CN103849644A - Sorghum germ inoculation transgenic technology - Google Patents
Sorghum germ inoculation transgenic technology Download PDFInfo
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- CN103849644A CN103849644A CN201210522442.3A CN201210522442A CN103849644A CN 103849644 A CN103849644 A CN 103849644A CN 201210522442 A CN201210522442 A CN 201210522442A CN 103849644 A CN103849644 A CN 103849644A
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Abstract
The invention discloses a sorghum germ inoculation transgenic technology and belongs to the technical field of biology. The technology comprises the following steps: sterilizing sorghum seeds, puncturing seed embryos by using a steel needle, inoculating agrobacterium which carries a binary vector, soaking the seeds in an inoculation solution, enhancing infect, performing dark box culture on the seeds, promoting in-vivo proliferation diffusion of the agrobacterium infect, transforming into normal culture to grow seedlings, and performing transgenic identification. The method is simple, convenient, easy to operate and low in cost.
Description
Technical field
A kind of Chinese sorghum plumule inoculation transgenic technology belongs to biological technical field, says that more specifically a kind of way that adds the inoculation of inoculation immersion bubble by Chinese sorghum embryo being carried out to stab inoculation improves the method for the efficiency of transgenosis inoculation.
Background technology
Chinese sorghum transgenic technology is to adopt biotechnological means transformation sorghum variety, cultivate there is high yield, the most effectively technique means of high-quality, the good characteristic new variety such as pest-resistant, disease-resistant.At present the transgenosis of Chinese sorghum generally all adopts the method for During Agrobacterium callus, and the method first will be carried out callus of induce, then During Agrobacterium, then with the method acquisition transgenic seedling of tissue culture.Because Chinese sorghum tissue culture difficulty is larger, therefore the efficiency of the method is lower, application difficult.
Summary of the invention
The object of the invention is to look for another way, adopt a kind of new technological line to carry out the genetically modified inoculation processing of Chinese sorghum, to reaching easy to implement the method, simple to operate, expense is cheap, respond well technique effect.
The present invention is achieved in that
1. a Chinese sorghum plumule inoculation transgenic technology is characterised in that, taking sorghum seeds as carrier, carry out acupuncture and add the inoculation repeatedly of inoculation immersion bubble, it comprises, and binary vector inoculation, sorghum seeds sterilization, sterilized water seed soaking, the inoculation of Chinese sorghum kind, inoculation liquid seed soaking, camera bellows are cultivated, 7 steps of growing nursery and culture.
2. binary vector inoculation: the Agrobacterium that proceeds to binary vector is inoculated in the YEP liquid nutrient medium of 100mL and cultivates 2 days.
3. sorghum seeds sterilization: choose 100 full sorghum seeds and first use 70% ethanol disinfection 3min, then, with 84 thimerosal sterilization 30min, sterilization finishes rear with aqua sterilisa rinsing 3-4 time, washing and sterilizing liquid completely.
4. sterilized water seed soaking: seed is proceeded in aqua sterilisa to soak at room temperature 12 hours.
5. Chinese sorghum inoculation: soaked seed is taken out and is placed in sterilized thorn embryonic plate or culture dish, dip OD with draw point
600it is 0.6 Agrobacterium inoculation liquid thorn plumule.
6. inoculation liquid seed soaking: the seed having stung is placed in the Agrobacterium inoculation liquid that contains 400 μ mol/L Syringylethanones and is soaked 1.5 hours.
7. camera bellows is cultivated: the seed of handling well is placed in to germination box, 22 DEG C of dark cultivations 4 days of constant incubator.
8. growing nursery and culture: the seed simple grain after sprouting is broadcast to growing nursery and culture in nutrition pot, can carry out the qualification of transfer-gen plant in the time there is 3 leaves.
Beneficial effect of the present invention: a kind of Chinese sorghum plumule inoculation transgenic technology has been avoided after traditional During Agrobacterium callus the technological line of tissue culture again, there is compared with the conventional method following advantage: first, required time reduces about 1 month, During Agrobacterium method generally needs 3-4 month from callus of induce to obtaining transgenosis, and present method starts only to complete and need 2 months to qualification from inoculation; The second, present method is not high to technical requirements, and Chinese sorghum tissue culture difficulty is large, and easy brownization of callus is therefore high to experimenter's technical requirements, and present method does not relate to tissue culture procedures, therefore greatly reduces technical difficulty; The 3rd, the required expense of present method is less, existing transgenic technology need to be by plant tissue culture technique, and all ingredients of tissue culture is more expensive, and need foundation group training chamber and be equipped with a large amount of plant and instrument just can carry out the work, and the present invention has exempted the cost of this part, save substantial contribution.
Embodiment:
Below in conjunction with specific embodiment, the present invention is conducted further description.
Embodiment 1: S1390 carrier is transferred in the plant of Chinese sorghum strain U68
1. binary vector inoculation: the Agrobacterium that proceeds to S1390 binary vector is inoculated in the YEP liquid nutrient medium of 100mL and cultivates 2 days.
2. sorghum seeds sterilization: choose 300, Chinese sorghum strain U68 seed and first use 70% ethanol disinfection 3min, then, with 84 thimerosal sterilization 30min, sterilization finishes rear with aqua sterilisa rinsing 3-4 time, washing and sterilizing liquid completely.
3. sterilized water seed soaking: seed is proceeded in aqua sterilisa to soak at room temperature 12 hours.
4. Chinese sorghum inoculation: soaked U68 seed is taken out and is placed in sterilized thorn embryonic plate or culture dish, dip OD with draw point
600it is 0.6 Agrobacterium inoculation liquid thorn plumule.
5. inoculation liquid seed soaking: the seed having stung is placed in the Agrobacterium inoculation liquid that contains 400 μ mol/L Syringylethanones and is soaked 1.5 hours.
6. camera bellows is cultivated: the seed of handling well is placed in to germination box, 22 DEG C of dark cultivations 4 days of constant incubator.
7. growing nursery and culture: the seed simple grain after sprouting is broadcast to growing nursery and culture in nutrition pot, can carry out the qualification of transfer-gen plant in the time there is 3 leaves.
In 216 strain U68 seedling, 23 strain transfer-gen plants are obtained by above step.Last 50 days, reduce about 30 days than existing transgenic technology.
Embodiment 2: SRnai carrier is transferred in the plant of Chinese sorghum strain 3042B
1. binary vector inoculation: the Agrobacterium that proceeds to SRnai binary vector is inoculated in the YEP liquid nutrient medium of 100mL and cultivates 2 days.
2. sorghum seeds sterilization: choose 500, Chinese sorghum strain 3042B seed and first use 70% ethanol disinfection 3min, then, with 84 thimerosal sterilization 30min, sterilization finishes rear with aqua sterilisa rinsing 3-4 time, washing and sterilizing liquid completely.
3. sterilized water seed soaking: seed is proceeded in aqua sterilisa to soak at room temperature 12 hours.
4. Chinese sorghum inoculation: soaked 3042B seed is taken out and is placed in sterilized thorn embryonic plate or culture dish, dip OD with draw point
600it is 0.6 Agrobacterium inoculation liquid thorn plumule.
5. inoculation liquid seed soaking: the seed having stung is placed in the Agrobacterium inoculation liquid that contains 400 μ mol/L Syringylethanones and is soaked 1.5 hours.
6. camera bellows is cultivated: the seed of handling well is placed in to germination box, 22 DEG C of dark cultivations 4 days of constant incubator.
7. growing nursery and culture: the seed simple grain after sprouting is broadcast to growing nursery and culture in nutrition pot, can carry out the qualification of transfer-gen plant in the time there is 3 leaves.
In 457 strain 3042B seedling, 87 strain transfer-gen plants are obtained by above step.Last 50 days, reduce about 30 days than existing transgenic technology.
Claims (8)
1. a Chinese sorghum plumule inoculation transgenic technology is characterised in that, taking sorghum seeds as carrier, carry out acupuncture and add the inoculation repeatedly of inoculation immersion bubble, it comprises, and binary vector inoculation, sorghum seeds sterilization, sterilized water seed soaking, the inoculation of Chinese sorghum kind, inoculation liquid seed soaking, camera bellows are cultivated, 7 steps of growing nursery and culture.
2. binary vector inoculation: the Agrobacterium that proceeds to binary vector is inoculated in the YEP liquid nutrient medium of 100mL and cultivates 2 days.
3. sorghum seeds sterilization: choose 100 full sorghum seeds and first use 70% ethanol disinfection 3min, then, with 84 thimerosal sterilization 30min, sterilization finishes rear with aqua sterilisa rinsing 3-4 time, washing and sterilizing liquid completely.
4. sterilized water seed soaking: seed is proceeded in aqua sterilisa to soak at room temperature 12 hours.
5. Chinese sorghum inoculation: soaked seed is taken out and is placed in sterilized thorn embryonic plate or culture dish, dip OD with draw point
600it is 0.6 Agrobacterium inoculation liquid thorn plumule.
6. inoculation liquid seed soaking: the seed having stung is placed in the Agrobacterium inoculation liquid that contains 400 μ mol/L Syringylethanones and is soaked 1.5 hours.
7. camera bellows is cultivated: the seed of handling well is placed in to germination box, 22 DEG C of dark cultivations 4 days of constant incubator.
8. growing nursery and culture: the seed simple grain of sprouting is broadcast to growing nursery and culture in nutrition pot, and 3 leaf phases can be carried out the qualification of transfer-gen plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210522442.3A CN103849644A (en) | 2012-12-07 | 2012-12-07 | Sorghum germ inoculation transgenic technology |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210522442.3A CN103849644A (en) | 2012-12-07 | 2012-12-07 | Sorghum germ inoculation transgenic technology |
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| CN103849644A true CN103849644A (en) | 2014-06-11 |
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| CN201210522442.3A Pending CN103849644A (en) | 2012-12-07 | 2012-12-07 | Sorghum germ inoculation transgenic technology |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699379A (en) * | 2019-12-02 | 2020-01-17 | 山西省农业科学院生物技术研究中心 | Agrobacterium-mediated genetic transformation method for inducing callus by using mature sorghum embryo as explant |
| CN111454984A (en) * | 2020-03-04 | 2020-07-28 | 深圳大学 | Plant gene transformation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321662A (en) * | 2011-07-14 | 2012-01-18 | 中国科学院植物研究所 | Method for transforming stem tips of plants and special tool thereof |
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2012
- 2012-12-07 CN CN201210522442.3A patent/CN103849644A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321662A (en) * | 2011-07-14 | 2012-01-18 | 中国科学院植物研究所 | Method for transforming stem tips of plants and special tool thereof |
Non-Patent Citations (2)
| Title |
|---|
| JIANZHONG LIN ET AL.: "piercing and vacuum infiltration of the mature embryoa simplified method for agrobacterium-mediated transformation of indica rice", 《PLANT CELL REP》, 20 May 2009 (2009-05-20) * |
| 朱莉等: "高粱遗传转化研究进展", 《生物技术通报》, no. 1, 31 December 2011 (2011-12-31) * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699379A (en) * | 2019-12-02 | 2020-01-17 | 山西省农业科学院生物技术研究中心 | Agrobacterium-mediated genetic transformation method for inducing callus by using mature sorghum embryo as explant |
| CN110699379B (en) * | 2019-12-02 | 2023-09-29 | 山西省农业科学院生物技术研究中心 | Agrobacterium-mediated genetic transformation method for inducing callus by taking sorghum mature embryo as explant |
| CN111454984A (en) * | 2020-03-04 | 2020-07-28 | 深圳大学 | Plant gene transformation method and application thereof |
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Application publication date: 20140611 |