CN104604539A - Grafting method for arabidopsis - Google Patents
Grafting method for arabidopsis Download PDFInfo
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- CN104604539A CN104604539A CN201510049787.5A CN201510049787A CN104604539A CN 104604539 A CN104604539 A CN 104604539A CN 201510049787 A CN201510049787 A CN 201510049787A CN 104604539 A CN104604539 A CN 104604539A
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- grafting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
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Abstract
The invention discloses a grafting method for arabidopsis. According to the method, an arabidopsis wild type aseptic seed sprouted seedling is used as a stock, and an arabidopsis mutant aseptic seed sprouted seedling is used as a scion. The method has the advantages of being easy to carry out and operate, small in damage, high in survival rate, low in cost and the like. A new approach is provided for the arabidopsis grafting technology, a theoretical basis is laid for breed improvement of forest fruits, vegetables and other plants, and a direct scientific research system is provided for plant grafting in molecular mechanism and other related theoretical researches.
Description
Technical field
The present invention relates to the grafting method of a kind of small body type plant, particularly relate to a kind of grafting method of arabidopsis.
Background technology
Grafting is the one of the vegetative reproduction in vegetative propagation, is that the function after utilizing plant injured with callus is carried out.It is as an agriculture production technology, has been widely used in agricultural, forestry and gardening plant production and scientific research and development.Start so far at the beginning of last century, particularly 70, the eighties, people have carried out large quantity research to grafting basic theory.But some mechanism problem is still very undistinct at present.
Arabidopsis (Arabidopsis thaliana) is a kind of small-sized weeds of Cruciferae, because it has short, the feature such as individuality is little, genome is little breeding time, for a long time by the model plant as genetics, molecular biology, Developmental Biology research.2000 the end of the year arabidopsis gene group complete sequence be disclosed, complete the higher plant of full gene group sequencing as first, the gene structure of arabidopsis and functional analysis also one of focus becoming scientific research gradually.
Based on the particular advantages of arabidopsis as model plant, it is widely used in the correlative studys such as much botany, agronomy and forestry, its achievement in research deepens the understanding to other plant Related Research Domain and understanding by contributing to, and even directly can be used to guide improvement and the production of important agriculture and forestry plant.But through retrieval, in the research method of many relevant arabidopsiss, have no the report of the method about arabidopsis grafting.
Summary of the invention
For existing and deficiency, the problem to be solved in the present invention is the grafting method proposing a kind of arabidopsis.
The grafting method of arabidopsis of the present invention, step is:
1) stock prepares: under arabidopsis seed asepsis environment after disinfecting, be seeded on 1/2MS solid culture medium, sealing is placed on 4 DEG C of cultivations and carries out vernalization in 2-3 days, cultivates under proceeding to long-day conditions, growing after cotyledon until seedling is rootstock seedling, for grafting;
2) scion prepares: by Arabidopsis Mutants seed through step 1) identical disinfecting and cultivating, growing after cotyledon until seedling is scion seedling, for grafting;
3) grafting under gnotobasis;
4) graft is cultivated: graft proceeds to short-day and cultivates, and treats wound healing, grows two panels true leaf;
5) grafting is transplanted: the grafting survived is transplanted in culture matrix, nutrient solution, and the long-day cultivates, until grafting yields positive results, and results seed;
It is characterized in that:
Step 1) and 2) the described method to the surface of the seed disinfection is: by seed 70% Ethanol Treatment 5 ± 2 minutes, after aqua sterilisa cleaning twice, disinfect 10 ± 2 minutes with 2.6%NaClO, with distilled water cleaning 4-6 time;
Step 1), 2) and 5) described long-day conditions is: the photoperiod is 16 h light/8 h dark, temperature under illumination condition is 22-24 DEG C, temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
Step 3) method of described grafting is: on rootstock seedling is hypocotylar, 1/3 place's scissors is cut off, and makes underground part remain with the hypocotyl of 2/3 as stock; On scion seedling is hypocotylar, 1/3 place's scissors is cut off, and makes overground part remain with the hypocotyl of 1/3 as scion; The formation laminating merging of scion and two, stock being hindered face is attached to culture dish and speckles with on the culture dish inwall of the globule, sealing;
Step 4) described short-day condition is: the photoperiod is 8 h light/16 h dark, and the temperature under illumination condition is 22-24 DEG C, and the temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
Described step 5) described in culture matrix be vermiculite; Nutrient solution is the solution after B5 Nutrient medium dilute with water 3 times.Described graft and transplantation process is: after grafting survives, first open culture dish mouth 1/3, it is tamed, keep short-day state, after 3-4 days, with tweezers, regrowth is taken out, clean root medium, do not injure root hair as far as possible, be then transplanted in sterilized vermiculite culturing pot, keep long-day state training orientation.
In the grafting method of above-mentioned arabidopsis: step 3) described in the fast type surgical scissors of spy of the preferred long 145mm of scissors.The medical science gun tweezers of the preferred long 260mm of tweezers.Described seed disinfection and the process of grafting, preferably operate under superclean bench gnotobasis.
The invention provides a kind of method of arabidopsis grafting, for stock with arabidopsis wild type aseptic seed germination seedling, with the grafting method that Arabidopsis Mutants aseptic seed germination seedling is scion, be intended to utilize arabidopsis wild type and a large amount of Arabidopsis Mutants to carry out grafting, the stock and the scion that disclose plant graft make molecule mechanism, mutually for the breed improvement of the plants such as woods fruit, vegetables provides theoretical foundation.Method of the present invention have easy to implement the method, simple to operate, injure the features such as little, survival rate is high, cost is low, for arabidopsis graft technology provides new way, for plant graft provides direct scientific research system in correlation theory researchs such as molecule mechanisms.
Accompanying drawing explanation
Fig. 1 is the arabidopsis grafting on medium.
Fig. 2 is the arabidopsis grafting before transplanting.
Embodiment
Below in conjunction with instantiation, the present invention is described in further details.
Be scion and arabidopsis Landsberg erecta (Ler) seed seedling with arabidopsis Columbia-0 (Col-0) seed seedling be that stock carries out grafting for example, seed disinfection and grafting procedures all operate under superclean bench gnotobasis.
Concrete, the grafting method step of arabidopsis is:
1) stock prepares: under arabidopsis seed asepsis environment after disinfecting, be seeded on 1/2MS solid culture medium, sealing is placed on 4 DEG C of cultivations and carries out vernalization in 2-3 days, cultivates under proceeding to long-day conditions, growing after cotyledon until seedling is rootstock seedling, for grafting;
2) scion prepares: by Arabidopsis Mutants seed through step 1) identical disinfecting and cultivating, growing after cotyledon until seedling is scion seedling, for grafting;
Wherein, the above-mentioned method to the surface of the seed disinfection is: by seed 70% Ethanol Treatment 5 ± 2 minutes, after aqua sterilisa cleaning twice, disinfects 10 ± 2 minutes, with distilled water cleaning 4-6 time with 2.6%NaClO; Described long-day conditions is: the photoperiod is 16 h light/8 h dark, and the temperature under illumination condition is 22-24 DEG C, and the temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
3) grafting under gnotobasis;
Wherein, the method for described grafting is: on rootstock seedling is hypocotylar, 1/3 place's scissors is cut off, and makes underground part remain with the hypocotyl of 2/3 as stock; On scion seedling is hypocotylar, 1/3 place's scissors is cut off, and makes overground part remain with the hypocotyl of 1/3 as scion; The formation laminating merging of scion and two, stock being hindered face is attached to culture dish and speckles with on the culture dish inwall of the globule, sealing;
Above-mentioned scissors selects the fast type surgical scissors of the spy of long 145mm, and tweezers select the medical science gun tweezers of long 260mm.
4) graft is cultivated: graft proceeds to short-day and cultivates, and treats wound healing, grows two panels true leaf;
Wherein, described short-day condition is: the photoperiod is 8 h light/16 h dark, and the temperature under illumination condition is 22-24 DEG C, and the temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
5) grafting is transplanted: the grafting survived is transplanted in culture matrix, nutrient solution, and the long-day cultivates, until grafting yields positive results, and results seed;
Wherein, described culture matrix is vermiculite; Nutrient solution is the solution after B5 Nutrient medium dilute with water 3 times.Described long-day conditions is: the photoperiod is 16 h light/8 h dark, and the temperature under illumination condition is 22-24 DEG C, and the temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%; Described graft and transplantation process is: after grafting survives, first open culture dish mouth 1/3, it is tamed, keep short-day state, after 3-4 days, with tweezers, regrowth is taken out, clean root medium, do not injure root hair as far as possible, be then transplanted in sterilized vermiculite culturing pot, keep long-day state training orientation.
The formula following (unit mg/L) of above-mentioned B5 Nutrient medium:
| Composition | Working concentration (mg/L) | |
| Macroelement | Potassium nitrate KNO 3 | 2500 |
| MgSO 4·7H 2O | 250 | |
| CaCl 2·2H 2O | 150 | |
| (NH 4) 2SO 4 | 134 | |
| NaH 2PO 4·H 2O | 150 | |
| Trace element | KI | 0.75 |
| H 3BO 3 | 3.0 | |
| MnSO 4·4H 2O | 10 | |
| ZnSO 4·7H 2O | 2.0 | |
| Na 2MoO 4·2H 2O | 0.25 | |
| CoCl 2·6H 2O | 0.025 | |
| CuSO 4·5H 2O | 0.025 | |
| Molysite | Na 2-EDTA | 37.3 |
| FeSO 4·7H 2O | 27.8 |
The formula of above-mentioned MS solid culture medium is as follows:
| Macroelement composition | Molecular weight | Working concentration (mg/L) |
| Potassium nitrate KNO 3 | 101.21 | 1900 |
| Ammonium nitrate NH 4NO 3 | 80.04 | 1650 |
| Potassium dihydrogen phosphate KH 2PO 4 | 136.09 | 170 |
| Magnesium sulfate MgSO 4·7H 2O | 246.47 | 370 |
| Calcium chloride CaCl 2·2H 2O | 147.02 | 440 |
| Trace element | ||
| Potassium iodide KI | 166.01 | 0.83 |
| Boric acid H 3BO 3 | 61.83 | 6.2 |
| Manganese sulphate MnSO 4·4H 2O | 223.01 | 22.3 |
| Zinc sulphate ZnSO 4·7H 2O | 287.54 | 8.6 |
| Sodium molybdate Na 2MoO 4·2H 2O | 241.95 | 0.25 |
| Copper sulphate CuSO 4·5H 2O | 249.68 | 0.025 |
| Cobalt chloride CoCl 2·6H 2O | 237.93 | 0.025 |
| Molysite | ||
| Disodium ethylene diamine tetraacetate Na2.EDTA | 372.25 | 37.25 |
| Ferrous sulfate FeSO 4·7H 2O | 278.03 | 27.85 |
| Organic principle | ||
| Inositol | 100 | |
| Glycine | 2 | |
| Thiamine hydrochloride VB1 | 0.1 | |
| Puridoxine hydrochloride VB6 | 0.5 | |
| Nicotinic acid VB5 or VPP | 0.5 | |
| Sucrose sucrose | 342.31 | 30g/L |
| Agar agar | 7g/L |
Claims (2)
1. a grafting method for arabidopsis, step is:
1) stock prepares: under arabidopsis seed asepsis environment after disinfecting, be seeded on 1/2MS solid culture medium, sealing is placed on 4 DEG C of cultivations and carries out vernalization in 2-3 days, cultivates under proceeding to long-day conditions, growing after cotyledon until seedling is rootstock seedling, for grafting;
2) scion prepares: by Arabidopsis Mutants seed through step 1) identical disinfecting and cultivating, growing after cotyledon until seedling is scion seedling, for grafting;
3) grafting under gnotobasis;
4) graft is cultivated: graft proceeds to short-day and cultivates, and treats wound healing, grows two panels true leaf;
5) grafting is transplanted: the grafting survived is transplanted in culture matrix, nutrient solution, and the long-day cultivates, until grafting yields positive results, and results seed;
It is characterized in that:
Step 1) and 2) the described method to the surface of the seed disinfection is: by seed 70% Ethanol Treatment 5 ± 2 minutes, after aqua sterilisa cleaning twice, disinfect 10 ± 2 minutes with 2.6%NaClO, with distilled water cleaning 4-6 time;
Step 1), 2) and 5) described long-day conditions is: the photoperiod is 16 h light/8 h dark, temperature under illumination condition is 22-24 DEG C, temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
Step 3) method of described grafting is: on rootstock seedling is hypocotylar, 1/3 place's scissors is cut off, and makes underground part remain with the hypocotyl of 2/3 as stock; On scion seedling is hypocotylar, 1/3 place's scissors is cut off, and makes overground part remain with the hypocotyl of 1/3 as scion; The formation laminating merging of scion and two, stock being hindered face is attached to culture dish and speckles with on the culture dish inwall of the globule, sealing;
Step 4) described short-day condition is: the photoperiod is 8 h light/16 h dark, and the temperature under illumination condition is 22-24 DEG C, and the temperature under dark condition is 20-22 DEG C, and light intensity is 6000-8000lux, and relative moisture is greater than 70%;
Described step 5) described in culture matrix be vermiculite; Nutrient solution is the solution after B5 Nutrient medium dilute with water 3 times.
2. the grafting method of arabidopsis according to claim 1, is characterized in that: step 3) described in scissors be the fast type surgical scissors of spy of long 145mm.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105706873A (en) * | 2016-02-03 | 2016-06-29 | 青岛农业大学 | Grafting method for model arabidopsis thaliana |
| CN106613376A (en) * | 2017-02-09 | 2017-05-10 | 山东师范大学 | Aseptic grafting method of salt mustard |
| CN106665133A (en) * | 2017-01-16 | 2017-05-17 | 山东师范大学 | Sterile grafting method of Arabidopsis/thellungiella halophila |
| CN106856998A (en) * | 2017-02-27 | 2017-06-20 | 山东师范大学 | A kind of method of utilization salt mustard inverse molecule mechanism resistance to the grafting architectural study plant of arabidopsis |
| CN108713406A (en) * | 2018-06-07 | 2018-10-30 | 齐鲁师范学院 | The engrafting method of arabidopsis and its application in restoring Arabidopsis Mutants fertility |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105706873A (en) * | 2016-02-03 | 2016-06-29 | 青岛农业大学 | Grafting method for model arabidopsis thaliana |
| CN105706873B (en) * | 2016-02-03 | 2019-03-08 | 青岛农业大学 | The engrafting method of one mode arabidopsis |
| CN106665133A (en) * | 2017-01-16 | 2017-05-17 | 山东师范大学 | Sterile grafting method of Arabidopsis/thellungiella halophila |
| CN106665133B (en) * | 2017-01-16 | 2020-05-05 | 山东师范大学 | Sterile grafting method of arabidopsis thaliana/Thellungiella halophila |
| CN106613376A (en) * | 2017-02-09 | 2017-05-10 | 山东师范大学 | Aseptic grafting method of salt mustard |
| CN106613376B (en) * | 2017-02-09 | 2020-06-30 | 山东师范大学 | Sterile grafting method for Thellungiella halophila |
| CN106856998A (en) * | 2017-02-27 | 2017-06-20 | 山东师范大学 | A kind of method of utilization salt mustard inverse molecule mechanism resistance to the grafting architectural study plant of arabidopsis |
| CN106856998B (en) * | 2017-02-27 | 2020-06-26 | 山东师范大学 | Method for researching plant stress tolerance molecular mechanism by using grafting system of Thellungiella halophila and Arabidopsis thaliana |
| CN108713406A (en) * | 2018-06-07 | 2018-10-30 | 齐鲁师范学院 | The engrafting method of arabidopsis and its application in restoring Arabidopsis Mutants fertility |
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