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Method of improving gene transfer efficiency into plant cells

A plant cell and transgenic technology, applied in plant cells, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as unclear effect and popularization, and achieve the effect of improving transgenic efficiency and high transgenic efficiency.

Inactive Publication Date: 2002-10-30
JAPAN TOBACCO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is only a step forward from the previously widely used disk method (Horsch et al., 1985 (Reference (19)), it is not an improved new method
In addition, the effect and generalization of this method are still unclear, and it cannot be used as a general method.

Method used

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  • Method of improving gene transfer efficiency into plant cells
  • Method of improving gene transfer efficiency into plant cells
  • Method of improving gene transfer efficiency into plant cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 (1) test organization and test strain

[0058] The Japanese rice variety "Morning Light" was used as the experimental material, and its immature embryos were used. The seeds of immature embryos were collected within 1-2 weeks after flowering and prepared according to the method of (Hiei, Y. et al., 1994 (reference (13)). That is, immature seeds were removed after flowering 7-12 days The glumes were fixed with 70% ethanol for 30 seconds, carried out aseptic treatment with 1% sodium hypochlorite solution for 10 minutes, and then the immature embryos were taken out for testing. The callus derived from the immature embryos was obtained by inoculating the immature embryos In Gelrite 2N6 medium containing 4g / l (Hiei et al. (1994) (reference (13)), N6 inorganic salts and vitamins (Chu C.C.1978 (reference (8)), 1g / l phenol Proteoaminoacids, 2 mg / l 2,4-D)) were cultured for 2 weeks.

[0059] LBA4404(pIG121Hm) (Hiei, Y. et al., 1994 (reference (13))), LBA4404(pNB131)...

Embodiment 2

[0073] The immature corn embryos with a size of 1.2 mm (variety A188, collected from the Institute of Biological Resources, Ministry of Agriculture, Forestry and Fisheries) were aseptically sterilized, dissected and taken out, and put into a centrifuge tube containing 2 ml of LS-inf liquid medium. Wash once with the same liquid medium. Then add 2.0 ml of new same medium. Soak the centrifuge tube in a 46°C water bath and heat for 3 minutes. Centrifuge at 20KG and 4°C for 30 minutes with a cooling separator. The heat treatment and the centrifugation treatment are carried out after the above heat treatment and then the above centrifugation treatment. The control group was kept at room temperature for the same time. After various treatments, the culture medium was removed and added to the LS-inf medium containing 100 μM acetosyringone (concentration was about 1×10 9 cfu / ml) 1 ml of a suspension of Agrobacterium tumefaciens LBA4404 (pSB131) (Ishida et al. 1996 (Reference (18)))...

Embodiment 3

[0080]Fully mature seeds [Agrostis palustris cv. Pencross (Snow Brand Seedling Co., Ltd.)] were sterilized and inoculated with MS inorganic salts, MS vitamins, 4 mg / l dicamba, 0.5 mg / l 6BA, 0.7 g On a medium (TG2 medium) of proline / l, MES 0.5 g / l, sucrose 20 g / l, and gelrite (pH 5.8) 3 g / l, they were cultured at 25° C. in the dark. The induced callus was subcultured on the medium with the same composition to expand the embryogenic callus. The obtained embryogenic callus was transferred to gelrite-free TG2 liquid medium (TG2L), and cultured with shaking at 25° C. in the dark to obtain suspension culture cells. On the 3rd to 4th day after continuing the culture, transfer the suspension cultured cells into a 2ml centrifuge tube containing TG2L medium, wash once with the same medium, and then add 2ml of new medium. Place the centrifuge tube in a 46°C water bath for 5 minutes. The medium was removed and a new same medium was added, followed by centrifugation at 5,000 rpm and 4° C...

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Abstract

A method for improving the transgenic efficiency of plant cells is disclosed. This method is easier to carry out transgene than the previous Agrobacterium method, has high efficiency and does not harm cell tissues. In the method of the present invention, the transgenic efficiency of plant cells can be improved by centrifuging plant cells or plant tissues while transgenic by Agrobacterium bacteria.

Description

technical field [0001] The invention relates to a method for improving transgenic efficiency of plant cells. technical background [0002] The advantage of the Agrobacterium transgenic method is that the transgenic efficiency is high, the gene copy number is small, and it will not fragment the specific region of T-DNA, because it can obtain transgenic bodies with small variation after a short period of cultivation. Therefore, the Agrobacterium transgenic method is widely used in various plants, and it is the most effective method for plant transgenic. [0003] Although the Agrobacterium method is a very good method for plant transgenics, in fact the success of this transgene and the transduction efficiency all depend on the species of the plant, the genotype and the plant tissue used, because of these factors they will produce very different results. Large differences (Potrykus et al. 1998 (ref. (36))). That is to say, in addition to some plants that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/82C12N15/84
CPCC12N15/8201C12N15/8205C12N15/8206C12N5/04
Inventor 樋江井佑弘笠冈启介石田佑二
Owner JAPAN TOBACCO INC
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