Culture medium for acer elegans tissue culture

Abstract

The invention discloses a culture medium for acer elegans tissue culture, and belongs to the technical field of plant tissue culture. The culture medium comprises a double system of a germination culture medium with the pH value of 5.5-5.8 and a rooting culture medium with the pH value of 5.8-6.3. Wherein the germination culture medium takes potassium nitrate, calcium nitrate terahydrate, magnesium chloride hexahydrate and monopotassium phosphate as basic components, an ammonium-free solution system is innovatively adopted, a redox buffer system formed by ascorbic acid and cysteine is combined, and plant growth regulators such as TDZ and gibberellin are added. The rooting culture medium promotes root development through potassium nitrate and calcium nitrate tetrahydrate in a specific ratio in cooperation with IBA and phloroglucinol, and cane sugar is adopted as a carbon source and supplemented with activated carbon. By strictly controlling the molar ratio of Ca to Mg (2-2.5: 1) and the synergistic effect of paclobutrazol and other components, the browning occurrence rate of explants and the occurrence rate of glass seedlings are greatly reduced, and reliable technical support is provided for acer elegans germplasm resource preservation and industrial seedling raising.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture, in particular to a culture medium for tissue culture of Acer elegans. Background Art

[0002] The elm maple is a tree species of exceptional ornamental value. Currently, it is propagated primarily through traditional methods such as seeding, cuttings, and grafting. However, these methods suffer from low propagation coefficients, long cycles, and strong seasonality, making it difficult to meet market demand for large quantities of high-quality seedlings. Tissue culture, as a rapid propagation method capable of producing large numbers of genetically uniform plants in a short period of time, has become an important method for elm maple propagation.

[0003] Numerous tissue culture methods for Acer elegans have been reported in the prior art. For example, Chinese Invention Patent Publication No. CN111011220A discloses a tissue culture and rapid propagation method for Acer elegans. This method uses WPM medium as the basis. However, during actual Acer elegans tissue culture, the inventors discovered that vitrification and browning of Acer elegans were very common when using this medium. Consequently, a new culture medium for Acer elegans tissue culture was needed. Summary of the Invention

[0004] The object of the present invention is to provide a culture medium which is not prone to browning and vitrification during the tissue culture of Acer elegans.

[0005] To achieve the above object, the present invention provides a culture medium for tissue culture of Acer elegans, comprising a germination medium with a pH of 5.5 to 5.8 and a rooting medium with a pH of 5.8 to 6.3. The germination medium comprises the following components: 1300-1800 mg / L potassium nitrate, 650-750 mg / L calcium nitrate tetrahydrate, 250-300 mg / L magnesium chloride hexahydrate, and 60-100 mg / L potassium dihydrogen phosphate; The rooting medium comprises the following components: 450-600 mg / L potassium nitrate, 200-250 mg / L calcium nitrate tetrahydrate, 80-100 mg / L magnesium chloride hexahydrate, and 20-35 mg / L potassium dihydrogen phosphate. The molar ratio of Ca concentration to Mg concentration in the germination medium is (2-2.5):1, and the molar ratio of Ca concentration to Mg concentration in the rooting medium is (2-2.5):1.

[0006] The culture medium for tissue culture of the maple of the present invention completely removes ammonium nitrogen and adopts nitrate nitrogen to provide the nitrogen source for the maple of the present invention, with the nitrate nitrogen accounting for 100%. This can fundamentally avoid the occurrence of vitrified seedlings during tissue culture. In addition, the calcium content of the culture medium for tissue culture of the maple of the present invention is much higher than that of the commonly used MS culture medium. At the same time, the culture medium strictly controls the calcium-magnesium ratio of the culture medium, and promotes lignin deposition through the synergistic effect of calcium and magnesium.

[0007] More preferably, the culture medium for tissue culture of Acer truncatum provided by the present invention is suitable for the variety "Siming Meiwu".

[0008] Preferably, the germination medium further comprises the following components: 15-25 mg / L manganese chloride tetrahydrate, 5-10 mg / L zinc sulfate heptahydrate, 1-1.5 mg / L boric acid, and 0.1-0.2 mg / L sodium molybdate dihydrate.

[0009] In addition to the aforementioned macroelements, the germination culture medium also includes trace elements to ensure normal cell differentiation.

[0010] Preferably, the germination medium further comprises the following components: 100-200 mg / L inositol, 200-350 mg / L glutamine, 30-60 mg / L ascorbic acid, 15-25 mg / L cysteine hydrochloride, 0.5-1.5 mg / L nicotinic acid, and 0.5-1.5 mg / L thiamine.

[0011] The present invention adopts the synergistic effect of ascorbic acid and cysteine, which can effectively reduce the production of phenolic substances and neutralize the oxidation products of phenolic substances, thereby blocking the occurrence of browning.

[0012] Preferably, the germination medium further comprises the following components: 0.5-1 mg / L TDZ, 0.05-1 mg / L salicylic acid, 0.03-0.06 mg / L gibberellin, and 0.05-0.2 mg / L zeatin.

[0013] The germination medium of the present invention uses TDZ to promote bud differentiation of callus tissue, and its differentiation-promoting effect on Acer truncatum is far superior to that of traditional 6-BA. At the same time, the germination medium of the present invention is additionally added with zeatin, which can effectively induce bud formation and reduce the risk of vitrification.

[0014] Preferably, the germination medium further comprises the following components: 30-40 g / L of maltose and 0.1-0.8 g / L of activated carbon.

[0015] Salicylic acid and activated carbon are specially added to the germination medium of the present invention to reduce the production of phenolic substances and adsorb phenolic oxidants, thereby blocking the occurrence of browning. At the same time, maltose is used in the germination medium of the present invention to replace the sucrose in the traditional culture medium, which can be more easily utilized by the maple while maintaining the osmotic pressure.

[0016] Preferably, the rooting medium further comprises the following components: 15-25 mg / L manganese chloride tetrahydrate, 5-10 mg / L zinc sulfate heptahydrate, 1-1.5 mg / L boric acid, and 0.1-0.2 mg / L sodium molybdate dihydrate.

[0017] Preferably, the rooting medium further comprises the following components: 1-1.5 mg / L IBA, 3-5 mg / L phloroglucinol, and 0.1-0.5 mg / L paclobutrazol.

[0018] The rooting culture medium of the present invention can effectively inhibit the generation of redundant callus tissue while inducing the formation of root tip tissue of Acer elegans through the cooperation of IBA and phloroglucinol, thereby further reducing the generation of vitrified seedlings.

[0019] Preferably, the rooting medium further comprises the following components: 13-18 g / L of sucrose and 0.1-0.8 g / L of activated carbon.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention adopts a "zero ammonium" culture medium system, which eliminates the occurrence of vitrified seedlings from the root of the culture medium composition. At the same time, the present invention forms a redox buffer system through ascorbic acid + cysteine, which, in combination with phloroglucinol in the rooting stage, can effectively reduce the production of phenolic substances. In addition, the present invention adds activated carbon to the culture medium to absorb quinone substances produced by phenolic oxidation, thereby avoiding the occurrence of browning. Using the culture medium provided by the present invention to carry out tissue culture of Acer elegans can effectively improve the success rate of Acer elegans tissue culture, providing reliable technical support for the preservation of Acer elegans germplasm resources and industrialized seedling cultivation. DETAILED DESCRIPTION

[0021] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.

[0022] It should be noted that the endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0023] As described in the background art, browning and vitrification are prone to occur when using existing Acer elegans tissue culture medium for tissue culture. In view of this, a specific embodiment of the present invention provides a culture medium for Acer elegans tissue culture, which is composed of a germination medium and a rooting medium, wherein the rooting medium is a culture medium in which the macroelements are 1 / 3 of the germination elements.

[0024] Specifically, the macronutrient composition of the germination medium includes: 1300-1800 mg / L potassium nitrate, 650-750 mg / L calcium nitrate tetrahydrate, 250-300 mg / L magnesium chloride hexahydrate, and 60-100 mg / L potassium dihydrogen phosphate; The macronutrient composition of the rooting medium includes: 450~600 mg / L potassium nitrate, 200~250 mg / L calcium nitrate tetrahydrate, 80~100 mg / L magnesium chloride hexahydrate, and 20~35 mg / L potassium dihydrogen phosphate.

[0025] Among them, the pH of the germination medium is 5.5~5.8, and the pH of the rooting medium is 5.8~6.3, which are adjusted with hydrochloric acid or sodium hydroxide.

[0026] More specifically, in the culture medium of the above embodiment, the molar ratio of Ca concentration to Mg concentration in the germination medium is (2-2.5):1, and the molar ratio of Ca concentration to Mg concentration in the rooting medium is (2-2.5):1.

[0027] More specifically, in the above embodiment, the germination medium further comprises the following components: 15-25 mg / L manganese chloride tetrahydrate, 5-10 mg / L zinc sulfate heptahydrate, 1-1.5 mg / L boric acid, and 0.1-0.2 mg / L sodium molybdate dihydrate.

[0028] More specifically, in the above embodiment, the germination medium further comprises the following components: 100-200 mg / L of inositol, 200-350 mg / L of glutamine, 30-60 mg / L of ascorbic acid, 15-25 mg / L of cysteine hydrochloride, 0.5-1.5 mg / L of niacin, and 0.5-1.5 mg / L of thiamine.

[0029] More specifically, in the above embodiment, the germination medium further includes the following components: 0.5-1 mg / L TDZ, 0.05-1 mg / L salicylic acid, 0.03-0.06 mg / L gibberellin, and 0.05-0.2 mg / L zeatin.

[0030] More specifically, in the above embodiment, the germination culture medium further includes the following components: 30-40 g / L of maltose and 0.1-0.8 g / L of activated carbon.

[0031] More specifically, in the above embodiment, the rooting medium further comprises the following components: 15-25 mg / L manganese chloride tetrahydrate, 5-10 mg / L zinc sulfate heptahydrate, 1-1.5 mg / L boric acid, and 0.1-0.2 mg / L sodium molybdate dihydrate.

[0032] More specifically, in the above embodiment, the rooting medium further comprises the following components: 1-1.5 mg / L IBA, 3-5 mg / L phloroglucinol, and 0.1-0.5 mg / L paclobutrazol.

[0033] More specifically, in the above embodiment, the rooting medium further includes the following components: 13-18 g / L of sucrose and 0.1-0.8 g / L of activated carbon.

[0034] The culture medium provided by the invention reduces the incidence of browning and the generation of vitrified seedlings by optimizing element ratios and hormone ratios.

[0035] The technical solutions of the present invention are further described below by specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional understandings are defined herein for the purpose of illustrating or facilitating reference. Such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters provided by the manufacturers.

[0036] Example 1 Tissue culture was performed using Acer truncatum tissue culture medium S1. Explant selection: In April 2022, select several non-lignified "Siming Rose Dance" stem tips with 1-2 axillary buds as explants. The explants are 3-5 cm in length and rinsed twice with sterile water. S2. Disinfection: Soak the explants in 75% ethanol for 10 seconds, remove them, rinse them three times with sterile water, transfer them to 0.1% mercuric chloride solution and soak them for 8 minutes, remove them, and rinse them five times with sterile water. S3: Cut the explant into small segments of approximately 1 cm in length, retaining one axillary bud in each segment. Use forceps to insert the explant vertically into germination medium with a pH of 5.6 and containing the following components: Agar, 1500 mg / L potassium nitrate, 680 mg / L calcium nitrate tetrahydrate, 270 mg / L magnesium chloride hexahydrate, 85 mg / L potassium dihydrogen phosphate, 18.9 mg / L manganese chloride tetrahydrate, 6.2 mg / L zinc sulfate heptahydrate, 0.03 mg / L copper chloride dihydrate, 1 mg / L boric acid, 0.12 mg / L sodium molybdate dihydrate, 150 mg / L inositol, 300 mg / L glutamine, 50 mg / L ascorbic acid, 20 mg / L cysteine hydrochloride, 1.0 mg / L nicotinic acid, 1.2 mg / L thiamine, 0.8 mg / L TDZ, 0.1 mg / L salicylic acid, 0.05 mg / L gibberellin, 0.1 mg / L zeatin, 35 g / L maltose, 0.5 g / L activated carbon; After inoculation, the culture flask was placed in a light incubator under the following conditions: a temperature of 25±1°C, a light intensity of 3000 lux, and a photoperiod of 16 h / d. After 2 weeks of culture, pale yellow callus tissue began to form at the base of the explant. After 1 week of continuous culture, the callus tissue gradually increased in size and showed a granular structure. After 1 week of subculture, green protrusions began to appear on the surface of the callus tissue. After 1 week of continuous culture, the green protrusions developed into obvious buds, forming clustered buds. Subculture was performed once a week for 2 consecutive times to obtain a large number of robust clustered buds. S4: inoculating the callus into a rooting culture medium with a pH of 5.8, which includes: plant gel, 500 mg / L potassium nitrate, 227 mg / L calcium nitrate tetrahydrate, 90 mg / L magnesium chloride hexahydrate, 28 mg / L potassium dihydrogen phosphate, 18.9 mg / L manganese chloride tetrahydrate, 6.2 mg / L zinc sulfate heptahydrate, 0.03 mg / L copper chloride dihydrate, 1-1.5 mg / L boric acid, 0.12 mg / L sodium molybdate dihydrate, 1.2 mg / L IBA, 5 mg / L phloroglucinol, 0.3 mg / L paclobutrazol, 15 g / L sucrose, and 0.6 g / L activated carbon; After inoculation, the culture dish was placed in a light incubator at 22 ± 1°C, 2500 lux, and 14 h / d. After 2 weeks of incubation, white rhizogenes began to appear at the base of the clustered buds. After another 2 weeks of incubation, the roots gradually elongated and branched, forming complete clustered seedlings. S5: The clustered seedlings are transplanted into the substrate soil for planting. The substrate soil includes 1 part vermiculite, 1 part perlite and 2 parts humus soil. After planting, the clustered seedlings are placed in the greenhouse for seedling hardening. The temperature is controlled at 18~25℃ and the relative humidity is kept above 80%. The light intensity is adjusted with a sunshade net. Spray all day for the first 7 days, and then gradually reduce the number of sprays. Normal management is carried out after 15 days. After 1 month of hardening, the plants grow healthily and are transferred to field cultivation.

[0037] Example 2 Tissue culture was performed using Acer truncatum tissue culture medium S1. Explant selection: In April 2023, select several non-lignified "Simingmeiwu" stem tips with 1-2 axillary buds as explants. The explants should be 3-5 cm in length and rinsed twice with sterile water. S2. Disinfection: Soak the explants in 75% ethanol for 10 seconds, remove them, rinse them three times with sterile water, transfer them to 0.1% mercuric chloride solution and soak them for 8 minutes, remove them, and rinse them five times with sterile water. S3: Cut the explant into small segments of approximately 1 cm in length, retaining one axillary bud in each segment. Use forceps to insert the explant vertically into germination medium with a pH of 5.6 and containing the following components: Agar, 1800 mg / L potassium nitrate, 750 mg / L calcium nitrate tetrahydrate, 300 mg / L magnesium chloride hexahydrate, 100 mg / L potassium dihydrogen phosphate, 25 mg / L manganese chloride tetrahydrate, 10 mg / L zinc sulfate heptahydrate, 1.5 mg / L boric acid, 0.2 mg / L sodium molybdate dihydrate, 100 mg / L inositol, 325 mg / L glutamine, 60 mg / L ascorbic acid, 23 mg / L cysteine hydrochloride, 1.2 mg / L niacin, 1.5 mg / L thiamine, 1 mg / L TDZ, 0.05 mg / L salicylic acid, 0.06 mg / L gibberellin, 0.2 mg / L zeatin, 30 g / L maltose, 0.1 g / L activated carbon; After inoculation, the culture flask was placed in a light incubator under the following conditions: a temperature of 25±1°C, a light intensity of 3000 lux, and a photoperiod of 16 h / d. After 2 weeks of culture, pale yellow callus tissue began to form at the base of the explant. After 1 week of continuous culture, the callus tissue gradually increased in size and showed a granular structure. After 1 week of subculture, green protrusions began to appear on the surface of the callus tissue. After 1 week of continuous culture, the green protrusions developed into obvious buds, forming clustered buds. Subculture was performed once a week for 2 consecutive times to obtain a large number of robust clustered buds. S4: inoculating the callus into a rooting culture medium with a pH of 5.8, which includes: phytagel, 600 mg / L potassium nitrate, 250 mg / L calcium nitrate tetrahydrate, 100 mg / L magnesium chloride hexahydrate, 33 mg / L potassium dihydrogen phosphate, 15 mg / L manganese chloride tetrahydrate, 5 mg / L zinc sulfate heptahydrate, 1 mg / L boric acid, 0.1 mg / L sodium molybdate dihydrate, 1.5 mg / L IBA, 3 mg / L phloroglucinol, 0.5 mg / L paclobutrazol, 13 g / L sucrose, and 0.1 g / L activated carbon; After inoculation, the culture dish was placed in a light incubator at 22 ± 1°C, 2500 lux, and 14 h / d. After 2 weeks of incubation, white rhizogenes began to appear at the base of the clustered buds. After another 2 weeks of incubation, the roots gradually elongated and branched, forming complete clustered seedlings. S5: The clustered seedlings are transplanted into the substrate soil for planting. The substrate soil includes 1 part vermiculite, 1 part perlite and 2 parts humus soil. After planting, the clustered seedlings are placed in the greenhouse for seedling hardening. The temperature is controlled at 18~25℃ and the relative humidity is kept above 80%. The light intensity is adjusted with a sunshade net. Spray all day for the first 7 days, and then gradually reduce the number of sprays. Normal management is carried out after 15 days. After 1 month of hardening, the plants grow healthily and are transferred to field cultivation.

[0038] Comparative Example 1 S1. Explant selection: In April 2023, select several non-lignified "Simingmeiwu" stem tips with 1-2 axillary buds as explants. The explants should be 3-5 cm in length and rinsed twice with sterile water. S2. Disinfection: Soak the explants in 75% ethanol for 10 seconds, remove them, rinse them three times with sterile water, transfer them to 0.1% mercuric chloride solution and soak them for 8 minutes, remove them, and rinse them five times with sterile water. S3: Cut the explant into small segments of approximately 1 cm in length, retaining one axillary bud in each segment. Use forceps to insert the explant vertically into germination medium with a pH of 6.0 and containing the following components: Agar, WPM+6-BA1.0mg / L+IBA0.01mg / L+PVP500mg / L+GA31.0mg / L; After inoculation, the culture bottle was placed in a light incubator under the same culture conditions as in Example 2; S4: Inoculate the callus into rooting culture. The pH of the rooting medium is 5.8, which includes: 1 / 2MS + IBA 3.0mg / L; After the inoculation is completed, the culture dish is placed in a light incubator and the culture strips are the same as in Example 2 to form complete clumps of seedlings; S5: The clustered seedlings are transplanted into the substrate soil for planting. The substrate soil includes 1 part vermiculite, 1 part perlite and 2 parts humus soil. After planting, the clustered seedlings are placed in the greenhouse for seedling hardening. The temperature is controlled at 18~25℃ and the relative humidity is kept above 80%. The light intensity is adjusted with a sunshade net. Spray all day for the first 7 days, and then gradually reduce the number of sprays. Normal management is carried out after 15 days. After 1 month of hardening, the plants grow healthily and are transferred to field cultivation.

[0039] The browning ratio of explants and the glass seedling ratio in Example 2 and Comparative Example 1 were respectively counted, and the results are shown in Table 1.

[0040] Table 1 As can be seen from Table 1, the culture medium provided by the present invention can effectively reduce the incidence of browning of explants and the production of vitrified seedlings.

[0041] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A culture medium for tissue culture of Acer elegans, characterized in that: Including germination medium with pH of 5.5~5.8 and rooting medium with pH of 5.8~6.3, The germination medium comprises the following components: 1300-1800 mg / L potassium nitrate, 650-750 mg / L calcium nitrate tetrahydrate, 250-300 mg / L magnesium chloride hexahydrate, and 60-100 mg / L potassium dihydrogen phosphate; The rooting medium comprises the following components: 450-600 mg / L potassium nitrate, 200-250 mg / L calcium nitrate tetrahydrate, 80-100 mg / L magnesium chloride hexahydrate, and 20-35 mg / L potassium dihydrogen phosphate. The molar ratio of Ca concentration to Mg concentration in the germination medium is (2-2.5):1, and the molar ratio of Ca concentration to Mg concentration in the rooting medium is (2-2.5):

1.

2. The medium for tissue culture of Acer elegans according to claim 1, wherein The germination culture medium further comprises the following components: 15-25 mg / L of manganese chloride tetrahydrate, 5-10 mg / L of zinc sulfate heptahydrate, 1-1.5 mg / L of boric acid, and 0.1-0.2 mg / L of sodium molybdate dihydrate.

3. The tissue culture medium for Acer elegans according to claim 1, wherein The germination culture medium further comprises the following components: 100-200 mg / L of inositol, 200-350 mg / L of glutamine, 30-60 mg / L of ascorbic acid, 15-25 mg / L of cysteine hydrochloride, 0.5-1.5 mg / L of nicotinic acid, and 0.5-1.5 mg / L of thiamine.

4. The tissue culture medium for Acer elegans according to claim 1, wherein The germination culture medium further comprises the following components: 0.5-1 mg / L TDZ, 0.05-1 mg / L salicylic acid, 0.03-0.06 mg / L gibberellin, and 0.05-0.2 mg / L zeatin.

5. The tissue culture medium for Acer elegans according to claim 1, wherein The germination culture medium further comprises the following components: 0.5-1 mg / L TDZ, 0.05-1 mg / L salicylic acid, 0.03-0.06 mg / L gibberellin, and 0.05-0.2 mg / L zeatin.

6. The medium for tissue culture of Acer elegans according to claim 1, wherein The rooting medium further comprises the following components: 15-25 mg / L of manganese chloride tetrahydrate, 5-10 mg / L of zinc sulfate heptahydrate, 1-1.5 mg / L of boric acid, and 0.1-0.2 mg / L of sodium molybdate dihydrate.

7. The tissue culture medium for Acer elegans according to claim 1, wherein The rooting culture medium further comprises the following components: 1-1.5 mg / L IBA, 3-5 mg / L phloroglucinol, and 0.1-0.5 mg / L paclobutrazol.

8. The medium for tissue culture of Acer elegans according to claim 1, wherein The rooting culture medium further comprises the following components: 13-18 g / L of sucrose and 0.1-0.8 g / L of activated carbon.

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