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Rapid preparation method of nuclear suspension suitable for determining chromosome ploidy of jujubes

A chromosome ploidy and cell nucleus technology is applied in the field of rapid preparation of cell nucleus suspension for determination of jujube chromosome ploidy, which can solve the problems of toxicity and high PI price, and achieve the effects of overcoming complicated steps, low cost and simple process.

Inactive Publication Date: 2018-12-11
HEBEI AGRICULTURAL UNIV.
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AI Technical Summary

Problems solved by technology

[0007] PI (propidium iodide) is a commonly used stain in flow cytometry ploidy identification. It is a non-specific dye that can perfectly combine with bases, but PI is expensive and toxic

Method used

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Embodiment Construction

[0019] First configure Tris-HCl lysate, take 2.422g Tris-HCl, 0.0813g MgCl 2 ·6H 2 O in a 100ml volumetric flask, add 0.5mL Triton-100, dilute to 100mL with ultrapure water, adjust the pH value to 7.5 with NaOH, and pre-cool in a refrigerator at 4°C; then configure PI dye solution, take 1mg PI dye Dissolve in 1mL of ultrapure water and store in a -20°C refrigerator. When used, take it out and thaw it, and place it at 4°C.

[0020] The young leaves of 'Linyi Lizao' and 'Chenguang' (the autotetraploid of 'Linyi Lizao') were selected from the field, put into sampling bags, brought back to the laboratory in an ice box and stored at 4 ℃; take 50 mg of young leaves, wash them with ultrapure water for 3 times, dry them, put them into a 4°C pre-cooled Petri dish, add 1mL of 4°C pre-cooled lysate, chop them quickly with a sharp blade, and then add 1mL Tris-HCl lysate, mix well and let stand for 1min; draw the lysate from the culture dish, filter it into a 3mL test tube with a 500-me...

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Abstract

The invention relates to a rapid preparation method of a nuclear suspension suitable for determining chromosome ploidy of jujubes. The rapid preparation method comprises: collecting young leaves, washing a plurality times with ultrapure water, placing into a pre-cooled culture dish, adding a pre-cooled Tris-MgCl2 lysate, quickly chopping with a cutter blade, adding a pre-cooled lysate, uniformly mixing, standing for a few minutes, filtering with a gauze, feeding into a centrifuge tube, adding RNase and a PI dye solution, and placing in a low-temperature dark place to obtain a nuclear suspension; and determining by using a flow cytometer, and obtaining the ploidy relationship by calculating the fluorescence intensity of the target peak. According to the present invention, the multiple stepsin the jujube chromosome sample preparation and the ploidy identification are improved, and the method is simple, rapid, accurate and low in cost, can be used for the rapid detection of the chromosome ploidy of large-batch jujubes, and is further suitable for Sour Jujube, Ziziphsu mauritiana Lam and other Ziziphus plants.

Description

technical field [0001] The invention belongs to the technical field of ploidy breeding, and in particular relates to a rapid preparation method of cell nucleus suspension suitable for jujube chromosome ploidy determination. Background technique [0002] jujube( Ziziphus jujuba Mill.) is a characteristic and dominant fruit tree native to my country, the largest dried fruit and one of the five dominant economic forests. However, the structure of jujube tree varieties is not reasonable enough at present, and there is a shortage of large-fruited, early-maturing, and disease-resistant varieties. It is urgent to accelerate the cultivation of new jujube tree varieties with excellent comprehensive properties. In jujube, due to the difficulty of hybrid breeding, ploidy breeding has become an important breeding method for jujube. With the deepening and mature application of jujube ploidy breeding research, it is becoming more and more important and urgent to quickly and accurately ...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N15/14
CPCG01N1/30G01N15/1434
Inventor 刘孟军刘平王利虎
Owner HEBEI AGRICULTURAL UNIV.
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