Eucalyptusurophylla*E. grandis flow-type detection method

A technology of flow detection and Eucalyptus grandis, applied in measuring devices, individual particle analysis, scientific instruments, etc., can solve problems such as restricting ploidy breeding and breeding technology, and achieve the effect of broadening application occasions and application scope and achieving good stability.

Inactive Publication Date: 2016-11-09
RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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  • Abstract
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AI Technical Summary

Problems solved by technology

However, because its nuclear lysis technology has not been developed and matured, its DNA ploidy identification (mainly used for ploidy breeding of eucalyptus), cell cycle, cell apoptosis and related metabolism have not been carried out, which seriously restricts its ploidy Development of breeding techniques such as breeding

Method used

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  • Eucalyptusurophylla*E. grandis flow-type detection method
  • Eucalyptusurophylla*E. grandis flow-type detection method
  • Eucalyptusurophylla*E. grandis flow-type detection method

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Preparation of lysate: first weigh 24.23g Tris, 0.81g MgCl with an analytical balance 2 ·6H 2 O, 0.82g EDTA Na 2 2H 2 O, 5.03g NaCl, 1.90g sodium metabisulfite, 19.21g citric acid, 10.00g PVP-10, put into a clean beaker that has been rinsed with double distilled water, add double distilled water, and stir with a glass rod until completely dissolved; then Pipette 10mL Triton X-100 and 5mL Tween 20 with a pipette gun, and use a 1L volumetric flask to make up the volume after they are all dissolved; finally, after measuring the pH value with a pH meter, adjust it with 1mol / L HCl solution and 10mol / L NaOH solution When the pH reaches the designated value of 7.5, store in a refrigerator at 4°C.

[0041] Pick 40 mg of 2 to 3 young leaves from the top of Eucalyptus macrocephalus DH32-29 cultivated seedlings, put the material in a petri dish, place the petri dish on ice, use a pipette gun to absorb 1mL of lysate at 0~4°C to immerse the material, and use a blade Cut the mate...

Embodiment 2

[0043] The lysate after the flow detection in Example 1 was placed in a foam box at 25°C under light-blocking conditions for 48 hours, and the flow detection was performed again. The histogram of the nuclear DNA is as follows: figure 2 As shown, its lysis evaluation parameters are shown in Table 1.

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Abstract

The invention belongs to the field of plant ploidy detection and discloses a Eucalyptusurophylla*E. grandis flow-type detection method. The method comprises 1, taking tender leaves of Eucalyptusurophylla*E. grandis DH32-29, adding the tender leaves into a lysate, cutting the tender leaves through a blade and carrying out cracking to obtain a cell cracking fluid suspension, 2, filtering the cell cracking fluid suspension, centrifuging the filtrate, removing the supernatant, adding a lysate into the filter residues so that cell nucleuses in the precipitates are re-suspended and filtering the suspension to obtain a filtrate, 3, adding a RNase enzyme solution into the filtrate obtained by the step 2 and carrying out enzymolysis to obtain an enzymatic hydrolysate, 4, adding a PI dye solution into the enzymatic hydrolysate obtained by the step 3 and carrying out dying to obtain a dyed solution, and 5, detecting the dyed solution through a flow cytometer. The method realizes instant sample preparation after material collection in the field. After preparation for 2 days (48h), the cell nucleus dyed solution is subjected to flow-type detection and analysis so that detection effect stability is ensured and the problem that the existing flow-type detection method needs immediate detection after collection is solved.

Description

technical field [0001] The invention belongs to the field of plant ploidy detection, in particular to a method for flow detection of Eucalyptus urophylla. Background technique [0002] Flow cytometry (Flow cytometry, FCM) is a new technology born in the 1950s, but it was not until the late 1980s that this technology was widely used in the field of plant science, and it was mainly used in nuclear DNA Content determination, DNA ploidy identification and cell cycle analysis and other research. The key point of whether this technology can be successfully used in plants is whether it can prepare single-cell (or single-nucleus) suspensions of plant materials. Researchers have been constantly improving the pre-treatment technology for cell detection in response to this problem, such as Heller (1973 ) uses hydrolytic enzymes to digest the cell wall of plants to release the nucleus, but this method is time-consuming, and later generations seldom continue to use this method; after th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 韩超徐建民
Owner RES INST OF TROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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