CN102119658A - Method for culturing aseptic seedlings of plants - Google Patents
Method for culturing aseptic seedlings of plants Download PDFInfo
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- CN102119658A CN102119658A CN2010105774782A CN201010577478A CN102119658A CN 102119658 A CN102119658 A CN 102119658A CN 2010105774782 A CN2010105774782 A CN 2010105774782A CN 201010577478 A CN201010577478 A CN 201010577478A CN 102119658 A CN102119658 A CN 102119658A
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- mother liquor
- seed germination
- macronutrient
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Abstract
The invention relates to a technology for culturing aseptic seedlings of plants and application thereof, particularly suitable for seedlet plants such as model plant arabidopsis thaliana, medicinal plant artemisia annua, fruit and vegetable tomato, pepper and the like. The method gets rid of the disturbances of other microbes in the environment and is particularly suitable for searching mutual action between the plants and the microbe. The method comprises steps of: preparing nutritious substrates for seed germination and seedling growth, sterilizing the surfaces of the plant seeds, and germinating the plant seeds. The method is a convenient and efficient g method for culturing aseptic seedlings of plants and has low cost; in addition, the method is free of sterilizing agent residual, thereby ensuring the reliability and accuracy of subsequent research results.
Description
Technical field
The present invention relates to a plant species aseptic seedling culture technique and an application thereof.Be particularly suitable for the seedlet plant, as model plant arabidopsis, medicinal plant artemisia annua, fruits and vegetables tomato, capsicum etc.Because of getting rid of the interference of other microorganisms in the environment, be particularly suitable for studying plant and microbial interaction.
Background technology
Since 20th century 70, the eighties, along with molecular biological rise, the biologist begins to seek a kind of model plant that is suitable for doing in heredity and molecular biology level scrutiny, and Cruciferae phanerogams arabidopsis (Arabidopsis thaliana) is found and is well suited for being used for Plant genetics, Developmental Biology and molecular biological research.Many important discoveries in the plant Related Research Domain in recent years, as the growth of root, bloom, hormone regulating and controlling and plant be to environmental stimulus (comprising biotic factor, abiotic component) reaction mechanism etc., nearly all be with arabidopsis as experiment material, so arabidopsis is described as botanic " fruit bat ".
Arabidopsis as the status of model plant, is determined by its characteristics in the life science field: growth cycle short (can produce first seed about 6 weeks); Plant is little, occupation of land is few, can adopt the laboratory standard program to cultivate; Producing many, the every strains of seed amount per generations can produce thousands of seeds; Full genome checks order, genetic background is clear; Obtainable a series of genetic mutants etc. are arranged.Arabidopsis more and more is subjected to researcher's favor in the plant science correlative study.Foreign literature wide coverage arabidopsis can be cultivated aseptic seedling under laboratory condition, but nearly all is in the culture matrix of plant gel as coagulating agent.Plant gel (phytagel agar) is more than 10 times (agar powder 250g-68.00 unit, the plant gel 250g-800.00 unit) that imported product, price are higher than the plain agar powder.In addition, arabidopsis seed very little (also littler) than the grains of sand, to plant skin thinner, accumulates in one in the aqueous solution easily.If use powerful disinfectants such as 0.1%HgCl
2, or as 2% liquor natrii hypochloritis of bibliographical information, disinfectant can permeate into by kind of a skin, difficult washing is removed, the residual meeting of disinfectant causes harmful effect to seed germination, not only greatly reduce emergence rate, and the residual growth that may the interfere with subsequent plant of disinfectant, be unfavorable for studying the reaction mechanism of arabidopsis to envirment factor (as: heavy metal ion, high salt etc.).
MS is the abbreviation of MS medium inventor Murashige and Skoog.
[1，2]
[1]Murashige?T?and?Skoog?F(1962)A?revised?medium?for?rapid?growth?and?bioassays?with?tobaccotissue?cultures.Physiol?Plant?15(3)：473497.
[2] Chen Yinliang, Chen Zhihong writes. the cell culture engineering. and Shanghai: Huadong Chemical College publishing house, p254,1992.
Summary of the invention
Technical problem: technical problem to be solved by this invention is not enough at above-mentioned prior art and easy, plant aseptic seedling cultural method efficiently that propose, and with low cost, no disinfectant is residual, guarantees follow-up scientific research result's reliability and accuracy.
Technical scheme: a plant species aseptic seedling cultural method of the present invention comprises preparation, plant seed processes for disinfecting surfaces, the plant seed germination of the nutrient matrix of seed germination and growth of seedling;
A. the preparation of the nutrient matrix of seed germination and growth of seedling: employing group training agar powder, add MS macronutrient mother liquor, MS micronutrient element mother liquor and MS mother liquid of iron salt, transfer pH to 5.8, autoclaving: 121 ℃, 20 minutes, it is standby that nutrient agar solidifies the back;
B. plant seed processes for disinfecting surfaces: plant seed places miniature centrifuge tube, successively with after 75%, 95% ethanolic solution and absolute ethyl alcohol sterilization, being aided with sterile water wash, sows on nutrient matrix;
C. plant seed germination: be sub-packed in the aseptic wide-mouth bottle after the nutrient matrix preparation of seed germination, every bottled 20-25mL is sowed at the plant seed after the surface sterilization on the nutrient matrix with the sterile working program; Wide-mouth bottle is after planting put between group training 22-23 ℃, and 16 hours every days, periodicity of illumination was cultivated, and became seedling until seed germination.
Described nutrient matrix is a MS agar, and every liter of culture matrix adds MS macronutrient mother liquor 40-60 milliliter, MS micronutrient element mother liquor 4-6mL, MS mother liquid of iron salt 4-6mL, and all the other are water, transfer pH to 5.8; Add agar powder 8g again in every liter of MS culture fluid, autoclaving 20 minutes, becomes MS agar.
Described MS macronutrient mother liquor is to concentrate 20 times MS macronutrient solution:
Take by weighing NH
4NO
3: 33g; KNO
3: 38g; CaCl
22H
2O:8.8g;
Mg SO
47H
2O:7.4g; KH
2PO
4: 3.4g; Be dissolved in 800mL d H
2Among the O, be settled to 1000mL, be MS macronutrient mother liquor.
Described MS micronutrient element mother liquor is to concentrate 200 times MS micronutrient element solution:
Take by weighing KI:0.166g; H
3BO
3: 1.24g; MnSO
44H
2O:4.46g;
ZnSO
47H
2O:1.72g; Na
2MoO
42H
2O:0.05g; CuSO
45H
2O:0.005g; CoCl
26H
2O:0.005g; Be dissolved in 800mL dd H
2Among the O, be settled to 1000mL, be MS
The micronutrient element mother liquor.
Described MS mother liquid of iron salt is to concentrate 200 times MS iron salt solutions:
Take by weighing Fe SO
47H
2O:5.56g; Na
2EDTA2H
2O:7.46g; Be dissolved in 800mL
Dd H
2Among the O, be settled to 1000mL, be the MS mother liquid of iron salt.
Beneficial effect: the present invention adopts sugar-free MS agar as the medium of cultivating aseptic seedling, and emergence rate is higher on the basis that reduces cost, and the arabidopsis aseptic seedling quality of acquisition is good, helps obtaining accurate believable experimental result.Moreover, because of adopting gentle surface disinfectant ethanol, greatly lowered the toxicity of disinfectant infiltration kind of skin, thereby improved seed germination rate embryo and endosperm.Have following advantage:
(1) adopt sugar-free MS agar as the medium of cultivating aseptic seedling, emergence rate is higher on the basis that reduces cost, the arabidopsis aseptic seedling well developed root system, the cauline leaf growing way that obtain are good, and avoid the anxiety of living contaminants in the air, insect bite, help follow-up introducing experiment and handle the factor, obtain accurate believable experimental result.
(2) adopt gentle surface disinfectant ethanol, in experiment, only add Different concentrations of alcohol solution, be aided with 3-4 washing, thoroughly flush away disinfectant, reduced behind disinfectant infiltration kind of the skin toxicity to embryo and endosperm, not only improve seed germination rate, and avoid the residual (as: Hg in mercury chloride or the clorox of disinfectant
2+, Na
+) to the interference of subsequent experimental (plant preventing from heavy metal ion, salt-resistance correlative study).
(3) in experiment during with the Different concentrations of alcohol solution disinfection, because of ethanolic solution proportion less than the arabidopsis seed, seed sinks down at the bottom of the miniature centrifuge tube easily, helps to topple over waste liquid, the ethanolic solution that more renews.
(4) seed after the last washing, be suspended in the 0.15% sterilization agar solution, draw seed suspension with the rifle head, evenly sow on MS agar [preceding method (ii)], can obtain well to sprout the effect of emerging, (i) [dries disinfection seed on aseptic filter paper with preceding method, evenly being sowed at the MS medium with oese] effect that obtains is suitable, in practical operation, method is i.e. 0.15% agar suspension method (ii), and is more easy, efficient, save time.
(5) the technology of the present invention also can be used for arabidopsis mutant body eds5, the cultivation of NahG aseptic seedling, and culture matrix has improvement slightly, adds 1% sucrose and get final product in aforementioned culture matrix.
Description of drawings
Fig. 1 is a plant aseptic seedling culture technique schematic diagram of the present invention.
Embodiment
For solving the problems of the technologies described above, the specific implementation method that the present invention adopts is:
A. the preparation of the nutrient matrix of seed germination and growth of seedling: employing group training agar powder, (MS is the abbreviation of MS medium inventor Murashige and Skoog to add MS, macronutrient mother liquor, MS micronutrient element mother liquor and MS mother liquid of iron salt down together), transfer pH to 5.8, autoclaving: 121 ℃, 20 minutes, it is standby that nutrient agar solidifies the back;
B. plant seed processes for disinfecting surfaces: plant seed places miniature centrifuge tube, successively with after 75%, 95% ethanolic solution and absolute ethyl alcohol sterilization, being aided with sterile water wash, sows on nutrient matrix;
-3-
C. plant seed germination: (specification: 250mL), every bottled 20-25mL is sowed at the plant seed after the surface sterilization on the nutrient matrix with the sterile working program to be sub-packed in aseptic wide-mouth bottle after the nutrient matrix preparation of seed germination.Wide-mouth bottle is after planting put between group training 22-23 ℃, and 16 hours every days, periodicity of illumination was cultivated, and became seedling until seed germination.
In the described nutrient matrix, every liter of MS macronutrient mother liquor 40-60 milliliter, MS micronutrient element mother liquor 4-6mL, MS mother liquid of iron salt 4-6mL that culture matrix adds, all the other are water, transfer pH to 5.8, this is the MS culture fluid; In every liter of MS culture fluid, add agar powder 8g again, autoclaving, 20 minutes, this was a MS agar.
Described MS macronutrient mother liquor is the MS macronutrient solution for concentrated 20 times:
Take by weighing NH
4NO
3: 33g; KNO
3: 38g; CaCl
22H
2O:8.8g;
MgSO
47H
2O:7.4g; KH
2PO
4: 3.4g; Be dissolved in 800mL dH
2Among the O, be settled to 1000mL, be MS macronutrient mother liquor (concentrating 20 times).
Described MS micronutrient element mother liquor is to concentrate 200 times MS micronutrient element solution:
Take by weighing KI:0.166g; H
3BO
3: 1.24g; MnSO
44H
2O:4.46g; ZnSO
47H
2O:1.72g; Na
2MoO
42H
2O:0.05g; CuSO
45H
2O:0.005g; CoCl
26H
2O:0.005g; Be dissolved in 800mLddH
2Among the O, be settled to 1000mL, be MS micronutrient element mother liquor (concentrating 200 times).
Described MS mother liquid of iron salt is to concentrate 200 times MS iron salt solutions:
For: take by weighing FeSO
47H
2O:5.56g; Na
2EDTA2H
2O:7.46g; Be dissolved in 800mL ddH
2Among the O, be settled to 1000mL, be MS mother liquid of iron salt (concentrating 200 times).
(1) adopts the coagulating agent of plain agar powder, and do not add carbon source (carbohydrate) in the MS medium, because contain seed germination and the required whole nutrition of growth of seedling in the endosperm of seed as the MS medium.Promptly adopt sugar-free MS agar as the medium of cultivating aseptic seedling.
(2) low temperature (4 ℃), the lucifuge processing in advance of arabidopsis seed is 2-3 days, to obtain the synchronization of seed germination.Above-mentioned arabidopsis seed is placed in the aseptic microcentrifugal tube (be the Eppendorf pipe, be called for short the Ep pipe), handle jolting 10min on the decolorization swinging table earlier with the 75% ethanolic solution 1mL that is added with several 10% Tween-20s.Use 1mL 95% ethanolic solution sterilization three times then instead, each 1min.Sterile water wash 3-4 time adds the rapid sucking-off of 300 μ L absolute ethyl alcohols.Next, (i) disinfection seed is dried on aseptic filter paper, evenly be sowed at the MS medium with oese; Perhaps, (ii) use sterile water wash 2-3 time again, after anhydrating, disinfection seed is suspended in 0.15% agar solution, draw seed suspension, evenly sow on the MS medium with the rifle head.Put between plant tissue culture 22-23 ℃, periodicity of illumination was cultivated in 16 hours, became seedling until seed germination.
Described plant aseptic seedling culture technique is at phanerogams, and seedlet plant particularly is suitable for carrying out in the laboratory usefulness of the research of stress resistance of plant, plant and microbial interaction, plant hormone signal transduction pathway; Also can be used for ecological correlative study, cultivate in indoor aseptic seedling earlier, be transplanted to then in the greenhouse earth culture, move to the land for growing field crops in case of necessity and experimentize.
Described plant aseptic seedling culture technique is specially adapted to seedlet plant aseptic seedling and cultivates,, kind skin little because of its seed approaches, with the inaccessible not only thorough disinfection of existing experimental technique operation but also there is not the residual purpose of disinfectant, and adopt aseptic seedling culture technique of the present invention can solve this difficult problem.
Claims (5)
1. a plant species aseptic seedling cultural method is characterized in that this method comprises preparation, plant seed processes for disinfecting surfaces, the plant seed germination of the nutrient matrix of seed germination and growth of seedling;
A. the preparation of the nutrient matrix of seed germination and growth of seedling: employing group training agar powder, add MS macronutrient mother liquor, MS micronutrient element mother liquor and MS mother liquid of iron salt, transfer pH to 5.8, autoclaving: 121 ℃, 20 minutes, it is standby that nutrient agar solidifies the back;
B. plant seed processes for disinfecting surfaces: plant seed places miniature centrifuge tube, successively with after 75%, 95% ethanolic solution and absolute ethyl alcohol sterilization, being aided with sterile water wash, sows on nutrient matrix;
C. plant seed germination: be sub-packed in the aseptic wide-mouth bottle after the nutrient matrix preparation of seed germination, every bottled 20-25mL is sowed at the plant seed after the surface sterilization on the nutrient matrix with the sterile working program; Wide-mouth bottle is after planting put between group training 22-23 ℃, and 16 hours every days, periodicity of illumination was cultivated, and became seedling until seed germination.
2. plant aseptic seedling cultural method according to claim 1, it is characterized in that described nutrient matrix is a MS agar, every liter of culture matrix adds MS macronutrient mother liquor 40-60 milliliter, MS micronutrient element mother liquor 4-6mL, MS mother liquid of iron salt 4-6mL, and all the other are water, transfer pH to 5.8; Add agar powder 8g again in every liter of MS culture fluid, autoclaving 20 minutes, becomes MS agar.
3. plant aseptic seedling cultural method according to claim 1 is characterized in that described MS macronutrient mother liquor is to concentrate 20 times MS macronutrient solution:
Take by weighing NH
4NO
3: 33g; KNO
3: 38g; CaCl
22H
2O:8.8g;
Mg SO
47H
2O:7.4g; KH
2PO
4: 3.4g; Be dissolved in 800mL d H
2Among the O, be settled to 1000mL, be MS macronutrient mother liquor.
4. plant aseptic seedling cultural method according to claim 1 is characterized in that described MS micronutrient element mother liquor is to concentrate 200 times MS micronutrient element solution:
Take by weighing Kl:0.166g; H
3BO
3: 1.24g; MnSO
44H
2O:4.46g;
ZnSO
47H
2O:1.72g; Na
2MoO
42H
2O:0.05g; CuSO
45H
2O:0.005g; CoCl
26H
2O:0.005g; Be dissolved in 800mL dd H
2Among the O, be settled to 1000mL, be MS micronutrient element mother liquor.
5. plant aseptic seedling cultural method according to claim 1 is characterized in that described MS mother liquid of iron salt is to concentrate 200 times MS iron salt solutions:
Take by weighing Fe SO
47H
2O:5.56g; Na
2EDTA2H
2O:7.46g; Be dissolved in 800mLdd H
2Among the O, be settled to 1000mL, be the MS mother liquid of iron salt.
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| CN2010105774782A CN102119658A (en) | 2010-12-07 | 2010-12-07 | Method for culturing aseptic seedlings of plants |
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| CN2010105774782A CN102119658A (en) | 2010-12-07 | 2010-12-07 | Method for culturing aseptic seedlings of plants |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004605A (en) * | 2012-12-29 | 2013-04-03 | 安徽科技学院 | Culture technique for aseptic pepper seedlings |
| CN104542304A (en) * | 2015-01-30 | 2015-04-29 | 浙江理工大学 | Method for culturing small seeds |
| CN106350452A (en) * | 2016-08-25 | 2017-01-25 | 兰州大学 | Separation method of grass endophytic fungi |
| CN107372119A (en) * | 2017-08-30 | 2017-11-24 | 河南质量工程职业学院 | A kind of Young Tomato Embryos cultural method and its culture medium |
| CN110667908A (en) * | 2019-10-21 | 2020-01-10 | 北部湾大学 | Tissue culture seedling packaging method suitable for long-distance transportation |
-
2010
- 2010-12-07 CN CN2010105774782A patent/CN102119658A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| DETLEF WEIGEL等: "《拟南芥实验手册（英文影印版）》", 31 March 2004 * |
| YELITAO1983: "请教关于拟南芥组培种子消毒的问题", 《HTTP://EMUCH.NET/HTML/200810/1013911.HTML》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004605A (en) * | 2012-12-29 | 2013-04-03 | 安徽科技学院 | Culture technique for aseptic pepper seedlings |
| CN104542304A (en) * | 2015-01-30 | 2015-04-29 | 浙江理工大学 | Method for culturing small seeds |
| CN106350452A (en) * | 2016-08-25 | 2017-01-25 | 兰州大学 | Separation method of grass endophytic fungi |
| CN107372119A (en) * | 2017-08-30 | 2017-11-24 | 河南质量工程职业学院 | A kind of Young Tomato Embryos cultural method and its culture medium |
| CN110667908A (en) * | 2019-10-21 | 2020-01-10 | 北部湾大学 | Tissue culture seedling packaging method suitable for long-distance transportation |
| CN110667908B (en) * | 2019-10-21 | 2022-08-02 | 北部湾大学 | Tissue culture seedling packaging method suitable for long-distance transportation |
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Application publication date: 20110713 |