA·2H2O) 37.3 mg / L; Ferrous sulfate (FeSO4·7H2O) 27.8 mg / L.

[0039] Basic organic components (consistent with MS medium formulation): myo-inositol 100 mg / L; glycine 2.0 mg / L; thiamine hydrochloride (vitamin B1) 0.1 mg / L; pyridoxine hydrochloride (vitamin B6) 0.5 mg / L; nicotinic acid (vitamin B3) 0.5 mg / L.

[0040] Carbon source and curing agent: sucrose 25g / L; agar 6.0g / L.

[0041] Plant growth regulator combination: Indole-3-butyric acid (IBA) 1.0 mg / L; 1-Naphthaleneacetic acid (NAA) 0.2 mg / L; Indole-3-acetic acid (IAA) 0.2 mg / L.

[0042] Functional additive: Silver nitrate (AgNO3) 3.0 mg / L.

[0043] Solvent and pH: The solvent is distilled water.

[0044] After being adjusted with a pH adjuster, the final pH value of the culture medium was 5.8.

[0045] Preparation method:

[0046] Mother liquor preparation:

[0047] Prepare concentrated stock solutions of macroelements (×10), microelements (×100), iron salts (×100), and organic components (×100) for MS medium, and store them at 4°C for later use.

[0048] Prepare stock solutions of indolebutyric acid (IBA, 1 mg / mL), naphthaleneacetic acid (NAA, 1 mg / mL), and indoleacetic acid (IAA, 1 mg / mL), respectively. The specific method is as follows: Weigh 10 mg of IBA, dissolve it with 1-2 drops of 1M NaOH solution, and then dilute to 10 mL with distilled water. The preparation method for NAA and IAA stock solutions is the same as for IBA. Store the prepared stock solutions at 4°C protected from light.

[0049] Prepare a stock solution of silver nitrate (AgNO3, 1 mg / mL). Weigh 10 mg of AgNO3, dissolve it in 10 mL of sterile distilled water, filter it through a 0.22 μm pore size aqueous microporous membrane for sterilization, dispense it into individual containers, and store it at 4 °C in the dark.

[0050] Culture medium preparation:

[0051] Measure approximately 800 mL of distilled water using a graduated cylinder and pour it into a clean 1 L beaker.

[0052] Calculate and measure the final concentrations of the MS mother liquor containing macro-elements, micro-elements, iron salts, and organic components, and add them sequentially to a beaker.

[0053] Weigh out 25g of sucrose and 6.0g of agar powder, and add them to the above solution.

[0054] Place the beaker on an electric magnetic stirrer and heat (about 80-90℃) while stirring until the agar is completely dissolved and the solution is clear.

[0055] Stop heating and continue stirring to allow the solution to cool naturally to approximately 55°C.

[0056] Add the pre-prepared IBA, NAA, and IAA stock solutions in sequence according to the final concentration.

[0057] Add sterilized silver nitrate (AgNO3) stock solution that has been filtered through a membrane.

[0058] Add distilled water to a final volume of approximately 950 mL and stir well.

[0059] pH adjustment and volume determination:

[0060] The pH of the culture medium was measured using a calibrated pH meter.

[0061] Adjust the pH of the culture medium precisely to 5.8 by adding 0.1M NaOH solution or 0.1M HCl solution dropwise while stirring.

[0062] Transfer the pH-adjusted culture medium to a 1L volumetric flask, bring the volume to 1000mL with distilled water, and shake well.

[0063] Dispensing and sterilization:

[0064] Dispense the culture medium, after bringing it to a final volume, into 150mL culture flasks or test tubes, with approximately 30-40mL dispensed into each flask.

[0065] Put the cap on (do not tighten) or seal it with sealing film (leaving a gap for ventilation).

[0066] The dispensed culture medium was placed in a high-pressure steam sterilizer and sterilized at 121°C and 0.1 MPa for 20 minutes.

[0067] Cooling and storage:

[0068] After the sterilization process is complete, allow the pressure in the sterilizer to drop to zero naturally before removing the culture medium.

[0069] In a clean bench or clean environment, tighten or completely seal the cap of the tissue culture flask while it is still hot.

[0070] Place the culture medium on a clean, level surface and allow it to cool and solidify at room temperature.

[0071] The solidified culture medium can be stored at 4-10℃ in the dark and is recommended to be used within 2 weeks.

[0072] Application effect verification:

[0073] Test materials: virus-free test-tube seedlings of the main potato varieties cultivated on the plateau (seedlings about 2-3 cm tall, with 2-3 unfolded leaves, no roots or very few roots).

[0074] Culture conditions: Aseptically inoculate the test-tube seedlings into the culture medium prepared in this embodiment, with 3 seedlings inoculated per bottle. Incubate in a culture room under the following conditions: 16-hour light / 8-hour dark cycle, light intensity 2000-2500 Lux, daytime temperature (during the light period) 23±1℃, nighttime temperature (during the dark period) 18±1℃, and relative humidity 60-70%.

[0075] Observation and statistics: After 4 weeks of cultivation, the effects were observed and the data were statistically analyzed.

[0076] Rooting status: The rooting rate reached 98.3%, with an average of 8.5±1.2 roots per plant and an average root length of 4.2±0.8cm. The roots are white or light yellow, robust, and have well-developed lateral roots.

[0077] Vitrification status: The vitrification rate was only 3.5%. The vast majority of plants had fully expanded leaves, a deep green color, and sturdy stems, showing normal and robust growth.

[0078] Overall growth: The average height of the plants increased to 6.5±0.9cm, the number of leaves increased, and the plants grew vigorously.

[0079] Example 2:

[0080] This embodiment is basically the same as the previous embodiment, except for the experimental design and culture medium formulation:

[0081] The experiment included four treatment groups with identical basal culture media, differing only in the type and concentration of plant growth regulators. The common basal formulation for all treatment groups was as follows (consistent with the MS basal components, carbon source, curing agent, and silver nitrate in Example 1):

[0082] Basic inorganic salts: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L; potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L; disodium ethylenediaminetetraacetate 37.3 mg / L, ferrous sulfate 27.8 mg / L.

[0083] Basic organic ingredients: Inositol 100mg / L, Glycine 2.0mg / L, Thiamine Hydrochloride 0.1mg / L, Pyridoxine Hydrochloride 0.5mg / L, Niacin 0.5mg / L.

[0084] Carbon source and curing agent: sucrose 25g / L, agar 6.0g / L.

[0085] Functional additive: Silver nitrate 3.0 mg / L.

[0086] Solvent and pH: Prepared with distilled water, pH adjusted to 5.8.

[0087] The differences in plant growth regulators among the treatment groups are as follows:

[0088] Control group A (conventional single hormone regimen): only indolebutyric acid (IBA) 1.0 mg / L was added.

[0089] Control group B (common dual-hormone regimen 1): Indolebutyric acid (IBA) 1.0 mg / L + naphthaleneacetic acid (NAA) 0.2 mg / L.

[0090] Control group C (common dual-hormone regimen 2): Indolebutyric acid (IBA) 1.0 mg / L + indoleacetic acid (IAA) 0.2 mg / L.

[0091] The present invention group (i.e., the scheme of Example 1, the combination of three hormones): added indolebutyric acid (IBA) 1.0 mg / L + naphthaleneacetic acid (NAA) 0.2 mg / L + indoleacetic acid (IAA) 0.2 mg / L.

[0092] Culture medium preparation method: The preparation method of the culture medium for each treatment group is completely consistent with the method in Example 1, and is briefly described below:

[0093] Prepare the basic culture medium (containing MS components, sucrose, and agar) according to the common basic formula.

[0094] After the culture medium has cooled to about 55°C, the corresponding plant growth regulator stock solution is added according to the hormone formula of each treatment group (the preparation and storage methods of IBA, NAA, and IAA stock solutions are the same as in Example 1).

[0095] Add sterile silver nitrate stock solution (concentration 3.0 mg / L) that has been filtered through a 0.22 μm filter membrane to the culture medium of all treatment groups.

[0096] Adjust the pH to 5.8 using 0.1M NaOH or HCl.

[0097] After being dispensed, the product is sterilized by high-pressure steam at 121°C for 20 minutes, then cooled and solidified for later use.

[0098] Experimental Materials and Methods:

[0099] Test materials: Same as in Example 1, selected virus-free test-tube seedlings of highland potatoes with consistent growth status (seedling height 2-3cm, with 2-3 leaves, no roots or primary roots <2mm).

[0100] Inoculation and Culture: In a sterile laminar flow hood, the basal cuts of the test-tube seedlings were inoculated into the four culture media mentioned above. Each treatment group was inoculated with 30 bottles, one seedling per bottle. After inoculation, the seedlings were cultured under the same conditions in the same culture room as in Example 1: a photoperiod of 16h / 8h (light / dark), light intensity of 2000-2500 Lux, daytime temperature of 23±1℃, nighttime temperature of 18±1℃, and relative humidity of 60-70%.

[0101] Observation and data statistics: After 4 weeks of cultivation, the following indicators were measured and statistically analyzed for all plants:

[0102] Rooting rate: The percentage of plants that produce adventitious roots ≥1cm in length out of the total number of inoculated plants.

[0103] Root count: Count the number of adventitious roots per seedling and calculate the average value.

[0104] Root morphology: Observe and record the thickness, length, color, and lateral root development of the roots. Randomly select 10 plants, measure the length of the longest root and the diameter at the base using calipers, and calculate the average value.

[0105] Plant growth status: Measure plant height (from the surface of the culture medium to the growing point), count the number of new leaves, and observe stem thickness, leaf color and spread.

[0106] After 4 weeks of cultivation, the growth performance and statistical data of each treatment group are shown in the table below:

[0107] Treatment group (plant growth regulators) Rooting rate (%) Average number of roots (per plant) Average root length (cm) Root thickness (mm) Lateral root development Average plant height (cm) Description of plant growth status Control group A (IBA 1.0 mg / L) 91.7 5.2±1.0 3.5±0.7 0.45±0.05 generally 5.8±0.7 Rooting is slow, roots are thin and long, and some plants are slightly weak. Control group B (IBA+NAA) 95.0 6.8±1.2 3.1±0.6 0.58±0.06 relatively developed 6.0±0.8 The roots are relatively short and thick, and the seedlings are relatively strong, but some roots are prone to developing callus tissue. Control group C (IBA+IAA) 93.3 5.9±1.1 4.5±0.9 0.48±0.05 generally 6.2±0.9 The roots are relatively long but thin, and the plant tends to grow excessively tall. This invention group (IBA+NAA+IAA) 98.3 8.5±1.2 4.2±0.8 0.62±0.07 developed 6.5±0.9 The root system is robust, with numerous roots of appropriate length and thickness; the plant as a whole is healthy, without excessive growth or callus formation.

[0108] Experimental results show that:

[0109] Rooting induction ability: The rooting rate of the present invention group (IBA+NAA+IAA) was the highest (98.3%), and the average number of roots (8.5) was significantly higher than that of all control groups.

[0110] Root quality: The root system of this invention group showed the best performance in terms of thickness (0.62 mm) and lateral root development, while maintaining a suitable length (4.2 cm), achieving robust growth. In contrast, the roots of control group B (IBA+NAA) were thicker but shorter; the roots of control group C (IBA+IAA) were longer but thinner; and the roots of control group A (IBA alone) were not superior in either quantity or quality.

[0111] Overall plant growth: The test-tube seedlings cultured in this invention have a moderate average plant height, sturdy stems, and deep green, spreading leaves, exhibiting the best overall growth state.

[0112] Synergistic effect: This invention combines the characteristics of IBA inducing root primordia, NAA promoting root thickening, and IAA promoting root elongation. The combination of the three hormones produces a synergistic effect, and the effect is significantly better than single or arbitrary dual hormone combination schemes in promoting the number of roots, optimizing root structure (thickness, length, lateral roots) and improving the overall robustness of plants.

[0113] Example 3:

[0114] This embodiment is basically the same as the previous embodiment, except for the experimental design and culture medium formulation:

[0115] The experiment included two treatment groups with identical basal culture medium components and plant growth regulator combinations, differing only in the addition of silver nitrate (AgNO3). The common basal formulation for all treatment groups is as follows:

[0116] Basic inorganic salts: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L; potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L; disodium ethylenediaminetetraacetate 37.3 mg / L, ferrous sulfate 27.8 mg / L.

[0117] Basic organic ingredients: Inositol 100mg / L, Glycine 2.0mg / L, Thiamine Hydrochloride 0.1mg / L, Pyridoxine Hydrochloride 0.5mg / L, Niacin 0.5mg / L.

[0118] Carbon source and curing agent: sucrose 25g / L, agar 6.0g / L.

[0119] Plant growth regulator combination: indolebutyric acid (IBA) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.2 mg / L, indoleacetic acid (IAA) 0.2 mg / L.

[0120] Solvent: Distilled water.

[0121] pH value: 5.8.

[0122] The differences in silver nitrate addition among the treatment groups are as follows:

[0123] Control group D (without silver nitrate): No silver nitrate (AgNO3) was added.

[0124] The present invention group (containing silver nitrate): silver nitrate (AgNO3) 3.0 mg / L was added.

[0125] Culture medium preparation method:

[0126] Preparation of stock solutions: The preparation and storage methods for the stock solutions of each component of MS medium and the stock solutions of plant growth regulators (IBA, NAA, IAA) are the same as in Example 1.

[0127] Preparation of basic culture medium: According to the common basic formula, weigh or measure the corresponding stock solution, sucrose and agar, dissolve them in distilled water, and heat and stir until completely dissolved.

[0128] Additives and pH adjustment: After the culture medium has cooled to about 55°C, add the pre-prepared IBA, NAA and IAA stock solutions to the culture medium of all treatment groups and stir well.

[0129] Only the culture medium of the present invention group was supplemented with a stock solution of silver nitrate (AgNO3, 1 mg / mL) that had been sterilized by filtration through a 0.22 μm filter membrane, to bring the final concentration to 3.0 mg / L. No stock solution of silver nitrate was added to the culture medium of control group D.

[0130] The pH of the two culture media was precisely adjusted to 5.8 using either 0.1M NaOH solution or 0.1M HCl solution.

[0131] Dispensing and sterilization: Dispense the pH-adjusted culture medium into tissue culture bottles, autoclave at 121℃ and 0.1MPa for 20 minutes, and then cool and solidify for later use.

[0132] Experimental Materials and Methods:

[0133] Test materials: Same as in Examples 1 and 2, virus-free test-tube seedlings of highland potatoes with consistent growth status were selected.

[0134] Inoculation and culture conditions:

[0135] Inoculation: Under aseptic conditions, the test-tube seedlings were inoculated into the two culture media mentioned above, with 30 bottles inoculated for each treatment group, and one seedling per bottle.

[0136] Culture conditions (vitrification induction environment): To more clearly observe the effect of silver nitrate, this example uses culture conditions that easily induce vitrification for verification. Specifically, the culture room temperature is maintained at 25±1℃ during the day and 20±1℃ at night; the relative humidity is increased to 75-80%; the light intensity is appropriately reduced to 1500-1800 Lux, and the photoperiod remains 16 hours of light / 8 hours of darkness.

[0137] Observation, statistics, and evaluation criteria:

[0138] Observation period: Regular observation begins after 2 weeks of culture, preliminary statistics are performed in the 3rd week, and final data statistics are performed after 4 weeks of culture.

[0139] Criteria for determining vitrified seedlings (a seedling is considered vitrified if it meets one of the following characteristics):

[0140] a. The plant, or parts thereof, is translucent or water-soaked.

[0141] b. The leaves are swollen, thickened, and brittle, with a light green or yellowish-green color, and the leaf edges are curled or rolled downwards.

[0142] C. The stem nodes do not elongate significantly, and the stem is fragile and translucent.

[0143] d. Poor root development or waterlogged appearance.

[0144] Statistical indicators:

[0145] Vitrification rate: The percentage of vitrified seedlings out of the total number of seedlings surveyed.

[0146] Normal seedling growth indicators: Statistical analysis of rooting rate, average number of roots, average root length and average plant height of non-vitrified plants (method as in Example 2).

[0147] Morphological observation: The differences between the two groups of plants in leaf morphology, stem characteristics and root status were recorded and compared in detail.

[0148] After 4 weeks of cultivation, the growth performance and statistical data of each treatment group are as follows:

[0149] Treatment group (silver nitrate added) Vitrification rate (%) Rooting rate of normal seedlings (%) Average number of roots per normal seedling (roots / plant) Average root length of normal seedlings (cm) Average height of normal seedlings (cm) Description of main morphological features <![CDATA[Control group D (without AgNO 3） > 36.7 89.5 6.2±1.3 3.8±0.9 5.9±1.1 Most plants exhibited typical vitrification symptoms: leaves appeared water-soaked, translucent, and swollen; stems were fragile and bright green; and roots were short or water-soaked. Normal seedlings also generally showed paler leaves and weaker plants. <![CDATA[This invention group (containing 3.0 mg / L of AgNO3)]]> 3.5 98.3 8.5±1.2 4.2±0.8 6.5±0.9 Vitrification was effectively controlled. The vast majority of plants had dark green, flat, and normally thick leaves; the stems were sturdy and a healthy green; the roots were white or pale yellow, thick and well-developed. The plants were generally robust.

[0150] Note: The indicators of the normal seedlings in control group D were derived from statistics of the plants that did not undergo vitrification (accounting for 63.3%).

[0151] Experimental results show that:

[0152] The decisive influence on vitrification: Under culture conditions that easily induce vitrification, the vitrification rate of the control group D without silver nitrate was as high as 36.7%; while the vitrification rate of the present invention group with 3.0 mg / L silver nitrate was only 3.5%, showing a highly significant difference. This directly proves that silver nitrate plays a key role in effectively preventing and significantly reducing the incidence of vitrification in high-altitude potato test-tube seedlings.

[0153] The effect on overall plant health and root development: The group of this invention not only had a low vitrification rate, but its normal seedlings also showed significantly better rooting rate, root quantity and quality, and plant height than the normal seedlings in control group D. This indicates that the addition of silver nitrate not only alleviated physiological obstacles but also created an internal environment more conducive to robust plant growth and root development.

[0154] Mechanism of action: Silver nitrate, as an inhibitor of ethylene action, may reduce the excessive accumulation of ethylene induced by culture stress (such as the characteristics of high-altitude varieties and the pressure of culture environment) and the physiological damage caused by it (such as inhibited cell wall synthesis, chlorophyll degradation, and excessive cell water content) by blocking the signal transduction or biosynthesis pathway of ethylene. This effectively alleviates the vitrification phenomenon and indirectly promotes the normal differentiation and growth of plants.

[0155] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A formulation for a high-altitude potato tissue culture medium, characterized in that, Includes the following components and concentration ranges: Basic inorganic salts, wherein the basic inorganic salts are composed of macroelements, microelements, and iron salts, wherein: The macroelements consist of the following components: potassium nitrate 1800-2000 mg / L, ammonium nitrate 1500-1800 mg / L, potassium dihydrogen phosphate 150-200 mg / L, magnesium sulfate 350-400 mg / L, and calcium chloride 400-480 mg / L. The trace elements consist of the following components: potassium iodide 0.7-0.9 mg / L, boric acid 5.5-6.5 mg / L, manganese sulfate 20.0-25.0 mg / L, zinc sulfate 8.0-9.0 mg / L, sodium molybdate 0.20-0.30 mg / L, copper sulfate 0.02-0.03 mg / L, and cobalt chloride 0.02-0.03 mg / L. The iron salt is sodium ferric ethylenediaminetetraacetate, which is chelated from disodium ethylenediaminetetraacetate at a concentration of 36.0-38.0 mg / L and ferrous sulfate at a concentration of 27.0-28.5 mg / L. The basic organic components consist of the following: inositol 90-110 mg / L, glycine 1.5-2.5 mg / L, thiamine hydrochloride 0.08-0.12 mg / L, pyridoxine hydrochloride 0.4-0.6 mg / L, and nicotinic acid 0.4-0.6 mg / L; Carbon source and curing agent: sucrose 20-30 g / L, agar 5.5-7.0 g / L; Plant growth regulator combination: indolebutyric acid 0.5-1.5 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, indoleacetic acid 0.1-0.3 mg / L; Functional additive: Silver nitrate 1.0-5.0 mg / L; And water; The pH value of the culture medium is 5.6-5.

8.

2. The formulation of a high-altitude potato tissue culture medium according to claim 1, characterized in that, In the plant growth regulator combination, the concentration of indolebutyric acid is 1.0 mg / L, the concentration of naphthaleneacetic acid is 0.2 mg / L, and the concentration of indoleacetic acid is 0.2 mg / L.

3. The formulation of a high-altitude potato tissue culture medium according to claim 1, characterized in that, The concentration of silver nitrate is 2.0-3.0 mg / L.

4. The formulation of a high-altitude potato tissue culture medium according to claim 1, characterized in that, The culture medium is prepared by the following steps: S1. Dissolve the basic inorganic salt components, basic organic components, sucrose and agar in water, heat and stir until completely dissolved to obtain the basic culture medium; S2. After the temperature of the basic culture medium drops to 50-60℃, add the plant growth regulator combination and silver nitrate to it and mix well; S3. Adjust the pH of the mixture obtained in step S2 to 5.6-5.8; S4. Dispense and sterilize the culture medium obtained in step S3, and then cool it to obtain the final product.

5. The formulation of a high-altitude potato tissue culture medium according to claim 4, characterized in that, In step S2, the silver nitrate is added as a mother liquor that has been sterilized by filtration through a 0.22 μm filter membrane.

6. The formulation of a high-altitude potato tissue culture medium according to claim 4, characterized in that, In step S4, the sterilization conditions are: high-pressure steam sterilization at 121°C for 15-20 minutes.

7. The formulation of a high-altitude potato tissue culture medium according to claim 1, characterized in that, The contents of the basic inorganic salt components, basic organic components, and iron salts are the same as those of the corresponding components in the MS medium.

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