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Method for detecting nuclear DNA content of Camellia plant

A camellia and cell nucleus technology, which is applied in the biological field, can solve the problems of high requirements for plant materials, low success rate, and increased error variation of measurement results, so as to achieve good experimental repeatability, avoid limitations, and improve accuracy. Effect

Inactive Publication Date: 2018-06-15
RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

By adding a large amount of antioxidant (DTT) and tannin-binding substance (PVP-10), the WPB buffer solution solves the problem of precipitation and background fluorescence of tissue lysate to some extent, but the success rate of using this technique is not high , and the results of the determination have large errors, and the specific defects are as follows: (1) This method has high requirements for plant materials, and very young tissues must be used for experiments; , even young leaves or shoots contain a large amount of secondary metabolites, especially tannins that can combine with the DNA dyes used, resulting in increased error variation in measurement results; (3) the nuclear lysate contains High concentration of PVP-10 (1%, mass volume ratio) and DTT (20mM), resulting in viscous lysate, easy to block the pipeline system of flow cytometer

Method used

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  • Method for detecting nuclear DNA content of Camellia plant
  • Method for detecting nuclear DNA content of Camellia plant
  • Method for detecting nuclear DNA content of Camellia plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 prepares the test nucleus lysis solution of camellia callus by the nucleus lysis solution of the present invention

[0060] 1. Select wild camellia (Camellia japonica) materials for disinfection treatment and callus induction

[0061] (1) Select about 15-year-old wild camellia materials planted in the Institute of Subtropical Forestry, Chinese Academy of Forestry, and cut 5 branches in good growth condition.

[0062] (2) Select 20 healthy leaves without disease spots and yellowing from the obtained branches, put them in a 500ml beaker, put them into running water and soak them for 5 minutes, then rinse them repeatedly 5 times.

[0063] (3) The leaves are cut into small pieces with a particle size substantially smaller than 1 cm with a blade.

[0064] (4) In the ultra-clean workbench, transfer the small pieces to an Erlenmeyer flask containing 200 ml of 70%-75% (volume ratio) ethanol, place it for 30 seconds, and rinse with sterile water 3 times.

[0065] ...

Embodiment 2

[0073] Example 2 Preparation of the test cell nucleus lysis solution of camellia tender leaves by WPS lysate

[0074] (1) The young leaves of wild camellia were selected, washed with tap water, dried and placed on ice.

[0075] (2) Cut the leaves into thin strips with a width of about 1 mm with a sharp scalpel blade, and place them in a plastic petri dish with a diameter of 5 cm.

[0076] (3) Add 2ml of WPB buffer (Tris 0.2M, MgCl 2 4mM, EDTA-Na2 2mM, NaCl 86mM, dithiothreitol DTT 20mM, polyvinylpyrrolidone PVP-10 1% (mass volume ratio), Triton X-100 1% (volume ratio), PH=7.5), make blade The strips are completely submerged in the lysate (e.g. figure 2 shown on the left).

[0077] (4) After shaking gently, place the culture dish on ice for 15 minutes, pass the cell nucleus lysate through a nylon filter with a pore size of 40 microns, and transfer it to a 2ml round-bottomed plastic tube.

[0078] (5) According to the volume of the filtrate, add 1 / 10 volume of propidium io...

Embodiment 3

[0079] Example 3 Flow Cytometry Result Analysis Comparison

[0080] (1) Turn on the flow cytometry instrument (BD Acurri C6), first use deionized water as a blank sample, and check the flow status of the instrument pipeline.

[0081] (2) Place the nuclei lysis solution to be tested obtained in Examples 1 and 2 on the flow cytometer, start slow loading until the total number of particles is collected to more than 80,000, and stop loading. Use the FL-2 and SSC of the flow cytometer to circle the distribution area of ​​the nucleus, and use the distribution of the fluorescent signal to calculate the staining of the nuclear DNA (such as image 3 shown). image 3 The markers of M4, M5, M7, and M8 in (b) and (d) are the stained regions of Camellia nuclear DNA, M4 and M7 represent diploid nuclei, and M5 and M8 represent tetraploid nuclei. It can be seen by comparison that the DNA fluorescence signal distribution of the nuclei lysed by the callus is more concentrated, the background ...

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Abstract

The invention provides a method for detecting the nuclear DNA content of a Camellia plant. The method comprises the following steps: (1) cutting a Camellia plant blade or stem segment to obtain an explant; (2) performing disinfecting pretreatment on the explant; (3) inducing calluses; (4) processing the induced calluses to achieve nucleus separation, and filtering the processed calluses; (5) staining a filtrate obtained in step (4) to prepare a nuclear lysis solution to be detected; and (5) detecting the nuclear DNA content of the nuclear lysis solution to be detected through a flow cytometer.The method has application significance in ploidy inspection and genome size determination of the Camellia plant, and provides a new analytical technique for polyploid breeding of the Camellia plant.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a method for analyzing the DNA content of the cell nucleus by using the callus tissue of the genus Camellia. Background technique [0002] Camellia species are rich in resources, including many woody plants with important economic value. Studies have shown that the DNA content (ploidy) of plant nuclei is correlated with traits such as morphology and resistance, making ploidy breeding an important way to obtain new varieties and new germplasm. [0003] Flow cytometry (Flow Cytometry) is an analytical technique emerging at the end of the 20th century, and it is also one of the most important means for analyzing the DNA content of plant nuclei. Using flow cytometry to accurately and efficiently detect the nuclear DNA content of Camellia plants is a key technology for ploidy breeding of Camellia plants. However, the tissues of Camellia plants contain complex seco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6806C12Q1/6851A01H4/00
CPCA01H4/00A01H4/001C12Q1/6806C12Q1/6851C12Q1/6895C12Q2600/13C12Q2565/626
Inventor 殷恒福杨稳吕焘李纪元李辛雷范正琪
Owner RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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