CN108812313A - A kind of minimal medium of Plant Tissue Breeding - Google Patents
A kind of minimal medium of Plant Tissue Breeding Download PDFInfo
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- CN108812313A CN108812313A CN201810623423.7A CN201810623423A CN108812313A CN 108812313 A CN108812313 A CN 108812313A CN 201810623423 A CN201810623423 A CN 201810623423A CN 108812313 A CN108812313 A CN 108812313A
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- minimal medium
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- nitrate
- sulfate
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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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- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract
The invention discloses a kind of plant tissue culture media basal culture medium ZS,It is related to a kind of field of plant reproduction,The minimal medium ZS is to contain potassium nitrate 3075mg in the ZS minimal medium of every 1000mL,Ammonium phosphate 578mg,Calcium nitrate 491mg,Ammonium chloride 160mg,Magnesium nitrate 222mg,Ammonium sulfate 396mg,Potassium iodide 0.83mg,Boric acid 6.2mg,Manganese sulfate 22.3mg,Zinc sulfate 8.6mg,Sodium molybdate 0.25mg,Copper sulphate 0.025mg,Cobalt chloride 0.025mg,Inositol 100mg,Thiamine hydrochloride (VB1) 0.1mg,Puridoxine hydrochloride (VB6) 0.5mg,Niacin 0.5mg,Disodium ethylene diamine tetraacetate 37.3mg,Ferrous sulfate 27.8mg.Tissue culture effect identical with MS culture medium can be obtained in the case where not using ammonium nitrate using the minimal medium.
Description
Technical field
The present invention relates to plant tissue culture numerous fields fastly, and in particular to a kind of minimal medium of Plant Tissue Breeding.
Background technique
Murshige and Skoog culture medium (MS, 1962) is the plant tissue culture media being widely used at present, and ammonium nitrate is
The important component of the culture medium.Ammonium nitrate is a kind of strong oxidizer, when meeting combustible kindling, can encourage the intensity of a fire；With combustible powder
End mixing, can occur intense reaction and explode；It can also explode when by sharp pounding or sharply heating.Its with reducing agent, have
Such as mixing such as sulphur, phosphorus or metal powder of machine object, combustibles can form explosive mixture, be the raw material for manufacturing explosive.Because it is deposited
In huge security risk, in recent years, the such article of state supervision is increasingly stringenter, and market circulation is by national departments concerned
The difficult of ammonium nitrate is bought in limitation, laboratory, or even purchase is made troubles less than ammonium nitrate to research and production.But
Ammonium nitrogen and nitrate nitrogen are existed simultaneously in ammonium nitrate molecule, is necessary to plant growth, it is extensive in Plant Tissue Breeding
Using.Therefore, it is badly in need of one kind and had not only been able to maintain the performance of original MS culture medium, but also is avoided that basic using the plant tissue of ammonium nitrate
Culture medium.Currently, CN102224802A proliferation medium for strawberry, open one kind can replace the culture medium of ammonium nitrate, but should
Culture medium is only applicable to proliferation medium for strawberry, does not have versatility, is not suitable for promoting.
Summary of the invention
For the deficiencies in the prior art, the invention discloses a kind of minimal mediums of Plant Tissue Breeding, should
Culture medium can be suitably used for a variety of frequently seen plants tissue cultures, and obtain the effect same or similar with MS culture medium.
In order to solve the above technical problems, the technical scheme is that：
The present invention provides a kind of minimal medium ZS of Plant Tissue Breeding, contains in every 1000mL culture medium：Potassium nitrate
3075mg, ammonium phosphate 578mg, calcium nitrate 491mg, ammonium chloride 160mg, magnesium nitrate 222mg, ammonium sulfate 396mg, potassium iodide
0.83mg, boric acid 6.2mg, manganese sulfate 22.3mg, zinc sulfate 8.6mg, sodium molybdate 0.25mg, copper sulphate 0.025mg, cobalt chloride
0.025mg, inositol 100mg, thiamine hydrochloride (VB1) 0.1mg, puridoxine hydrochloride (VB6) 0.5mg, niacin 0.5mg, ethylenediamine
Tetraacethyl disodium 37.3mg, ferrous sulfate 27.8mg.
Specific invention process of the invention is as follows：
(1) according to the content (table 1) of a great number of elements in MS culture medium, the ion concentration (table 2) of each element is calculated；
The composition and content of a great number of elements in 1 MS culture medium of table
The various ion concentrations of a great number of elements in 2 MS culture medium of table
| Ion | Ion concentration (mmol/L) |
| K+ | 20.04 |
| NO3- | 39.40 |
| NH4 + | 20.61 |
| PO4 3- | 1.25 |
| Ca2+ | 2.99 |
| Mg2+ | 1.50 |
| SO4 2- | 1.50 |
| Cl- | 3.00 |
(2) remove ammonium nitrate (NH4NO3), it finds suitable chemical reagent and is combined substitution (table 3)；
The present invention maintains NO3 -、NH4 +、Ca2+、Mg2+、Cl-The concentration of plasma is constant, has properly increased K+、PO4 3-、
SO4 2-The concentration (table 4) of ion, this is key point place of the invention.Inventor is shown by many experiments：The present invention is mentioned
The ZS medium component of confession forms and content, breaks up in callus induction, the bud of tobacco, takes root and potato test tube seedling culture
Etc., effect similar with MS culture medium can be obtained.
The ZS culture medium a great number of elements of the present invention of table 3 composition and content
| Chemical reagent | Chemical molecular formula | Molecular weight | Dosage (mg/L) | Reagent concentration mmol/L |
| Potassium nitrate | KNO3 | 101.11 | 3075 | 30.42 |
| Ammonium phosphate | (NH4)3PO4 | 203.13 | 578 | 3.88 |
| Calcium nitrate | Ca(NO3)2·4H2O | 236.18 | 707 | 2.99 |
| Ammonium chloride | NH4Cl | 53.49 | 160 | 3.00 |
| Magnesium nitrate | Mg(NO3)2·6H2O | 256.40 | 384 | 1.50 |
| Ammonium sulfate | (NH4)2SO4 | 132.14 | 396 | 3.00 |
The various ion concentrations of a great number of elements in 4 ZS culture medium of table
| Ion | Ion concentration (mol/L) |
| K+ | 30.42* |
| NO3 - | 39.40 |
| NH4 + | 20.61 |
| PO4 3- | 3.88* |
| Ca2+ | 2.99 |
| Mg2+ | 3.00 |
| SO4 2- | 3.00* |
| Cl- | 3.00 |
Note：* the ion concentration to change.
(3) other elements and content are constant in MS culture medium.
The present invention fully considers N, P, K ratio, nitrate nitrogen and ammonium nitrogen ratio, and the pH value of culture medium, plant is to chlorine, sulphur etc.
The factors such as the demand of element realize ion concentration, interionic rationally match by changing the composition and content of chemical substance
Than the regulation of, charge number etc., difficulty is larger.Inventor reaches final culture medium by the optimization of tens of secondary chemical substances
The effect same or similar with MS culture medium.
Beneficial effects of the present invention are：
1, the minimal medium of the Plant Tissue Breeding of disclosure of the invention can be suitably used for a variety of frequently seen plants tissue cultures,
And obtain the effect same or similar with MS culture medium.
2, culture medium of the present invention is avoided using ammonium nitrate, therefore has the characteristics that safety is good, is convenient for large-scale promotion
It uses.
Detailed description of the invention
Fig. 1 is the contrast effect that tobacco healing tissue's induction, bud break up on two kinds of culture mediums of ZS and MS, A：Tobacco leaf
Callus induction；B：Tobacco adventitious buds differentiation；
Fig. 2 is the contrast effect figure of tobacco tissue-cultured seedling growth and Furcation defects on two kinds of culture mediums of ZS and MS, A：Tobacco tissue culture
Seedling growth；B：Tobacco tissue culture seedling rooting；
Fig. 3 is the contrast effect figure of Potato Plantlets in vitro on two kinds of culture mediums of MS and ZS.
Specific embodiment
The present invention is further illustrated in conjunction with the embodiments, and the following description is only intended to explain the invention, not to it
Content is defined.
Following table is the ingredient and content of herein described ZS minimal medium.(remaining is distilled water, and pH is 5.5~6.0)
5 ZS minimal medium component list (mg/L) of table
Embodiment 1：Tobacco leaf callus induction and shoot regeneration culture
Tobacco leaf callus induction and shoot regeneration culture medium are：ZS minimal medium+6-BA 3mg/L+NAA
0.2mg/L+ sucrose 30g/L+ agar powder 8g/L, pH 5.8.(remaining is distillation to the ingredient of ZS minimal medium as shown in table 5
Water, pH are 5.5~6.0)
The production method is as follows：Based on ZS minimal medium, it is separately added into 6-benzyladenine (BA), methyl α-naphthyl acetate
(NAA), sucrose and agar powder are uniformly mixed, and adjusting pH with the HCl of the KOH or 1mol/L of 1mol/L is 5.8；The ZS base of every 1L
Add 3mg 6-BA, 0.2mg NAA, 30g sucrose and 8g agar powder in basal culture medium.
The differentiation of tobacco healing tissue's induction and bud is carried out using the plant tissue culture media of aforementioned present invention, specifically
For：Tobacco leaf is taken, with 70% ethanol disinfection 30 seconds, then uses 0.1%HgCl2Disinfection 8-10 minutes, with aseptic water washing 4 times
Afterwards, it is cut into the fritter of about 1cm square；The tobacco leaf sheared is placed on culture medium and is cultivated；Condition of culture is：16 small time
According to 2000~3000LX of intensity of illumination, temperature is 25~26 DEG C；8 hours dark, temperature are 22~23 DEG C；Illumination and dark culture
Alternately.
As a result it is：After inoculation 20 days, blade is thickened, and a large amount of callus are generated around blade；Further after being commissioned to train
After supporting 20 days, a large amount of adventitious buds are generated on blade, 5~10 times of proliferation times, sprout is healthy and strong, and bud color is light green, vane extension.Such as figure
Shown in 1A, B.
Comparative example 1 substitutes ZS minimal medium with conventional MS culture medium, remaining is completely the same as embodiment 1.
As a result it is：After inoculation 20 days, blade is thickened, and a large amount of callus are generated around blade；Further after being commissioned to train
After supporting 20 days, a large amount of adventitious buds are generated on blade, 5~10 times of proliferation times, sprout is healthy and strong, and bud color is light green, vane extension.With ZS
Culture medium is substantially similar.As shown in Figure 1A, B.
Embodiment 2：Tobacco tissue culture seedling rooting
Tobacco tissue-cultured seedling root media is：1/2ZS minimal medium+sucrose 20g/L+ agar powder 8g/L, pH 5.8.
The production method is as follows：Based on 1/2ZS minimal medium, it is separately added into sucrose and agar powder, is uniformly mixed, is used
It is 5.8 that the HCl of the KOH or 1mol/L of 1mol/L, which adjust pH,；Add 20g sucrose and 8g agar in the 1/2ZS minimal medium of every 1L
Powder.The ingredient of ZS minimal medium is as shown in table 5 (remaining is distilled water, and pH is 5.5~6.0)
Taking root for tobacco tissue-cultured seedling is carried out using the plant tissue culture media of aforementioned present invention, specially：To tobacco
Regeneration bud it is long to 1cm high when, cut to be inoculated into above-mentioned root media and cultivate；Condition of culture is：Illumination in 16 hours, illumination are strong
2000~3000LX is spent, temperature is 25~26 DEG C；8 hours dark, temperature are 22~23 DEG C；Illumination and dark culture are alternately.
As a result it is：After regeneration bud is inoculated into root media 30 days, the intact plant of 8~10cm of plant height can be grown up to；Plant
Stalwartness, vane extension, leaf color is dark green, well developed root system.As shown in Figure 2.
Comparative example 2 substitutes ZS minimal medium with conventional MS culture medium, remaining is completely the same as embodiment 2.
As a result it is：After regeneration bud is inoculated into root media 30 days, the intact plant of 8~10cm of plant height can be grown up to；Plant
Stalwartness, vane extension, leaf color is dark green, well developed root system.It is substantially similar to ZS culture medium.As shown in Figure 2.
Embodiment 3：Potato test tube seedling culture
Potato test tube seedling culture medium is：ZS minimal medium+white sugar 30g/L+ agar powder 6g/L, pH 5.8.
The production method is as follows：Based on ZS minimal medium, it is separately added into white sugar and agar powder, is uniformly mixed, is used
It is 5.8 that the HCl of the KOH or 1mol/L of 1mol/L, which adjust pH,；Add 30g white sugar and 6g agar powder in the ZS minimal medium of every 1L.
The ingredient of ZS minimal medium is as shown in table 5 (remaining is distilled water, and pH is 5.5~6.0)
The culture of potato test tube seedling is carried out using the plant tissue culture media of aforementioned present invention, specially：
Eugonic potato virus-free plantlet is selected, in superclean bench, clip stem segment size is relatively uniform, each stem section
With 1-2 leaf bud, culture medium is accessed, each culture bottle connects 15 stem sections, is placed in culture in culturing room's (incubator)；Cultivate item
Part is：Illumination in 14 hours, 2000~3000LX of intensity of illumination, temperature are 25~26 DEG C；10 hours dark, temperature are 22~23
℃；Illumination and dark culture are alternately.
As a result it is：After culture 20 days, Virus-free Tube Potato Plantlets growth is vigorous, and blade is big and leaf color is dark green, and stem is sturdy,
Well developed root system, 7~8cm of plant height.As shown in Figure 3 and shown in table 6.
Comparative example 3 substitutes ZS minimal medium with conventional MS culture medium, remaining is completely the same as embodiment 3.
As a result it is：After culture 15 days, Virus-free Tube Potato Plantlets growth is vigorous, and blade is big and leaf color is dark green, and stem is sturdy,
Well developed root system, 7~8cm of plant height.Detoxic seedling plant height, the number of blade, number of taking root, average root long, stem thickness, it is effective in terms of with
ZS culture medium is substantially similar.As shown in Fig. 3 and table 6.
Influence of the 6 ZS and MS culture medium of table to Potato Plantlets in vitro
| Culture medium | Plant height/cm | The number of blade/piece | It takes root number/item | Average root long/cm | Stem thickness/mm | Effectively section Turns Per Knot |
| ZS | 7.62±0.25 | 8.33±0.22 | 2.92±0.15 | 10.85±0.32 | 1.08±0.15 | 5.25±0.24 |
| MS | 7.59±0.31 | 8.30±0.27 | 2.89±0.17 | 9.97±0.29 | 1.06±0.12 | 5.16±0.28 |
Although above-mentioned do in conjunction with attached drawing with regard to optimal culture medium prescription of the invention and preferable several specific embodiments
Illustrate, but is not to be construed as limiting the scope of the invention.Those skilled in the art should understand that in technical side of the invention
On the basis of case, the various modifications or variations that can be made by those skilled in the art with little creative work still in this hair
In bright protection scope.
Claims (5)
1. a kind of minimal medium ZS of Plant Tissue Breeding, which is characterized in that contain in the ZS minimal medium of every 1000mL
The raw material of following mass parts：Potassium nitrate 3075mg, ammonium phosphate 578mg, calcium nitrate 491mg, ammonium chloride 160mg, magnesium nitrate
222mg, ammonium sulfate 396mg, potassium iodide 0.83mg, boric acid 6.2mg, manganese sulfate 22.3mg, zinc sulfate 8.6mg, sodium molybdate
0.25mg, copper sulphate 0.025mg, cobalt chloride 0.025mg, inositol 100mg, thiamine hydrochloride (VB1) 0.1mg, puridoxine hydrochloride
(VB6) 0.5mg, niacin 0.5mg, disodium ethylene diamine tetraacetate 37.3mg, ferrous sulfate 27.8mg.
2. application of the minimal medium ZS described in claim 1 in plant tissue culture.
3. application according to claim 2, which is characterized in that the application is in tobacco healing tissue's induction, bud differentiation
In application.
4. application according to claim 2, which is characterized in that the application is in the growth of tobacco tissue-cultured seedling and Furcation defects
Using.
5. application according to claim 2, which is characterized in that the application is the application in Potato Plantlets in vitro.
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| CN201810623423.7A CN108812313B (en) | 2018-06-15 | 2018-06-15 | Basic culture medium for plant tissue culture |
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| CN201810623423.7A CN108812313B (en) | 2018-06-15 | 2018-06-15 | Basic culture medium for plant tissue culture |
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| CN108812313B CN108812313B (en) | 2020-02-11 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110754366A (en) * | 2019-12-04 | 2020-02-07 | 西南大学 | Eurytopic plant tissue culture medium and 1/2 culture medium |
| CN111937745A (en) * | 2020-08-13 | 2020-11-17 | 江苏宝德农业科技有限公司 | Tissue culture method for rapid propagation of tissue culture seedlings for field planting in production process of detoxified miniature potatoes |
| CN112136688A (en) * | 2020-08-12 | 2020-12-29 | 中国热带农业科学院热带生物技术研究所 | Plant tissue culture medium and preparation method thereof |
| CN112352679A (en) * | 2020-11-19 | 2021-02-12 | 广州市卉通农业科技有限公司 | Culture medium for efficient production of double-line arrowroot tissue culture seedlings and preparation method thereof |
| CN112931206A (en) * | 2021-03-04 | 2021-06-11 | 华南农业大学 | Plant culture medium free of easily-exploding compound and application thereof |
| CN114916443A (en) * | 2022-05-27 | 2022-08-19 | 广西特色作物研究院 | Method for differentiating cluster buds from excellent single-plant callus of Guangxi sweet tea |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
-
2018
- 2018-06-15 CN CN201810623423.7A patent/CN108812313B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102224802A (en) * | 2011-04-19 | 2011-10-26 | 浙江大学 | Proliferation medium for strawberry |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110754366A (en) * | 2019-12-04 | 2020-02-07 | 西南大学 | Eurytopic plant tissue culture medium and 1/2 culture medium |
| CN110754366B (en) * | 2019-12-04 | 2022-03-08 | 西南大学 | Eurytopic plant tissue culture medium and 1/2 culture medium |
| CN112136688A (en) * | 2020-08-12 | 2020-12-29 | 中国热带农业科学院热带生物技术研究所 | Plant tissue culture medium and preparation method thereof |
| CN111937745A (en) * | 2020-08-13 | 2020-11-17 | 江苏宝德农业科技有限公司 | Tissue culture method for rapid propagation of tissue culture seedlings for field planting in production process of detoxified miniature potatoes |
| CN115568419A (en) * | 2020-08-13 | 2023-01-06 | 江苏宝德农业科技有限公司 | 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 |
| CN112352679A (en) * | 2020-11-19 | 2021-02-12 | 广州市卉通农业科技有限公司 | Culture medium for efficient production of double-line arrowroot tissue culture seedlings and preparation method thereof |
| CN112931206A (en) * | 2021-03-04 | 2021-06-11 | 华南农业大学 | Plant culture medium free of easily-exploding compound and application thereof |
| CN114916443A (en) * | 2022-05-27 | 2022-08-19 | 广西特色作物研究院 | Method for differentiating cluster buds from excellent single-plant callus of Guangxi sweet tea |
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