A Flow Cytometry Identification Method for the Progeny of Homozygous Lines Obtained from Wheat Haploid Breeding

Abstract

Belonging to the biotechnology field, the invention discloses a flow identification method for homozygous line progenies obtained by wheat haploid breeding. According to the method, the concentrationsof citric acid and sodium chloride in a buffer solution mother liquor are optimized to 400-440g / L and 160-200g / L, nonylphenol polyoxyethylene ether is employed to substitute the common stabilizers and dispersants, and Hoechst 33258 with a concentration of 100-300mg / L is adopted to substitute DAPI and PI as the fluorescent dye, and the screen mesh of filtration and the centrifugal rotation speed are optimized to 350-450meshes and 1000-3000r / min respectively. Compared with the prior art, the method provided by the invention shortens the identification time, simplifies the identification steps,has more reasonable buffer configuration, can acquire more effective cells and remove more cell fragments, avoids detection interference, and improves the identification precision.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for identifying the offspring of homozygous lines obtained from wheat haploid breeding. Background technique [0002] Wheat is a monocotyledonous grass plant that is widely grown all over the world. As a staple food for humans, the caryopsis of wheat can be ground into flour to make bread, steamed buns, biscuits, noodles and other foods; fermented to make beer, alcohol, liquor or biofuel. Wheat is one of the three major grains and is used almost entirely for food. The haploid breeding technology in the wheat breeding method has significant advantages in shortening the breeding period and improving the breeding efficiency. After the obtained haploid is doubled by chromosome doubling, dominant diploid plants or polyploid plants can be obtained. [0003] In the existing diploid or polyploid plant identification technology, to identify whether the plant obtained by wheat haplo...

AI Technical Summary

Problems solved by technology

The entire identification process has many steps and is cumbersome, and the buffer preparation and cell enzymatic hydrolysis used take a long time

Another example is the patent No. 201610578478.1 of the invention "A buffer solution suitable for ploidy identification of various plants using flow cytometry and its preparation and use". The concentration of sodium chloride cannot achieve the best buffering effect; the Tween-20 reagent added to the buffer solution is relatively inefficient in dispersing free cell nuclei; staining agents such as DAPI and PI have certain damage to the activity of cells, affecting the cell viability. The proliferation activity of the cell suspension is unreasonable, so that the sieved filtrate contains more nuclei sticking together, which is not conducive to instrument detection and affects the detection accuracy; the filtrate is directly added to the staining solution, The number of effective cells that can be used for identification is low, which affects the detection accuracy

These problems affect the stability and dispersion of the cells and nuclei to be identified, which is not conducive to the final identification

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