CN115568419A - Rapid propagation liquid culture medium for potato tissue culture seedlings for field planting and rapid propagation method for tissue culture seedlings for field planting of virus-free miniature potatoes - Google Patents
Rapid propagation liquid culture medium for potato tissue culture seedlings for field planting and rapid propagation method for tissue culture seedlings for field planting of virus-free miniature potatoes Download PDFInfo
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- CN115568419A CN115568419A CN202211294501.6A CN202211294501A CN115568419A CN 115568419 A CN115568419 A CN 115568419A CN 202211294501 A CN202211294501 A CN 202211294501A CN 115568419 A CN115568419 A CN 115568419A
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- 235000002595 Solanum tuberosum Nutrition 0.000 title claims abstract description 46
- 244000061456 Solanum tuberosum Species 0.000 title claims abstract description 46
- 239000001963 growth medium Substances 0.000 title claims abstract description 44
- 235000012015 potatoes Nutrition 0.000 title claims abstract description 21
- 238000009630 liquid culture Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 title claims abstract description 14
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 20
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229920000742 Cotton Polymers 0.000 claims abstract description 12
- 229920001525 carrageenan Polymers 0.000 claims abstract description 12
- 235000010418 carrageenan Nutrition 0.000 claims abstract description 12
- 239000000679 carrageenan Substances 0.000 claims abstract description 11
- 229940113118 carrageenan Drugs 0.000 claims abstract description 11
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims abstract description 11
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 10
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 5
- 239000001110 calcium chloride Substances 0.000 claims abstract description 5
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 5
- 238000005520 cutting process Methods 0.000 claims abstract description 5
- 238000009835 boiling Methods 0.000 claims abstract description 4
- 239000010413 mother solution Substances 0.000 claims abstract description 4
- 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 3
- 229930006000 Sucrose Natural products 0.000 claims abstract description 3
- 238000004519 manufacturing process Methods 0.000 claims description 19
- 238000012136 culture method Methods 0.000 claims description 15
- 238000005286 illumination Methods 0.000 claims description 8
- 239000012452 mother liquor Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 230000001902 propagating effect Effects 0.000 claims description 4
- 229930191978 Gibberellin Natural products 0.000 claims description 3
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 claims description 3
- 239000003448 gibberellin Substances 0.000 claims description 3
- 206010021033 Hypomenorrhoea Diseases 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000003630 growth substance Substances 0.000 claims description 2
- 238000004904 shortening Methods 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims 3
- 239000002609 medium Substances 0.000 claims 3
- 238000004161 plant tissue culture Methods 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 description 8
- 239000007787 solid Substances 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 6
- 230000003203 everyday effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000003349 gelling agent Substances 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- -1 generally speaking Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
- A01H4/002—Culture media for tissue culture
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation liquid culture medium of a tissue culture seedling for field planting and a rapid propagation method of a tissue culture seedling for field planting of potato virus-free mini potatoes. The invention provides a tissue culture seedling rapid propagation liquid culture medium for potato field planting and a tissue culture seedling rapid propagation method for potato virus-free miniature potato field planting, 1) ammonium nitrate in an MS culture medium formula is replaced by ammonium chloride, and calcium chloride is replaced by calcium nitrate; 2) Replacing carrageenan with ginned cotton; 3) The consumption of each mother solution and white sugar is reduced by 10 to 30 percent; 4) Directly filling without boiling, wherein the filling amount of a 240 type culture bottle is 20-25 ml; 5) Propagation: 1-2 blades of the mother seedling are cut for one section, cuttage is not needed after the cutting, and the mother seedling is scattered lightly and shaken evenly; tissue culture: the tissue culture was performed at 3300Lx light intensity. The method of the invention improves the propagation speed by more than 30 percent, shortens the tissue culture time by 7 days and reduces the cost by more than 35 percent.
Description
The application is a divisional application of a tissue culture method for rapid propagation of tissue culture seedlings for permanent planting in the production process of miniature potato virus-free potatoes, wherein the application date is 13/08/2020, and the application number is 202010811475.4.
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation liquid culture medium for potato tissue culture seedlings for field planting and a rapid propagation method for the tissue culture seedlings for field planting in the production process of potato virus-free miniature potatoes.
Background
Ammonium nitrate is an important component of the existing culture medium, because of the explosion danger, ammonium nitrate is forbidden at present, the problem of plant tissue culture is brought after the state prohibits selling ammonium nitrate, the medicines required by the culture medium cannot be filled, the nutrient elements required by the plant cannot be met, and the seeking of substitutes and the using amount of the substitutes becomes urgent. In addition, mass production of tissue culture seedlings requires a large amount of culture medium, and therefore, how to rapidly produce the culture medium is also critical.
The production of the potato virus-free miniature potato has strong seasonality and is directly related to yield and quality. Therefore, a large amount of seedlings for field planting need to be produced in a short field planting proper period, and the soldiers need to be cultivated for thousands of days. Enterprises for producing detoxified miniature potatoes in large-scale factories face that the suitable planting time of tissue culture seedlings is short, the tissue culture seedlings cannot prepare seeds in advance like other crops, the seeds can be produced in advance for one month, operators cannot bring one person to work at random, skilled workers are needed, and the enterprises are greatly burdened and cost if enough workers are kept all the year round.
Disclosure of Invention
In view of the state of the prior art, the invention aims to provide a liquid culture medium for fast propagation of potato tissue culture seedling field planting and a tissue culture method for fast propagation of tissue culture seedling for field planting in the production process of potato virus-free miniature potatoes. In particular from the point of view of the culture medium.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a liquid culture medium for field planting and rapid propagation of potato tissue culture seedlings, which is characterized in that an MS culture medium is improved, ammonium nitrate in the MS culture medium is replaced by ammonium chloride, calcium chloride is replaced by calcium nitrate, the dosage of the ammonium chloride is 1300 +/-200 mg/L, the dosage of the calcium nitrate is 350mg/L or the dosage of the ammonium chloride is 650mg/L, and the dosage of the calcium nitrate is 800mg/L.
The invention has the technical scheme that a tissue culture method for rapid propagation of tissue culture seedlings for field planting in the production process of virus-free miniature potatoes, and the formula of a culture medium is adjusted; the consumption of mother liquor is reduced; preparing and filling a culture medium; inoculating and propagating; tissue culture;
the method comprises the following steps:
1) Ammonium nitrate (forbidden in China) in the MS culture medium formula is replaced by ammonium chloride, and calcium chloride is replaced by calcium nitrate;
2) Replacing carrageenan with ginned cotton;
3) The consumption of each mother solution and white sugar is reduced by 10 to 30 percent;
4) Filling directly at 100 ℃ for more than two minutes (without boiling), wherein the filling amount of a 240-type culture bottle is 20-25 mL;
5) Propagation: 1-2 blades of the mother seedling are cut for one section, cuttage is not needed after the cutting, the seedling is scattered lightly and shaken evenly, and the orientation of the stem section is not needed.
6) Tissue culture: the illumination intensity of tissue culture is 3300Lx, the illumination for the first 5 days is 10 + -1 h, and the illumination for the later 5 days is changed into 16 + -2 h; the temperature of the tissue culture was: the light period is 23 +/-1 ℃, and the dark period is 17 +/-1 ℃; culturing for 15-20 days to obtain the seedling and field planting.
Preferably, the amount of the ammonium chloride used in step 1) is: 1300 +/-200 mg/L, the dosage of calcium nitrate is: 350mg/L.
Preferably, the type 240 culture flask used in the step 2) uses 0.15 +/-0.05 g of ginned cotton.
Preferably, the amount of the mother liquor used in the step 3) is reduced by 20% + -0.05.
Preferably, the filling amount of the 240 type culture bottle in the step 4) is 20-25 mL, and the consumption is reduced by 25%.
Preferably, 25 stem segments are inoculated per bottle in step 5), and cutting is not required.
After the tender shoots are formed, the tender shoots are sprayed by adopting a solution of 100-500 mg/1000mL of gibberellin serving as growth regulator. Gibberellin 100 or 500mg/1000mL solution is formulated with 5 or 15wt% alcohol.
The invention provides an application of the rapid propagation liquid culture medium or the tissue culture method in the technical scheme in one or more of the following (1) to (3);
(1) The seedling yield for field planting in the production of the miniature potatoes is improved;
(2) The seedling cost for field planting in the production of the miniature potatoes is reduced;
(3) Shortening the seedling period of the tissue culture seedling.
Preferably, the potatoes comprise potato detoxified mini-potato varieties; the potato virus-free miniature potato variety comprises Atlantic, dutch potato, YOUJIN 885, and Zhongshui five or 226.
The invention has the beneficial effects that: according to the method, the formula of the culture medium is adjusted, the carrageenan is replaced by the ginned cotton, the culture medium is rapidly prepared, the cuttage links in the inoculation process are reduced, the inoculation speed is increased, and the inoculation speed is increased from 200 bottles inoculated by each worker every day to more than 300 bottles; in addition, due to the good fluidity of the nutrient elements of the liquid culture, the tissue culture seedlings grow rapidly, the seedling period is shortened, 20-25 days are needed originally, and only about 15 days are needed at present. Can realize the batch rapid propagation of the seedlings for field planting in the production of the miniature potatoes, simultaneously save the medicine and the electric energy, reduce the cost by 33 percent and increase the yield by 30 percent. The inoculation efficiency is improved mainly because one process, namely cuttage, is reduced. The culture medium preparation efficiency is improved because the culture medium does not need to be boiled, and the time is saved. The culture time is shortened because the water culture is equivalent to that after the carrageenan is replaced by the lint, and the solid culture is realized by the carrageenan. In water culture, nutrient elements are easier to absorb and grow quickly. In general, the tissue culture method of the present invention is actually liquid culture. The prior art adopts solid culture, namely, carrageenan or agar and other gelling agents are used for boiling, which consumes much time. However, liquid culture has a defect that contamination of endophytes cannot be distinguished, so the method can only be used for the last propagation before field planting. The requirement of using a large number of seedlings in the planting period is met; another advantage that comes is: the workload of workers is reduced, the solid culture medium is 210 bottles every day, and the liquid culture medium is 330 bottles every day. The cost can be reduced. The invention can quickly meet the requirement of seasonal planting seedlings in the production process of the miniature detoxified potatoes, and fewer inoculators complete large-area production seedlings.
Detailed Description
The invention provides a tissue culture method for rapid propagation of tissue culture seedlings for field planting in the production process of miniature detoxified potatoes, which comprises the following steps:
1) Adjusting the formula of the culture medium; 2) Replacing carrageenan with ginned cotton; 3) The consumption of mother liquor is reduced; 4) The culture medium is not required to be boiled in the preparation process, and is directly filled, so that the time is saved; 5) Inoculating and propagating; 6) And (4) tissue culture.
The potato detoxified miniature potato variety for production is a main domestic circulating variety at present, and comprises the following specific varieties: atlantic, netherlands, eujin 885, zhongshu five, 226.
The invention firstly adjusts the formula of the culture medium to adapt to the current market supply and demand conditions. Ammonium nitrate (a dangerous substance) is strictly forbidden to be sold in the market at present, and mass production is difficult to buy, so ammonium chloride is adopted for replacing ammonium nitrate, but calcium chloride in other elements is changed into calcium nitrate in consideration of the damage of chloride ions to potatoes and the tolerance of the potatoes to the chloride ions, and the dosage is determined through multiple groups of tests. In the invention, the ginned cotton is used for replacing the carrageenan, generally speaking, the culture medium is solid, the carrageenan, the agar and the like are used as gelling agents, firstly, the stem section of the potato virus-free seedling is fixed like soil, and secondly, the microbial pollution is conveniently checked. The invention aims at the production seedlings in the field planting stage, the culture medium adopts a liquid culture medium, and the ginned cotton (cotton) only plays a role of supporting and floating the stem section, so that the stem section is not submerged in the nutrient solution, and the number of glass seedlings is reduced. 0.15g of lint for type 240 flask.
The reduction of the dosage of each mother solution of the culture medium is based on the poor fluidity of nutrient components in the solid culture medium, which is not beneficial to absorption and low in utilization rate; the liquid culture medium has good fluidity of nutrient components and high utilization rate, so the method properly reduces the nutrient components and reduces the cost under the condition of meeting the growth requirement. The dosage is reduced by 20 percent through a plurality of tests.
Because the invention uses the ginned cotton to replace the carrageenin, the preparation of the culture medium does not need to be boiled, thereby saving a great deal of time (one pot of 50L of solid culture medium needs to be boiled for 2 hours). In the invention, the filling amount of the culture medium is reduced by one fourth compared with the solid, and 25mL of a 240-type culture bottle is needed, so that the lint is just immersed in the nutrient solution.
In the inoculation process of the invention, the cut stem segments do not need to be cut one by using tweezers, and can be uniformly distributed on cotton by only slightly scattering or shaking, and the front surface of the stem segment does not need to be upward, so that the inoculation speed is greatly improved.
The invention can meet the requirement only by giving 10h of illumination in the initial stage of the stem tissue culture. After the formation of shoots, the shoots were exposed to light for 16 h. Illumination intensity 3300lx.
The temperature for tissue culture is 23 ℃ in the photoperiod and 17 ℃ before darkness.
The tissue culture time of the invention is 15-20 days, and the requirements of field planting can be met.
The tissue culture method provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Examples
The invention provides a tissue culture method for rapid propagation of tissue culture seedlings for field planting in the production process of miniature detoxified potatoes, which comprises the following steps:
1) Adjusting the formula of the culture medium; 2) Replacing carrageenan with ginned cotton; 3) The consumption of mother liquor is reduced; 4) Preparing and filling a culture medium; 5) Inoculating and propagating; 6) And (5) tissue culture.
A typical modified MS medium is formulated as follows:
TABLE 1 modified MS Medium formulation
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. The utility model provides a potato tissue culture seedling rapid propagation liquid medium for field planting which characterized in that, the liquid medium improves the MS culture medium, replaces the ammonium nitrate of MS culture medium with ammonium chloride, and the calcium chloride is replaced with calcium nitrate, the ammonium chloride quantity is 1300 + -200 mg/L, the calcium nitrate quantity is 350mg/L or the ammonium chloride quantity is 650mg/L, the calcium nitrate quantity is 800mg/L.
2. A tissue culture method for rapid propagation of tissue culture seedlings for field planting in the production process of virus-free miniature potatoes is characterized by comprising the steps of adjusting the formula of a culture medium; the consumption of mother liquor is reduced; preparing and filling a culture medium; inoculating and propagating; tissue culture;
the method comprises the following specific steps:
1) Using the liquid medium of claim 1;
2) Replacing carrageenan with ginned cotton;
3) The consumption of each mother solution and white sugar is reduced by 10 to 30 percent;
4) Directly filling without boiling, wherein the filling amount of a 240-type culture bottle is 20-25 ml;
5) Propagation: 1-2 leaves of the mother seedling are cut for one section, cuttage is not needed after the cutting is finished, and the mother seedling is scattered lightly and shaken evenly;
6) Tissue culture: the illumination intensity of tissue culture is 3300Lx, the illumination for the first 5 days is 10 + -1 h, and the illumination for the later 5 days is changed into 16 + -2 h; the temperature of the tissue culture was: the light period is 23 +/-1 ℃, and the dark period is 17 +/-1 ℃; culturing for 15-20 days to obtain the seedling and field planting.
3. The tissue culture method of claim 2, wherein the lint for type 240 flask in step 2) is 0.15 ± 0.05g.
4. The tissue culture method of claim 2, wherein the amount of the mother liquor used in the step 3) is reduced by 20% ± 0.05.
5. The tissue culture method according to claim 2, wherein the filling amount of the 240-type culture flask in the step 4) is 20-25 ml, and the consumption is reduced by 25%.
6. The tissue culture method of claim 2, wherein 25 stem segments are inoculated per bottle in step 5) without cutting.
7. The tissue culture method of claim 2, wherein the growth regulator gibberellin 100-500 mg/1000mL solution is sprayed on the tender shoots after the tender shoots are formed.
8. Use of the rapid propagation liquid culture medium of claim 1 or the tissue culture method of any one of claims 2 to 7 in one or more of the following (1) to (3);
(1) The yield of seedlings for field planting in the production of the miniature potatoes is improved;
(2) The seedling cost for field planting in the production of the miniature potatoes is reduced;
(3) Shortening the seedling period of the tissue culture seedling.
9. Use according to claim 8 or 9, wherein the potatoes comprise potato-detoxified mini-potato varieties; the potato virus-free miniature potato variety comprises Atlantic, dutch potato, YOUJIN 885, and Zhongshui five or 226.
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| CN202211294501.6A CN115568419A (en) | 2020-08-13 | 2020-08-13 | Rapid propagation liquid culture medium for potato tissue culture seedlings for field planting and rapid propagation method for tissue culture seedlings for field planting of virus-free miniature potatoes |
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| CN202211294501.6A CN115568419A (en) | 2020-08-13 | 2020-08-13 | Rapid propagation liquid culture medium for potato tissue culture seedlings for field planting and rapid propagation method for tissue culture seedlings for field planting of virus-free miniature potatoes |
| CN202010811475.4A CN111937745A (en) | 2020-08-13 | 2020-08-13 | Tissue culture method for rapid propagation of tissue culture seedlings for field planting in production process of detoxified miniature potatoes |
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| CN115843686A (en) * | 2022-12-03 | 2023-03-28 | 瑞盈优品(广东)农业科技有限责任公司 | Culture medium for sweet potato tissue culture and preparation method thereof |
| CN116114600A (en) * | 2022-12-06 | 2023-05-16 | 江苏宝德农业科技有限公司 | Application of paper in potato detoxification mini-potato production and rapid propagation and field planting method of tissue culture seedlings for potato detoxification mini-potato production |
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Application publication date: 20230106 |