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Improved corn pollen magnetic transfection method

A technology of corn pollen and magnetic transfer, applied in the field of plant genetic engineering, can solve problems such as technical obstacles and restrict commercial application, and achieve the effects of maintaining vitality, improving seed setting rate, maintaining state and integrity

Active Publication Date: 2022-02-25
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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Problems solved by technology

Technical barriers to most elite maize inbred lines limit their commercial application, especially in the development of advanced breeding techniques such as precise genome editing directly in elite crop germplasm

Method used

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  • Improved corn pollen magnetic transfection method
  • Improved corn pollen magnetic transfection method
  • Improved corn pollen magnetic transfection method

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Experimental program
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Effect test

Embodiment Construction

[0030] An improved maize pollen magnetic transfection method:

[0031] Nanomagnetic beads (MNP, product number 9006) and magnetic plates (product number 9008-96) were purchased from Chemicell, Germany.

[0032] The main inbred lines of maize are 178, HZ178, Jing 92 and Zheng 58.

[0033] The specific maize pollen magnetic transfection process is as follows: figure 1 shown.

[0034] (1) Extract a large amount of high-purity plasmid DNA for transfection, and use ddH 2Adjust the concentration of O to 1000±50ng / μL, aliquot into small tubes, store at -20°C, avoid repeated freezing and thawing, and take out the plasmid DNA and thaw to room temperature before magnetic transfection.

[0035] (2) According to the flowering situation of the corn inbred line and the transformation requirements of the plasmid, estimate the transformation test that needs to be carried out on the day; let the nano-magnetic beads MNP stand at room temperature for 10 minutes, take a 200 μL PCR tube, write ...

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Abstract

The invention discloses an improved corn pollen magnetic transfection method. The method comprises the following steps: (1) preparing plasmid DNA for transfection; (2) combining nanomagnetic beads MNP with the plasmid DNA under a room temperature condition to form an MNP-DNA compound; (3) collecting fresh pollen of corn in a full-bloom stage, and quickly taking the pollen back to a room under a water-proof ice box preservation condition; (4) uniformly mixing corn pollen preservation liquid with sieved corn pollen, and performing trepanning pretreatment under a low-temperature condition; (5) adding the MNP-DNA compound into a trepanning pretreatment solution, conducting gentle and uniform mixing, and carrying out transfecting on a low-temperature pre-cooled magnetic plate; and (6) after the transfection is finished, carrying a pollen suspension to a field by using an ice box, and directly pollinating corn ears. According to the method, the corn planted in the field is directly utilized for transformation, only conventional refrigeration equipment is needed, operation is easy and convenient, and cost is low, so the method is suitable for all corn varieties.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to an improved maize pollen magnetic transfection method capable of maintaining pollen viability and improving transformation efficiency. Background technique [0002] Corn is a widely grown crop worldwide to meet people's growing food, feed and fuel needs. Although the yield and quality of maize have been significantly improved through traditional breeding in the past two hundred years, it still faces severe challenges from various biotic and abiotic stresses. To address these challenges, genetically modified maize has been developed to introduce desirable traits such as insect resistance, herbicide resistance, drought tolerance, cold tolerance, and improved nutritional quality. Efficient genetic transformation is the key to the development of transgenic maize. Since the first maize transformation based on protoplast electroporation was successful in 1988, sci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/02A01H6/46
CPCC12N15/8207A01H1/027C12N15/8206C12N15/82A01H1/02B82Y5/00
Inventor 张中保吴忠义郑登俞王作平张春李向龙
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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