Efficient akebia trifoliate ploidy detection method

A technology of Akebia clover and detection method, which is applied in the direction of measuring devices, instruments, particle and sedimentation analysis, etc., and can solve problems such as inaccurate results and strong interference peaks in identification results

Pending Publication Date: 2021-09-07
SICHUAN AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

So far, there is no documented successful case of flow cytometry ploidy identification related to Akebia trifoliate in my country. Only Shi Xiaobing of Guizhou University used this technology in 2015, but due to the toxic effect of colchicum and the polyphenol content of Akebia trifoliata Many characteristics lead to strong interference peaks in the identification results and inaccurate results

Method used

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  • Efficient akebia trifoliate ploidy detection method

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Embodiment

[0055] The present invention provides a kind of high-efficiency Akebia clover ploidy detection method, comprises the following steps:

[0056] S1. Preparation of sample pretreatment reagent

[0057] The sample pretreatment preparation is composed of 500mL cell nucleus lysis buffer with pH=7.2 and 50mL staining buffer with pH=7.2. The cell nucleus lysis buffer should be stored in a constant temperature environment at 4°C. After the preparation of the sample pretreatment preparation is completed, it passes through a 500-mesh mesh sieve, that is, the pore size of the mesh sieve is 29.96 μm, so as to avoid large-volume impurities from blocking the injection hole.

[0058] Wherein, the nuclei lysis buffer formula is as follows:

[0059]

[0060]

[0061] The staining buffer recipe is as follows:

[0062]

[0063] S2. Sampling of materials to be tested

[0064] For the preparation of plant cell nucleus suspension, materials such as callus, hypocotyl, cotyledon, suspensio...

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Abstract

The invention discloses an efficient akebia trifoliate ploidy detection method. The method comprises the steps of preparing a sample pretreatment agent; to-be-detected material sampling: selecting materials such as calluses, hypocotyls, cotyledons, suspension culture cells, leaves and the like, repeatedly washing and cleaning by using sterilized double distilled water when the experimental materials are taken, selecting tender parts, taking 480 to 510mg of samples, and avoiding tissues such as plant veins, vascular bundles and the like with high metabolite content during sampling; preparing a cell nucleus suspension; performing instrument detection and quality control of a corresponding fluorescence channel; performing contrast and standard flow cytometry detection; and performing data comparison and ploidy determination. The method has the advantages that pretreatment steps are simplified, the cell nucleus suspension required by detection is efficiently prepared, RNA interference is eliminated, flow cytometry sample loading parameters are standardized, identification results are unified and standard, multiple methods are combined to guarantee accurate results, large-scale detection is achieved, results are visualized, and data transmission is convenient and fast.

Description

technical field [0001] The invention relates to the technical field of plant ploidy detection, in particular to an efficient method for detecting ploidy of Akebia trifoliate. Background technique [0002] Akebia trifoliata is a perennial woody vine of the genus Akebia in the family Akebia. The related research in China, Japan and Korea has developed rapidly, and the research fields involve cultivation technology, breeding technology and molecular basic research. As early as 1986, Chinese scholar Jiang Like analyzed the amino acids of its ripe fruit, and found in subsequent research that it is rich in free amino acids, oleanolic acid, flavonoids and saponins, etc., and is a new type of therapeutic fruit. In 2018, my country's Sichuan Province promulgated the "Sichuan Provincial August Melon Food Safety Local Standards" to boost the development of the Sanye Akebia industry. Its artificial planting area has accumulated more than 70,000 mu in Sichuan Province. Related deep-proce...

Claims

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Application Information

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IPC IPC(8): G01N15/14G01N15/10
CPCG01N15/1434G01N15/1012G01N2015/1006G01N2015/011
Inventor 罗培高符鹏杨皓张秋怡陈巍关雎尤华忠陈琛钟胜福杨淮王娴淑喻小娇何自行董晴
Owner SICHUAN AGRI UNIV
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