Agrobacterium-mediated transgenic method for mature seed embryos of corn

An Agrobacterium-mediated, seed embryo technology is applied in horticultural methods, botanical equipment and methods, angiosperms/flowering plants, etc. Effects of Limiting and Avoiding Maize Callus Tissue Differentiation and Plant Regeneration

Inactive Publication Date: 2012-05-02
GUANGXI UNIV
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  • Abstract
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Problems solved by technology

Zhang Rong et al. (2001) used immature embryos as direct recipients to successfully obtain transformants through the Agrobacterium-mediated met

Method used

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  • Agrobacterium-mediated transgenic method for mature seed embryos of corn
  • Agrobacterium-mediated transgenic method for mature seed embryos of corn
  • Agrobacterium-mediated transgenic method for mature seed embryos of corn

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Experimental program
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Effect test

preparation example Construction

[0035] (3) Preparation and wounding of naked embryos of mature corn seeds;

[0036] (4) Agrobacterium-mediated gene transformation of naked and wounded corn mature seed embryos in step (2);

[0037] (5) Re-screening of herbicide resistance of regenerated seedlings;

[0038] (6) Transplanting the surviving seedlings after the herbicide screening treatment;

[0039] (7) Polymerase chain reaction (PCR) identification of maize transgenic plants.

[0040] Such as figure 1 As shown, the plasmid vector pGSA1252::RA33G4 for plant gene expression constructed in step (1);

[0041] In order to verify the method of the present invention, we used the common plant expression vector plasmid pGSA1252 and our own cloned RA33G4 gene as examples for the example operation process of the cloning construction step of step (1) of the method of the present invention. RA33G4 gene is an early drought-induced protein gene we cloned from corn; pGSA1252 plasmid itself contains a chloramphenicol-resistant gene and a...

Embodiment 1

[0050] Construction of plasmid vector pGSA1252::RA33G4 for plant gene expression

[0051] Plasmid DNA was extracted according to the routine method of the laboratory. Enzyme digestion and DNA ligation methods were performed in accordance with the instructions provided by the manufacturer of the enzyme used. The extracted DNA of pGSA1252 was digested with Ncol and Spel, and then ligated with the DNA fragment of RA33G4 gene that was also digested with Ncol and Spel under the action of T4DNA ligase to form a transgene expression plasmid vector pGSA1252::RA33G4 .

[0052] Construction of Agrobacterium tumefaciens strain LBA4404 containing plant gene expression plasmid vector pGSA1252::RA33G4.

[0053] All involved implementation processes are carried out under aseptic conditions.

[0054] (1) A single colony of Agrobacterium LBA4404 without plasmid was inoculated into a common YEB medium in the laboratory containing rifampicin at an appropriate concentration, and cultured at 28°C on a 2...

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Abstract

The invention discloses an agrobacterium-mediated transgenic method for mature seed embryos of corn. The method comprises the following steps of: (1) constructing a plasmid vector expressed by plant genes; (2) constructing an agrobacterium tumefaciens strain containing the plasmid vector expressed by the plant genes in step (1); (3) preparing and wounding bare embryos of mature seeds of the corn; (4) performing genetic transformation on the agrobacterium-mediated, bare and wounded embryos of the mature seeds of the corn in step (2); (5) re-screening the herbicide resistance of regenerated seedlings; (6) transplanting the seedlings which survive after herbicide screening treatment; and (7) performing polymerase chain reaction (PCR) identification on transgenic corn plants. By the method, the problems that the periods of tissue differentiation of maize calluses and plant regeneration are long and the root regeneration is difficult are solved; and the season limitation on the reported transgenic manipulation in which maize rataria is taken as a receptor is effectively avoided, and the time of transformation operation is shortened.

Description

Technical field [0001] The invention relates to the field of maize gene transformation engineering, in particular to an Agrobacterium-mediated transgenic method of maize mature seed embryos. Background technique [0002] Corn is one of the main food crops. Compared with rice, wheat, sorghum and other crops, corn (Zea mays L.) has a short cultivation history, but its production has developed the fastest. The world's demand for corn is also increasing day by day. In order to meet demand, improve certain traits, and increase yield, transgenic technology is an effective means to solve the shortage of genetic resources in conventional breeding and the slow speed of selection. [0003] Maize transgenic technology has been greatly developed in recent years. A series of transformation technologies such as Agrobacterium-mediated method, gene bombardment method, PEG method, electric shock method, silicon carbide fiber-mediated method and pollen tube introduction method have been established,...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 李有志潘虹樊宪伟
Owner GUANGXI UNIV
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