CN107006369A - A kind of radix tetrastigme tissue culture culture medium and preparation method thereof - Google Patents
A kind of radix tetrastigme tissue culture culture medium and preparation method thereof Download PDFInfo
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- CN107006369A CN107006369A CN201710298056.3A CN201710298056A CN107006369A CN 107006369 A CN107006369 A CN 107006369A CN 201710298056 A CN201710298056 A CN 201710298056A CN 107006369 A CN107006369 A CN 107006369A
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- radix tetrastigme
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- 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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Abstract
The invention discloses a kind of radix tetrastigme tissue culture culture medium and preparation method thereof, field of tissue culture is particularly belonged to, its preparation method comprises the following steps：(1) pure water is added in container, boiling is heated to；(2) default component is sequentially added in container, and stirred, obtain mixed liquor；(3) pH of mixed solution is adjusted, and is dispensed and after sealing with conical flask, sterilizing in high-pressure steam sterilizing pan is positioned over, obtains the mixed liquor that sterilizing is completed；(4) mixed liquor for the completion that sterilizes is put into super-clean bench cooling, obtains radix tetrastigme tissue culture culture medium.Radix tetrastigme culture medium is prepared using method provided by the present invention and can obviously reduce radix tetrastigme explant by the possibility of living contaminants, so as to further increase the survival rate of radix tetrastigme explant, is conducive to the large area of radix tetrastigme to plant and promotes.
Description
Technical field
The invention belongs to field of tissue culture, specially a kind of radix tetrastigme tissue culture culture medium and preparation method thereof.
Background technology
Radix tetrastigme is the perennial liana that overgrows of Vitaceae, and whole strain can be used as medicine, and underground root tuber drug effect is more.Radix tetrastigme
Root tuber is sturdy, typically common to have spindle, ellipse or oval.Leaf alternate, petiole 2-4cm, palmately compound leaf.Vanelets are in narrow
Ellipse is about 3-7cm, wide about 1.5-3cm to narrow ovate-elliptic.Central lobule is bigger compared with both sides person, and leaf margin tool thorn-like is dredged
Tooth, leaf two sides is all hairless, or hair in middle arteries only below.Because the drug effect of radix tetrastigme is worth, therefore very worth progress three
Ye Qing Study on tissue culture.And it is urgent problem to be solved that radix tetrastigme survival rate is low during tissue culture.
The content of the invention
The invention provides a kind of radix tetrastigme tissue culture culture medium, the radix tetrastigme tissue culture culture medium can significantly improve radix tetrastigme block
The survival rate of root tissue.
To achieve the above object, the present invention provides following technical scheme:
A kind of preparation method of radix tetrastigme tissue culture culture medium, its preparation method comprises the following steps：
(1) pure water is added in container, boiling is heated to；
(2) default component is sequentially added in container, and stirred, obtain mixed liquor；
(3) pH of mixed solution is adjusted, and is dispensed with conical flask and after sealing, is positioned in high-pressure steam sterilizing pan and goes out
Bacterium, obtains the mixed liquor that sterilizing is completed；
(4) mixed liquor for the completion that sterilizes is put into super-clean bench cooling, obtains radix tetrastigme tissue culture culture medium.
Preferably, sterilization time is 45-60min in above-mentioned steps (3).
Preferably, above-mentioned radix tetrastigme tissue culture culture medium includes following component：Ammonium nitrate 0.8-1.2g, potassium nitrate 0.4-
0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.4-0.6g, sodium chloride 0.8-1.2g, glucose 1.2-1.4g, photocatalyst
0.1-0.5g, calcium chloride 0.2-0.3g, glycine 0.04-0.06g, auxin 0.01-0.02g, basic element of cell division 0.03-
0.05g, kinetin 0.01-0.02g, zeatin 0.01-0.02g, vitamin 0.04-0.08g, sucrose 1-3g and plant growth
Conditioning agent 0.04-0.09g.
Contain the inorganic nutrients thing needed in radix tetrastigme tissue culture procedures in above-mentioned culture medium：Epsom salt, chlorination
Calcium, ammonium nitrate, potassium dihydrogen phosphate, sodium chloride；Carbon source：Glucose；Organic additives：Glycine and vitamin；Growth regulator.
Above-mentioned five major classes nutriment is indispensable during radix tetrastigme tissue growth；
Meanwhile, 0.8-1.2g ammonium nitrate provides the nitrogen source of abundance for radix tetrastigme tissue growth, can effectively facilitate plant
The generation of protein, chlorophyll and nucleic acid in cell；0.4-0.6g potassium dihydrogen phosphates are also biology in radix tetrastigme histocyte
The formation of film, nucleotides and nucleic acid can promote there is provided sufficient P elements, and the potassium element that potassium dihydrogen phosphate is provided
Enter the transfer of nitrogen metabolism and carbohydrate, so as to accelerate the histiocytic growth of radix tetrastigme；0.2-0.3g calcium chloride
The calcium constituent of abundance is provided for the growth of radix tetrastigme histocyte wall；In addition, the culture medium also added epsom salt work
For magnesium source, accelerate the formation of chlorophyll；The Organic additives and growth regulator of addition can promote radix tetrastigme group in culture medium
Knit fast-growth.
Photocatalyst, i.e. nano titanium oxide 0.2-0.3g and nano zine oxide 0.2- are also added with above-mentioned culture medium
0.3g, photocatalyst will not only suppress the growth of radix tetrastigme cell, and can effectively kill from high-pressure steam sterilizing pan transfer
The microorganism infected during to super-clean bench, the ultraviolet-sterilization method in super-clean bench that compensate for can not kill miscellaneous bacteria inside culture medium
Defect.Miscellaneous bacteria is avoided to grow the growth of interference experiment personal observations radix tetrastigme tissue or being good for for miscellaneous bacteria influence radix tetrastigme explant
Kang Shengchang, so that the survival rate reduction of radix tetrastigme explant；Also, it is suitable also to be arranged in culture medium provided by the present invention
The plant growth regulator of parts by weight, the plant growth regulator can promote the induction differentiation of radix tetrastigme stem root tuber, so that
The radix tetrastigme explant survival rate is greatly increased.
More specifically, above-mentioned plant growth regulator includes：Auxin 0.01-0.02g, basic element of cell division 0.03-
0.05g, kinetin 0.01-0.02g and zeatin 0.01-0.02g.
Preferably, the photocatalyst is one or both of nano titanium oxide, nano zine oxide.
Preferably, the vitamin includes at least one of vitamin B1 and vitamin B2.
Preferably, described culture medium is by weight, including：Ammonium nitrate 0.8-1.2g, potassium nitrate 0.4-0.6g, sulfuric acid
Iron 0.2-0.3g, potassium dihydrogen phosphate 0.4-0.6g, sodium chloride 0.8-1.2g, glucose 1.2-1.4g, nano titanium oxide 0.2-
0.3g, nano zine oxide 0.2-0.3g, calcium chloride 0.2-0.3g, glycine 0.04-0.06g, auxin 0.01-0.02g, cell
Mitogen 0.03-0.05g, kinetin 0.01-0.02g, zeatin 0.01-0.02g, vitamin B1 0.02-0.04g, vitamin
B2 0.02-0.04g, epsom salt 0.2-0.4g, sucrose 1-3g and pure water 80-100g.
Above-mentioned radix tetrastigme tissue culture culture medium is specifically included by weight constituting：Ammonium nitrate 1.0g, potassium nitrate 0.5g, sulfuric acid
Iron 0.25g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, glucose 1.3g, nano titanium oxide 0.25g, nano zine oxide
0.25g, calcium chloride 0.25g, glycine 0.05g, auxin 0.015g, basic element of cell division 0.04g, kinetin 0.015g, corn
Plain 0.015g, vitamin B1 0.03g, vitamin B2 0.03g, epsom salt 0.3g, sucrose 2g and pure water 90g.
The preparation method of above-mentioned culture medium specifically includes following steps：
(1) 90ml pure water is added in container, boiling is heated to；
(2) ammonium nitrate 1.0g, potassium nitrate 0.5g, ferric sulfate 0.25g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, grape are taken
Sugared 1.3g, nano titanium oxide 0.25g, nano zine oxide 0.25g, calcium chloride 0.25g, glycine 0.05g, auxin
0.015g, basic element of cell division 0.04g, kinetin 0.015g, zeatin 0.015g, vitamin B1 0.03g, vitamin B2
0.03g and sucrose 2g are sequentially added in container, and are stirred, and obtain mixed liquor；
(3) 0.3g epsom salts are added in mixed solution, and are dispensed and after sealing with conical flask, high pressure steaming is positioned over
Sterilizing 50min in vapour autoclave, obtains the mixed liquor that sterilizing is completed；
(4) mixed liquor for the completion that sterilizes is put into super-clean bench cooling.
Compared with prior art, beneficial effects of the present invention are：
Radix tetrastigme culture medium is prepared using the above method and can obviously reduce radix tetrastigme explant by the possibility of living contaminants,
So as to further increase the survival rate of radix tetrastigme explant, be conducive to the large area of radix tetrastigme to plant and promote.
Embodiment
Technical scheme is described in further detail using embodiment below.
Embodiment 1
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 0.8g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.4g, sodium chloride 0.8g, Portugal
Grape sugar 1.2g, nano titanium oxide 0.2g, nano zine oxide 0.2g, calcium chloride 0.2g, glycine 0.04g, auxin 0.01g,
Basic element of cell division 0.03g, kinetin 0.01g, zeatin 0.01g, vitamin B1 0.02g, vitamin B2 0.02g, seven water sulphur
Sour magnesium 0.2g, sucrose 1g and pure water 80g.
Comparative example 1-1
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 0.8g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.4g, sodium chloride 0.8g, Portugal
Grape sugar 1.2g, calcium chloride 0.2g, glycine 0.04g, nano titanium oxide 0.2g, nano zine oxide 0.2g, vitamin B1
0.02g, vitamin B2 0.02g, epsom salt 0.2g, sucrose 1g and pure water 80g.
Comparative example 1-2
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 0.8g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.4g, sodium chloride 0.8g, Portugal
Grape sugar 1.2g, nano titanium oxide 0.2g, nano zine oxide 0.2g, calcium chloride 0.2g, glycine 0.04g, auxin 0.01g,
Basic element of cell division 0.03g, kinetin 0.01g, zeatin 0.01g, vitamin B1 0.02g, vitamin B2 0.02g, seven water sulphur
Sour magnesium 0.2g and pure water 80g.
Embodiment 2
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.0g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, Portugal
Grape sugar 1.3g, nano titanium oxide 0.25g, nano zine oxide 0.25g, calcium chloride 0.25g, glycine 0.05g, auxin
0.015g, basic element of cell division 0.04g, kinetin 0.015g, zeatin 0.015g, vitamin B1 0.03g, vitamin B2
0.03g, epsom salt 0.3g, sucrose 2g and pure water 90g.
Comparative example 2-1
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.0g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, Portugal
Grape sugar 1.3g, calcium chloride 0.25g, glycine 0.05g, nano titanium oxide 0.25g, nano zine oxide 0.25g, vitamin B1
0.03g, vitamin B2 0.03g, epsom salt 0.3g, sucrose 2g and pure water 90g.
Comparative example 2-2
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.0g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, Portugal
Grape sugar 1.3g, nano titanium oxide 0.25g, nano zine oxide 0.25g, calcium chloride 0.25g, glycine 0.05g, auxin
0.015g, basic element of cell division 0.04g, kinetin 0.015g, zeatin 0.015g, vitamin B1 0.03g, vitamin B2
0.03g, epsom salt 0.3g and pure water 90g.
Embodiment 3
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.2g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.6g, sodium chloride 1.2g, Portugal
Grape sugar 1.4g, nano titanium oxide 0.3g, nano zine oxide 0.3g, calcium chloride 0.3g, glycine 0.06g, auxin 0.02g,
Basic element of cell division 0.05g, kinetin 0.02g, zeatin 0.02g, vitamin B1 0.04g, vitamin B2 0.04g, seven water sulphur
Sour magnesium 0.4g, sucrose 3g and pure water 100g.
Comparative example 3-1
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.2g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.6g, sodium chloride 1.2g, Portugal
Grape sugar 1.4g, calcium chloride 0.3g, glycine 0.06g, nano titanium oxide 0.3g, nano zine oxide 0.3g, vitamin B1
0.04g, vitamin B2 0.04g, epsom salt 0.4g, sucrose 3g and pure water 100g.
Comparative example 3-2
The present embodiment provides a kind of radix tetrastigme tissue culture culture medium, by weight, by following material composition：
Ammonium nitrate 1.2g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.6g, sodium chloride 1.2g, Portugal
Grape sugar 1.4g, nano titanium oxide 0.3g, nano zine oxide 0.3g, calcium chloride 0.3g, glycine 0.06g, auxin 0.02g,
Basic element of cell division 0.05g, kinetin 0.02g, zeatin 0.02g, vitamin B1 0.04g, vitamin B2 0.04g, seven water sulphur
Sour magnesium 0.4g and pure water 100g.
Test case 1
It is 1~3 by the numbering of embodiment 1- embodiments 3, comparative example 1-1~1-2, comparative example 2-1~2-2 and comparative example 3-
The culture medium numbering that 1~3-2 is provided is a1~a2, b1~b2, c1~c2, will be derived from the three of Zhejiang A ＆ F University's Experimental Base
Leaf green grass or young crops root tuber histocyte is seeded on above-mentioned culture medium respectively, and every group sets 20 control experiment groups respectively；
Each culture medium is cultivated 20 days under 26 ± 1 DEG C of poor light conditions after inoculation, radix tetrastigme in more each culture medium is observed
The histiocytic growing state statistics of root tuber is as shown in table 1：
The radix tetrastigme root tuber tissue cell growth situation statistical form of table 1
Claims (9)
1. a kind of preparation method of radix tetrastigme tissue culture culture medium, it is characterised in that comprise the following steps：
(1) pure water is added in container, boiling is heated to；
(2) default component is sequentially added in container, and stirred, obtain mixed liquor；
(3) pH of mixed solution is adjusted, and is dispensed and after sealing with conical flask, sterilizing in high-pressure steam sterilizing pan is positioned over, obtains
The mixed liquor completed to sterilizing；
(4) mixed liquor for the completion that sterilizes is put into super-clean bench cooling, obtains radix tetrastigme tissue culture culture medium.
2. the preparation method of a kind of radix tetrastigme tissue culture culture medium according to claim 1, it is characterised in that in step (3)
Sterilization time is 45-60min.
3. a kind of preparation method of radix tetrastigme tissue culture culture medium according to claim 1, it is characterised in that the radix tetrastigme
Tissue culture culture medium includes following component：Ammonium nitrate 0.8-1.2g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, biphosphate
Potassium 0.4-0.6g, sodium chloride 0.8-1.2g, glucose 1.2-1.4g, photocatalyst 0.1-0.5g, calcium chloride 0.2-0.3g, glycine
0.04-0.06g, auxin 0.01-0.02g, basic element of cell division 0.03-0.05g, kinetin 0.01-0.02g, zeatin 0.01-
0.02g, vitamin 0.04-0.08g, sucrose 1-3g and plant growth regulator 0.04-0.09g.
4. a kind of radix tetrastigme tissue culture culture medium according to claim 3, it is characterised in that the plant growth regulator bag
Include：Auxin 0.01-0.02g, basic element of cell division 0.03-0.05g, kinetin 0.01-0.02g and zeatin 0.01-
0.02g。
5. a kind of radix tetrastigme tissue culture culture medium according to claim 3, it is characterised in that the photocatalyst is nano-silica
Change one or both of titanium, nano zine oxide.
6. a kind of radix tetrastigme tissue culture culture medium according to claim 3, it is characterised in that the vitamin includes vitamin
At least one of B1 and vitamin B2.
7. according to a kind of any described radix tetrastigme tissue culture culture mediums of claim 3-6, it is characterised in that by weight, including：
Ammonium nitrate 0.8-1.2g, potassium nitrate 0.4-0.6g, ferric sulfate 0.2-0.3g, potassium dihydrogen phosphate 0.4-0.6g, sodium chloride 0.8-
1.2g, glucose 1.2-1.4g, nano titanium oxide 0.2-0.3g, nano zine oxide 0.2-0.3g are calcium chloride 0.2-0.3g, sweet
Propylhomoserin 0.04-0.06g, auxin 0.01-0.02g, basic element of cell division 0.03-0.05g, kinetin 0.01-0.02g, zeatin
0.01-0.02g, vitamin B1 0.02-0.04g, vitamin B2 0.02-0.04g, epsom salt 0.2-0.4g, sucrose 1-
3g and pure water 80-100g.
8. a kind of radix tetrastigme tissue culture culture medium according to claim 7, it is characterised in that by weight composition, including：Nitre
Sour ammonium 1.0g, potassium nitrate 0.5g, ferric sulfate 0.25g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, glucose 1.3g, nano-silica
Change titanium 0.25g, nano zine oxide 0.25g, calcium chloride 0.25g, glycine 0.05g, auxin 0.015g, the basic element of cell division
0.04g, kinetin 0.015g, zeatin 0.015g, vitamin B1 0.03g, vitamin B2 0.03g, epsom salt 0.3g,
Sucrose 2g and pure water 90g.
9. the preparation method of culture medium according to claim 8, it is characterised in that comprise the following steps：
(1) 90ml pure water is added in container, boiling is heated to；
(2) ammonium nitrate 1.0g, potassium nitrate 0.5g, ferric sulfate 0.25g, potassium dihydrogen phosphate 0.5g, sodium chloride 1.0g, glucose are taken
1.3g, nano titanium oxide 0.25g, nano zine oxide 0.25g, calcium chloride 0.25g, glycine 0.05g, auxin 0.015g,
Basic element of cell division 0.04g, kinetin 0.015g, zeatin 0.015g, vitamin B1 0.03g, vitamin B2 0.03g and sugarcane
Sugared 2g is sequentially added in container, and is stirred, and obtains mixed liquor；
(3) 0.3g epsom salts are added in mixed solution, and is dispensed with conical flask and after sealing, is positioned over high steam and goes out
Sterilizing 50min in bacterium pot, obtains the mixed liquor that sterilizing is completed；
(4) mixed liquor for the completion that sterilizes is put into super-clean bench cooling.
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| CN201710298056.3A CN107006369A (en) | 2017-04-29 | 2017-04-29 | A kind of radix tetrastigme tissue culture culture medium and preparation method thereof |
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| CN107006369A true CN107006369A (en) | 2017-08-04 |
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| CN (1) | CN107006369A (en) |
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2017
- 2017-04-29 CN CN201710298056.3A patent/CN107006369A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 史清英: "三叶青茎段外植体组织培养体系的优化及印度梨形孢对其生长的影响", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 夏建明: "《染整助剂及其应用》", 30 September 2013, 中国纺织出版社 * |
| 张传海等: "《植物生物技术》", 30 September 2015, 合肥工业大学出版社 * |
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