Buffer liquid suitable for ploidy identification of various plants by flow cytometer, and preparation and use methods thereof

A technology of flow cytometry and ploidy identification, which is applied in the field of plant breeding, can solve the problems of many buffer components, narrow application range, complex preparation, etc., and achieve the effect of convenient application, low cost and simple preparation

Active Publication Date: 2016-12-21
SOUTHWEST UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of buffer suitable for utilizing flow cytometry to carry out the ploidy identification of various plants, and solve the problem that existing buffer components in the prior art are more, the preparation is relatively complicated, the cost is hig

Method used

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  • Buffer liquid suitable for ploidy identification of various plants by flow cytometer, and preparation and use methods thereof
  • Buffer liquid suitable for ploidy identification of various plants by flow cytometer, and preparation and use methods thereof
  • Buffer liquid suitable for ploidy identification of various plants by flow cytometer, and preparation and use methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the ploidy identification of citrus

[0044] Step 1, preparation of buffer:

[0045] (1) Weigh 88.2 g of trisodium citrate and 200 g of sodium chloride, dissolve them in 900 mL of double-distilled water, adjust the pH to 7.0 with hydrochloric acid, add double-distilled water to set the volume to 1000 mL, and obtain a buffer mother solution, which can be stored at room temperature for a long time.

[0046](2) When in use, dilute the buffer mother solution 20 times with double distilled water to obtain a working solution, containing 0.015mol / L trisodium citrate and 10% sodium chloride in mass concentration.

[0047] Step 2, the use of buffer:

[0048] (1) Take a little young leaves and place them in a plastic plate;

[0049] (2) Add 50 μL of working solution, chop the material with a sharp blade, add 1000 μL of buffer solution, shake slightly to mix;

[0050] (3) Pass the suspension through a 500-mesh sieve and let it stand for a while or add 50 μg / mL of ...

Embodiment 2

[0053] Embodiment 2, the ploidy identification of tobacco

[0054] Step 1, preparation of buffer:

[0055] (1) Weigh 88.2 g of trisodium citrate and 200 g of sodium chloride, dissolve them in 900 mL of double-distilled water, adjust the pH to 7.0 with hydrochloric acid, add double-distilled water to set the volume to 1000 mL, and obtain a buffer mother solution, which can be stored at room temperature for a long time.

[0056] (2) When in use, dilute the buffer mother solution 20 times with double distilled water to obtain cell hypertonic buffer, add Tween-20 to make the final concentration 0.25% (add 0.25 mL of Tween-20 per 100 mL of double distilled water 20), add β-mercaptoethanol so that the final concentration is 20mmol / L (add 0.14mL of β-mercaptoethanol to every 100mL of double-distilled water) to prepare a working solution, containing 0.015mol / L trisodium citrate, mass concentration 10% Sodium chloride, 0.25% Tween-20, 20mmol / L β-mercaptoethanol.

[0057] Step 2, the ...

Embodiment 3

[0062] Embodiment 3, the ploidy identification of kiwifruit

[0063] Step 1, preparation of buffer:

[0064] (1) Weigh 88.2 g of trisodium citrate and 200 g of sodium chloride, dissolve them in 900 mL of double-distilled water, adjust the pH to 7.0 with hydrochloric acid, add double-distilled water to set the volume to 1000 mL, and obtain a buffer mother solution, which can be stored at room temperature for a long time.

[0065] (2) When in use, dilute the buffer mother solution 20 times with double-distilled water to obtain cell hyperosmotic buffer, add Tween-20 to make the final concentration 0.1% (add 0.1mL Tween-20 per 100mL of double-distilled water 20), add β-mercaptoethanol, and add 0.1mL of β-mercaptoethanol per 100mL of double-distilled water to prepare a working solution.

[0066] Step 2, the use of buffer:

[0067] (1) Take a little young leaves and place them in a plastic plate;

[0068] (2) Add 200 μL of working solution, chop the material with a sharp blade, a...

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Abstract

The invention discloses a buffer liquid suitable for ploidy identification of various plants by a flow cytometer, and preparation and use methods thereof; the buffer liquid is composed of a buffer mother solution, Tween-20, and beta-mercaptoethanol; the preparation and use methods comprise the steps: firstly, diluting the buffer mother solution with double distilled water 20 times to obtain a cell ultra-infiltration buffer liquid, then adding 0.1-0.5 mL of Tween-20 in each 100 mL of the buffer liquid, and adding 0.1-0.2 mL of beta-mercaptoethanol in each 100 mL of the buffer liquid, wherein the buffer mother solution is composed of 0.3 mol/L trisodium citrate and sodium chloride with the mass concentration of 20%, and solvents are all double distilled water; then taking a plant tissue root tip; adding the buffer liquid, chopping, and mixing evenly; making the suspension pass through a 300-500 mesh screen, placing or immediately adding a dyeing liquid, and then performing sample loading and detecting. The buffer liquid has a few of components, has the advantages of simple preparation method and low cost, and is suitable for ploidy identification of various plants.

Description

technical field [0001] The invention belongs to the technical field of plant breeding, and relates to a buffer suitable for identifying ploidy of various plants by using a flow cytometer and a preparation and use method thereof. Background technique [0002] Plant ploidy identification is widely used in plant breeding and related basic research. In recent years, variety improvement through ploidy manipulation has become one of the hotspots in plant breeding and research. During the ploidy operation, it is necessary to identify the ploidy of a large number of materials and to classify and screen materials of different ploidy. [0003] Existing methods for ploidy identification mainly include chromosome preparation method, stomatal guard cell observation method, nucleus size observation method, flow cytometry detection method, etc., among which flow cytometry detection method is the fastest. [0004] In the detection of plant ploidy by flow cytometry, the application of buff...

Claims

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Application Information

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IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 党江波梁国鲁郭启高李晓林孙海艳何桥
Owner SOUTHWEST UNIVERSITY
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