CN111213586B - Anoectochilus roxburghii tissue culture medium and preparation method thereof - Google Patents
Anoectochilus roxburghii tissue culture medium and preparation method thereof Download PDFInfo
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- 239000003104 tissue culture media Substances 0.000 title claims abstract description 42
- 241000934230 Anoectochilus roxburghii Species 0.000 title claims abstract description 19
- 238000002360 preparation method Methods 0.000 title abstract description 16
- 241000719836 Anoectochilus formosanus Species 0.000 claims abstract description 60
- 239000001963 growth medium Substances 0.000 claims abstract description 54
- 244000286838 Eclipta prostrata Species 0.000 claims abstract description 47
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229930006000 Sucrose Natural products 0.000 claims abstract description 31
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 31
- 244000017020 Ipomoea batatas Species 0.000 claims abstract description 27
- 235000002678 Ipomoea batatas Nutrition 0.000 claims abstract description 27
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- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 claims abstract description 22
- 235000019345 sodium thiosulphate Nutrition 0.000 claims abstract description 22
- 239000005720 sucrose Substances 0.000 claims abstract description 22
- 229920001817 Agar Polymers 0.000 claims abstract description 20
- 239000008272 agar Substances 0.000 claims abstract description 20
- 229960005321 mecobalamin Drugs 0.000 claims abstract description 19
- JEWJRMKHSMTXPP-BYFNXCQMSA-M methylcobalamin Chemical compound C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O JEWJRMKHSMTXPP-BYFNXCQMSA-M 0.000 claims abstract description 19
- 235000007672 methylcobalamin Nutrition 0.000 claims abstract description 19
- 239000011585 methylcobalamin Substances 0.000 claims abstract description 19
- CJUUXVFWKYRHAR-UHFFFAOYSA-M 1-Naphthaleneacetic acid sodium salt Chemical compound [Na+].C1=CC=C2C(CC(=O)[O-])=CC=CC2=C1 CJUUXVFWKYRHAR-UHFFFAOYSA-M 0.000 claims abstract description 18
- 239000005972 6-Benzyladenine Substances 0.000 claims abstract description 18
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000011259 mixed solution Substances 0.000 claims description 42
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 33
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- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 2
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- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
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- 239000006013 carbendazim Substances 0.000 description 1
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
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- 238000010411 cooking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 201000003146 cystitis Diseases 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
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- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
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- 229940088597 hormone Drugs 0.000 description 1
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- 230000036039 immunity Effects 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
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- LPMXVESGRSUGHW-HBYQJFLCSA-N ouabain Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1C[C@@]2(O)CC[C@H]3[C@@]4(O)CC[C@H](C=5COC(=O)C=5)[C@@]4(C)C[C@@H](O)[C@@H]3[C@@]2(CO)[C@H](O)C1 LPMXVESGRSUGHW-HBYQJFLCSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an anoectochilus formosanus tissue culture medium and a preparation method thereof, wherein the culture medium comprises the following components: sucrose, agar, chitosan, eclipta extract, sodium thiosulfate, activated carbon, mecobalamin, purple sweet potato extract, 6-benzyladenine, indolebutyric acid and sodium naphthaleneacetate; also included is MS medium stock. The invention also provides a preparation method of the anoectochilus roxburghii tissue culture medium. According to the invention, by improving the formula and the preparation method of the anoectochilus formosanus tissue culture medium, pollutants in the culture medium are reduced, and meanwhile, nutrient substances are provided for tissue culture by using the culture medium, so that the problems of high pollution rate, influence of pesticide residue on the use value, easiness in browning and high tissue culture cost are effectively solved.
Description
Technical Field
The invention relates to the technical field of culture media, and particularly relates to an anoectochilus formosanus tissue culture medium and a preparation method thereof.
Background
Anoectochilus roxburghii (Anoectochilus roxburghii), also known as Jinsicao, is a perennial herb of the genus Kalimeris in the family Orchidaceae, is a traditional and rare Chinese medicine in China, has the effects of clearing heat, cooling blood, removing dampness, detoxifying and the like, is used for treating diseases such as diabetes, nephritis, cystitis and the like, and enjoys the name of 'drug king' in folk. For a long time, Anoectochilus formosanus is used for boiling water, making tea and cooking soup in Zhenan, Mintai and Yingtai for strengthening body constitution and nourishing. Modern researches show that the anoectochilus formosanus contains various components such as polysaccharide, flavonoid, cardiac glycoside and the like, and has the effects of enhancing the immunity of a human body, improving the resistance of the human body, preventing diseases from invading, enhancing and improving the physique and the like.
The pollution rate of the seedling bottle is high due to the influence of various factors such as environment, culture medium, artificial technology and the like in the tissue culture process of the anoectochilus formosanus, so that the tissue culture cost is improved. The pollution problem is always the main obstacle restricting the seedling breeding of anoectochilus formosanus at present, the pollution can cause the reduction of the proliferation efficiency, and the growth delay of culture materials can even cause the difficult transplantation and death of tissue culture seedlings. At present, the commonly used solution is to add bactericides such as antibiotics such as penicillin, chloramphenicol, tetracycline and even pesticides such as carbendazim and the like into a culture medium, although a certain sterilization effect is achieved, a large amount of pesticide residues exist, the medicinal value of the bactericide is influenced, the environment is also polluted, and the growth of anoectochilus roxburghii is also influenced by the addition of the antibiotics and the pesticides. Meanwhile, the growth period of the anoectochilus formosanus is longer, the anoectochilus formosanus is easy to brown, the tissue culture cost is higher, and the popularization and the planting of the anoectochilus formosanus are inconvenient.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the anoectochilus formosanus tissue culture medium and the preparation method thereof, by improving the formula and the preparation method of the anoectochilus formosanus tissue culture medium, pollutants in the culture medium are reduced, and meanwhile, nutrient substances are provided for tissue culture by utilizing the culture medium, so that the problems of high pollution rate, influence of pesticide residue on the use value, easiness in browning and high tissue culture cost are effectively solved.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows: provides a anoectochilus roxburghii tissue culture medium which comprises the following components in parts by weight: 25-30 parts of cane sugar, 3-6 parts of agar, 3-6 parts of chitosan, 15-20 parts of eclipta extract, 10-20 parts of sodium thiosulfate, 1-3 parts of activated carbon, 0.001-0.004 part of mecobalamine, 20-30 parts of purple sweet potato extract, 0.0006-0.001 part of 6-benzyl adenine, 0.0008-0.0015 part of indolebutyric acid and 0.0008-0.0012 part of sodium naphthalene acetate;
the culture medium also comprises MS culture medium mother liquor, and the mass volume ratio of the sucrose to the MS culture medium mother liquor is 25-30: 100-150 g/mL.
Further, the culture medium comprises the following components in parts by weight: 28 parts of cane sugar, 5 parts of agar, 5 parts of chitosan, 17 parts of eclipta extract, 15 parts of sodium thiosulfate, 2 parts of activated carbon, 0.003 part of mecobalamin, 25 parts of purple sweet potato extract, 0.0008 part of 6-benzyladenine, 0.0012 part of indolebutyric acid and 0.001 part of sodium naphthylacetate;
the culture medium further comprises MS culture medium mother liquor, and the mass-to-volume ratio of the sucrose to the MS culture medium mother liquor is 28:125 g/mL.
Further, the eclipta alba extract is prepared by the following method: crushing the eclipta, sieving with a sieve of 80-100 meshes, adding 4-5 wt% of citric acid aqueous solution, extracting for 3-5 hours at the temperature of 70-74 ℃, extracting for 2-4 times, combining extracting solutions for many times, and concentrating to 20% -25% of the original volume to obtain the eclipta extracting solution.
Furthermore, the weight ratio of the eclipta and the citric acid aqueous solution is 1: 4-6.
Further, the purple sweet potato extract is prepared by the following method: adding 2 times of water into the purple sweet potato, and squeezing to obtain juice; and then drying the juice, and crushing the dried juice into powder of 100-200 meshes, wherein the powder is the purple sweet potato extract.
Furthermore, the mesh number of the active carbon is 100-150 meshes.
The preparation method of the anoectochilus roxburghii tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution obtained in the step (3) at high temperature, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Further, in the step (3), a sodium hydroxide solution or a hydrogen chloride solution is adopted to adjust the pH value.
Further, in the step (4), during high-temperature sterilization, sterilization is carried out for 10-20 min at the temperature of 120-130 ℃ and under the pressure of 110-115 kPa.
Further, adding distilled water to a constant volume, wherein the sucrose concentration is 25-30 g/L.
In summary, the invention has the following advantages:
1. the invention provides a tissue culture medium of anoectochilus formosanus, which takes various substances such as sucrose, agar, chitosan, activated carbon and the like as raw material components, and improves the formula and the preparation method of the tissue culture medium of the anoectochilus formosanus, when the tissue culture medium is applied to the tissue culture process of the anoectochilus formosanus, the tissue culture medium can play roles of providing nutrient substances, preventing browning, promoting growth and preventing pollution, and bactericides such as penicillin, chloramphenicol and the like do not need to be added, so that the pesticide residue in the culture process is reduced, the use value of the anoectochilus formosanus is influenced, the pollution to the environment is also reduced, and the bactericides are prevented from influencing the growth of the anoectochilus formosanus; meanwhile, the culture medium can promote the rooting and sprouting of the anoectochilus formosanus in tissue culture, can shorten the growth period of the anoectochilus formosanus, reduce the tissue culture cost of the anoectochilus formosanus, ensure that the obtained cultured seedlings are not easy to die during transplanting, increase the survival rate of the anoectochilus formosanus and facilitate the popularization and the planting of the anoectochilus formosanus.
2. The culture medium has reasonable formula, the chitosan is easy to dissolve in a weak acid solvent, and the dissolved chitosan contains amino (NH) 2+ ) The amino group combined with negative electrons can play a role in inhibiting the activity of bacteria and can promote the healthy growth of anoectochilus formosanus; the sodium thiosulfate has an inhibiting effect on gram-positive bacteria and coliform groups, and can prevent the bacterial group pollution; the eclipta alba extract has an antibacterial effect on staphylococcus aureus, typhoid straw bacteria, dysentery sonnei and pseudomonas aeruginosa, no additional bactericide is added, pesticide residues are reduced, meanwhile, the beneficial substances contained in the eclipta alba extract are utilized to play a role in preventing browning, and the survival rate and the quality of anoectochilus formosanus are improved; the chitosan, the sodium thiosulfate and the eclipta extract have a synergistic effect, the activity of bacteria is inhibited by combining amino groups dissolved by the chitosan with negative electrons, and flora pollution is prevented by the sodium thiosulfate and the eclipta extract, so that the pollution rate and pesticide residues are reduced; the survival rate of anoectochilus formosanus is improved.
3. The activated carbon can completely adsorb growth regulating substances in the culture medium, changes the hormone amount in the culture medium and the ratio of cytokinin to auxin in the culture medium, has larger surface area and porosity, can adsorb certain oxygen, is beneficial to the growth of basal roots of anoectochilus roxburghii plants, and improves the growth amount of the plants; the eclipta alba extract and the active carbon can be cooperated to further enhance the effect of preventing browning.
4. Mecobalamin can inhibit the growth of infectious microbes, avoid infection and decay, improve the protoplasm activity of cells, promote cell division, accelerate the formation and differentiation of callus, and shorten the formation time of root systems; the purple sweet potato extract can promote the growth of roots and stems, and has an obvious promotion effect on the germination and development of anoectochilus formosanus buds; the purple sweet potato extract and the mecobalamin have synergistic effect, so that the formation and differentiation of callus can be accelerated, the growth of roots and stems can be promoted, the tissue culture time can be effectively shortened, the cost of each reagent is low, and the culture cost is reduced.
5. The sucrose can provide carbohydrate for the anoectochilus formosanus, is used as a carbon source and an energy source, and can also play a role in maintaining osmotic pressure of a culture medium; agar has a large temperature difference between the freezing point and the melting point, and is used as a coagulant for a culture medium to prepare a solid culture medium; the 6-benzyl adenine and the sodium naphthalene acetate can promote the formation of buds, induce the generation of calluses of the anoectochilus formosanus, effectively improve the adventitious bud induction rate and the number of adventitious buds of the anoectochilus formosanus and improve the plant quality of the anoectochilus formosanus; indolebutyric acid can induce the formation of a root protomer, promote cell differentiation and division, facilitate the generation of new roots and the differentiation of a vascular bundle system, promote the formation of adventitious roots of cuttings, shorten the culture period of anoectochilus roxburghii and improve the quality of anoectochilus roxburghii plants.
6. The culture medium disclosed by the invention is obvious in using effect, the preparation method is simple to operate and convenient to control, the anoectochilus formosanus tissue culture medium can be quickly prepared, and the problems of high pollution rate, influence of pesticide residues on the using value, easiness in browning and high tissue culture cost are effectively solved.
Detailed Description
Example 1
An anoectochilus formosanus tissue culture medium comprises the following components in parts by weight: 25 parts of cane sugar, 3 parts of agar, 3 parts of chitosan, 15 parts of eclipta extract, 10 parts of sodium thiosulfate, 0.001 part of mecobalamin, 20 parts of purple sweet potato extract, 1 part of 100-150 meshes of active carbon, 0.0006 part of 6-benzyl adenine, 0.00085 part of indolebutyric acid and 0.0008 part of sodium naphthaleneacetate; the eclipta alba extract is prepared by the following method: crushing eclipta, sieving with a 80-mesh sieve, adding 4 wt% of citric acid aqueous solution, extracting at 70 ℃ for 3 hours for 2 times, combining the extracting solutions for many times, and concentrating to 20-25% of the original volume to obtain the eclipta extracting solution, wherein the weight ratio of the eclipta to the citric acid aqueous solution is 1: 4; the culture medium also comprises MS culture medium mother liquor, and the mass volume ratio of the sucrose to the MS culture medium mother liquor is 25:100 g/mL.
The preparation method of the anoectochilus formosanus tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution III obtained in the step (3) for 10min at the temperature of 120 ℃ and under the pressure of 110-115 kPa, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Example 2
A tissue culture medium for anoectochilus formosanus comprises the following components in parts by weight: 26 parts of cane sugar, 4 parts of agar, 4 parts of chitosan, 16 parts of eclipta extract, 13 parts of sodium thiosulfate, 0.002 part of mecobalamin, 23 parts of purple sweet potato extract, 1 part of 100-150 meshes of active carbon, 0.0007 part of 6-benzyl adenine, 0.0009 part of indolebutyric acid and 0.0009 part of sodium naphthylacetate; the eclipta alba extract is prepared by the following method: crushing eclipta, sieving with a sieve of 80-100 meshes, adding 4 wt% of citric acid aqueous solution, extracting for 2 times at 71 ℃ for 3 hours with the weight ratio of 1:4, combining the extracting solutions for many times, and concentrating to 20-25% of the original volume to obtain the eclipta extracting solution; the culture medium also comprises MS culture medium mother liquor, and the mass-to-volume ratio of the sucrose to the MS culture medium mother liquor is 26:110 g/mL.
The preparation method of the anoectochilus formosanus tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, eclipta extract and purple sweet potato extract, adding into distilled water, stirring and dissolving to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution III obtained in the step (3) for 14min at the temperature of 123 ℃ under the pressure of 110-115 kPa, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Example 3
A tissue culture medium for anoectochilus formosanus comprises the following components in parts by weight: 28 parts of cane sugar, 5 parts of agar, 5 parts of chitosan, 17 parts of eclipta extract, 15 parts of sodium thiosulfate, 0.003 part of mecobalamin, 25 parts of purple sweet potato extract, 2 parts of activated carbon, 0.0008 part of 6-benzyladenine, 0.0012 part of indolebutyric acid and 0.001 part of sodium naphthylacetate; the eclipta alba extract is prepared by the following method: crushing eclipta, sieving with a sieve of 80-100 meshes, adding 4 wt% of citric acid aqueous solution, extracting 3 times at 73 ℃ for 4 hours with the weight ratio of 1:5, combining the extracting solutions for many times, and concentrating to 20-25% of the original volume to obtain the eclipta extracting solution; the culture medium also comprises MS culture medium mother liquor, and the mass volume ratio of the sucrose to the MS culture medium mother liquor is 28:125 g/mL.
The preparation method of the anoectochilus formosanus tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution III obtained in the step (3) for 15min at 125 ℃ under 110-115 kPa, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Example 4
A tissue culture medium for anoectochilus formosanus comprises the following components in parts by weight: 28 parts of cane sugar, 5 parts of agar, 5 parts of chitosan, 18 parts of eclipta extract, 18 parts of sodium thiosulfate, 0.004 part of mecobalamin, 30 parts of purple sweet potato extract, 2 parts of 100-150 meshes of active carbon, 0.0009 part of 6-benzyl adenine, 0.0013 part of indolebutyric acid and 0.0011 part of sodium naphthaleneacetate; the eclipta alba extract is prepared by the following method: crushing eclipta, sieving with a sieve of 80-100 meshes, adding 5wt% of citric acid aqueous solution, extracting 3 times at 73 ℃ for 4 hours with the weight ratio of 1:6, combining the extracting solutions for many times, and concentrating to 20-25% of the original volume to obtain the eclipta extracting solution; the culture medium also comprises MS culture medium mother liquor, and the mass volume ratio of the sucrose to the MS culture medium mother liquor is 28:140 g/mL.
The preparation method of the anoectochilus formosanus tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution III obtained in the step (3) for 18min at the temperature of 128 ℃ and under the pressure of 110-115 kPa, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Example 5
A tissue culture medium for anoectochilus formosanus comprises the following components in parts by weight: 30 parts of cane sugar, 6 parts of agar, 6 parts of chitosan, 20 parts of eclipta extract, 20 parts of sodium thiosulfate, 0.004 part of mecobalamin, 30 parts of purple sweet potato extract, 3 parts of 100-150 meshes of active carbon, 0.001 part of 6-benzyl adenine, 0.0015 part of indolebutyric acid and 0.0012 part of sodium naphthaleneacetate; the eclipta alba extract is prepared by the following method: crushing eclipta, sieving by a sieve of 80-100 meshes, adding 5wt% of citric acid aqueous solution, extracting for 5 hours at 74 ℃ for 4 times, combining extracting solutions for many times, and concentrating to 20% -25% of the original volume to obtain an eclipta extracting solution, wherein the weight ratio of the eclipta to the citric acid aqueous solution is 1: 6; the culture medium also comprises MS culture medium mother liquor, and the mass volume ratio of the sucrose to the MS culture medium mother liquor is 30:150 g/mL.
The preparation method of the anoectochilus roxburghii tissue culture medium comprises the following steps:
(1) weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
(2) adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthalene acetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
(3) mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III;
(4) and (4) sterilizing the mixed solution III obtained in the step (3) for 20min at the temperature of 30 ℃ and under the pressure of 110-115 kPa, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
Comparative example 1
Comparative example 1 differs from example 3 in that: anoectochilus roxburghii tissue culture medium lacks sodium thiosulfate and is the same as example 3.
Comparative example 2
Comparative example 2 differs from example 3 in that: the purple sweet potato extract is absent from the anoectochilus formosanus tissue culture medium, and the rest is the same as the example 3.
Comparative example 3
Comparative example 3 differs from example 3 in that: anoectochilus roxburghii tissue culture medium lacks mecobalamin, and the rest is the same as example 3.
Comparative example 4
A culture medium for reducing the tissue culture pollution rate of anoectochilus roxburghii comprises 1/2MS, 40-60 g/L banana, 0.8-1.2 g/L, NAA 0.8.8-1.2 mg/L active carbon, 0.4-0.8 mg/L6-BA, 22-30 g/L sucrose, 5.5-6.5 g/L agar powder, 40-60 g/L onion and 10-20 g/L crab shell powder.
Adopting the culture mediums obtained in the examples 1-5 and the comparative examples 1-4 to perform tissue culture on the anoectochilus formosanus, wherein the conditions such as illumination, humidity and the like are all the same, after three months of culture, 10 anoectochilus formosanus in each group are selected to measure the rooting speed, the length of a new root and the average fresh weight of each individual plant, and the rooting rate, the adventitious bud pollution rate and the survival rate are calculated, and the results are shown in table 1.
TABLE 1 statistical table of growth conditions of Anoectochilus roxburghii
As can be seen from Table 1, the anoectochilus formosanus obtained by tissue culture by using the culture medium of the invention has the advantages of high rooting speed, average new root length and average fresh weight of single plant which are superior to those of common culture media, high rooting rate, high survival rate and low pollution rate; meanwhile, comparison of comparative example 1 with example 3 demonstrates that sodium thiosulfate can play a role in preventing flora contamination; the comparison between the comparative example 2 and the example 3 shows that the purple sweet potato extract can promote the growth of rhizomes, promote the tissue formation and differentiation and shorten the root formation time; comparative example 3 and example 3 compare and demonstrate that mecobalamin can inhibit the growth of mixed bacteria, promote cell division, accelerate the formation and differentiation of callus and shorten the formation time of root system. Therefore, the culture medium can promote the rooting and sprouting of the anoectochilus formosanus in tissue culture, reduce the pollution rate, ensure that the obtained anoectochilus formosanus is not easy to die, increase the survival rate of the anoectochilus formosanus, have larger fresh weight, can obtain anoectochilus formosanus plants with better quality, and is convenient for the popularization and the planting of the anoectochilus formosanus.
While the present invention has been described in detail with reference to the specific embodiments thereof, it should not be construed as limited by the scope of the present patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
Claims (6)
1. The anoectochilus formosanus tissue culture medium is characterized by comprising the following components in parts by weight: 25-30 parts of cane sugar, 3-6 parts of agar, 3-6 parts of chitosan, 15-20 parts of eclipta extract, 10-20 parts of sodium thiosulfate, 1-3 parts of activated carbon, 0.001-0.004 part of mecobalamine, 20-30 parts of purple sweet potato extract, 0.0006-0.001 part of 6-benzyl adenine, 0.0008-0.0015 part of indolebutyric acid and 0.0008-0.0012 part of sodium naphthalene acetate;
the mass volume ratio of the sucrose to the MS culture medium mother liquor is 25-30: 100-150 g/mL;
the eclipta alba extract is prepared by the following method: crushing eclipta, sieving with a sieve of 80-100 meshes, adding 4-5 wt% of citric acid aqueous solution, extracting for 3-5 hours at the temperature of 70-74 ℃, extracting for 2-4 times, combining extracting solutions for many times, and concentrating to 20-25% of the original volume to obtain an eclipta extracting solution;
the purple sweet potato extract is prepared by the following method: adding 2 times of water into the purple sweet potato, and squeezing to obtain juice; then drying the juice, and crushing the dried juice into powder of 100-200 meshes, wherein the powder is the purple sweet potato extract;
the anoectochilus roxburghii tissue culture medium is prepared by the following method:
step (1): weighing sucrose, agar, chitosan, an eclipta extract and a purple sweet potato extract, and then adding distilled water to stir and dissolve to obtain a mixed solution I;
step (2): adding sodium thiosulfate, mecobalamin, 6-benzyladenine, indolebutyric acid and sodium naphthylacetate into the MS culture medium mother liquor, and stirring and dissolving to obtain a mixed solution II;
and (3): mixing the mixed solution I obtained in the step (1) and the mixed solution II obtained in the step (2), adding activated carbon, uniformly stirring, adding distilled water to a constant volume, and adjusting the pH value to 5.4-5.6 to obtain a mixed solution III; adding distilled water to a constant volume, wherein the sucrose concentration is 25-30 g/L;
and (4): and (4) sterilizing the mixed solution obtained in the step (3) at high temperature, cooling and subpackaging to obtain the anoectochilus formosanus tissue culture medium.
2. The anoectochilus formosanus tissue culture medium of claim 1, which comprises the following components in parts by weight: 28 parts of cane sugar, 5 parts of agar, 5 parts of chitosan, 17 parts of eclipta extract, 15 parts of sodium thiosulfate, 2 parts of activated carbon, 0.003 part of mecobalamin, 25 parts of purple sweet potato extract, 0.0008 part of 6-benzyladenine, 0.0012 part of indolebutyric acid and 0.001 part of sodium naphthylacetate;
the culture medium further comprises MS culture medium mother liquor, and the mass-to-volume ratio of the sucrose to the MS culture medium mother liquor is 28:125 g/mL.
3. The anoectochilus formosanus tissue culture medium according to claim 1, wherein the weight ratio of the eclipta and the citric acid aqueous solution is 1: 4-6.
4. The anoectochilus formosanus tissue culture medium according to claim 1 or 2, wherein the mesh number of the activated carbon is 100-150 meshes.
5. The tissue culture medium of Anoectochilus roxburghii according to claim 1, wherein the pH value is adjusted by using sodium hydroxide solution or hydrogen chloride solution in step (3).
6. The tissue culture medium of Anoectochilus roxburghii according to claim 1, wherein the sterilization in step (4) is performed at 120-130 ℃ under 110-115 kPa for 10-20 min.
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