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Dry plant tissue treatment method applied to flow cytometry

A technology of flow cytometry and plant tissue, which is applied in individual particle analysis, particle and sedimentation analysis, preparation of test samples, etc. It can solve problems such as difficult to extract nuclei, difficult to separate, and difficult to break plant cell membranes. Achieve the effect of being easily adsorbed by dyes and improving the scope of application

Active Publication Date: 2015-02-18
TAIZHOU UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although, the processing method of mature leaf is mentioned in the prior art, still do not relate to the method for processing and detecting dry plant tissue in the prior art, and because dry plant tissue is through dehydration process, plant cell tissue is basically separated from nucleus Stick together, it is difficult to separate, and the dehydrated plant cell membrane is difficult to break, it is difficult to extract the nucleus and ensure the staining effect, which affects the accuracy of flow cytometry analysis; on the other hand, due to the long storage time of dry plant tissue, The DNA in the nucleus is prone to breakage and degradation

Method used

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  • Dry plant tissue treatment method applied to flow cytometry
  • Dry plant tissue treatment method applied to flow cytometry
  • Dry plant tissue treatment method applied to flow cytometry

Examples

Experimental program
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Embodiment 1

[0044] This embodiment is the ploidy determination of Solidago canadensis, and the components of the cell nucleus extract used in this embodiment are:

[0045] Tris: 12mMmM; Na 2 EDTA: 1mM; spermine hydrochloride: 0.2mM; KCl: 70mM, NaCl: 10mM; β-mercaptoethanol: 10mM; Triton X-100: 0.3% (vol / vol); Adjusted with NaOH solution, the cell nucleus extract is pre-stored in a -20°C refrigerator for later use;

[0046] Put 50 mg of mature leaves of Solidago canadensis (leaves dried on silica gel) into a pre-cooled plastic petri dish, then add 1 mL of pre-cooled cell nucleus extract, and then use a double-sided blade to chop the leaves. The chopping process lasts for 10 minutes. For more than 10 minutes, first filter with a 30-micron nylon membrane to collect the filtrate containing the nucleus, and then perform a secondary chopping process on the remaining leaves, that is, use a double-sided blade to continue chopping the leaves. The chopping process lasts for more than 10 minutes un...

Embodiment 2

[0050] The present embodiment is the ploidy determination of the yellow warbler, and the components of the cell nucleus extract used in the present embodiment are:

[0051] Tris: 14mM; Na 2 EDTA: 1mM; spermine hydrochloride: 0.2mM; KCl: 70mM, NaCl: 15mM; β-mercaptoethanol: 20mM; Triton X-100: 0.5% (vol / vol); Adjusted with NaOH solution, the cell nucleus extract is pre-stored in a -20°C refrigerator for later use;

[0052] Put 50 mg of the mature leaves of the oriole (dried on silica gel) into a pre-cooled plastic petri dish, then add 1 mL of pre-cooled cell nucleus extract, and then use a double-sided blade to chop the leaves. The chopping process lasts for more than 10 minutes , first filter with a 30-micron nylon membrane, collect the filtrate containing the nucleus, and then perform a secondary chopping process on the remaining leaves, that is, use a double-sided blade to continue chopping the leaves. The chopping process lasts for more than 10 minutes until all the leaves...

Embodiment 3

[0056] This embodiment is the ploidy determination of Solidago canadensis, and the components of the cell nucleus extract used in this embodiment are:

[0057] Tris: 18mM; Na 2 EDTA: 3mM; spermine hydrochloride: 0.2mM; KCl: 70mM, NaCl: 25mM; β-mercaptoethanol: 20mM; Triton X-100: 0.2% (vol / vol); Adjusted with NaOH solution, the cell nucleus extract is pre-stored in a -20°C refrigerator for later use;

[0058] Put 45 mg of mature leaves of Solidago canadensis (leaves dried on silica gel) into a pre-cooled plastic petri dish, then add 1 mL of pre-cooled cell nucleus extract, and then chop the leaves with a double-sided blade with a thickness of 0.1 mm. The chopping process lasts for more than 10 minutes. First filter with a 30-micron nylon membrane to collect the filtrate containing the nucleus, and then perform a secondary chopping process on the remaining leaves, that is, use a double-sided blade with a thickness of 0.1mm to continue chopping the leaves. The crushing process...

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Abstract

The invention relates to a dry plant tissue treatment method applied to flow cytometry, belonging to the technical field of biological analysis and aiming at solving the problem that only a fresh sample can be analyzed in the prior art. The treatment method comprises the following steps: performing cell nucleus extraction, staining and analyzing, wherein a cell nucleus extracting solution used by cell nucleus extraction comprises the following components: 12-18mM of Tris, 1-3Mm of Na2EDTA, 0.2-0.3mM of spermine tetrahydrochloride, 70-90mM of KCl, 10-25mM of NaCl, 10-20mM of beta-mercaptoethanol and 0.2-0.5 percent of Triton X-100, and the pH value is 6.5-7.0. The method has the effects of preventing cell nucleus DNA degradation, improving the stability of cell nucleus and lowering probability of oxidation of cell nucleus, and can be applied to various dry plant tissues, and the application range is widened.

Description

technical field [0001] The invention relates to a treatment method suitable for flow cytometric analysis of dry plant tissues, belonging to the technical field of biological analysis. Background technique [0002] Flow cytometry (flow cytometry) is a technique that uses flow cytometry to perform rapid quantitative analysis and sorting of single cells or other biological particles in a liquid flow. In botanical research, it can be used for plant ploidy identification, nuclear DNA quantity determination, reproductive pathway identification, etc. [0003] At present, the conventional flow cytometry analysis of plant chromosome ploidy uses the root tips, leaves or delicate tissues of fresh plants, because it can avoid the high concentration of starch, polysaccharides and other metabolites in old tissues, and these Substances are distributed in the cytoplasm, which makes the cytoplasm closely adhere to the nucleus, which affects the purity of the nucleus. At the same time, it al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N1/30G01N15/10
Inventor 李钧敏高松陈露茜王蓓蕾
Owner TAIZHOU UNIV
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