Labeling method of apple pollen tube microfilament

A technology of apple pollen and labeling method, which is applied in biochemical equipment and methods, determination/inspection of microorganisms, etc., can solve problems such as poor labeling effect, and achieve the effect of high application value

Inactive Publication Date: 2015-04-22
BEIJING UNIV OF AGRI
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Problems solved by technology

In angiosperm Rosaceae, the labeling of pear pollen tube microfilaments has been reported. During the labeling process, the steps of en

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  • Labeling method of apple pollen tube microfilament

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Embodiment 1

[0022] A method for marking apple pollen tube filaments, comprising the following steps:

[0023] 1) Apple pollen culture: take out the pollen stored at -20°C and return to temperature, use the liquid culture method, weigh 10 mg of pollen, suspend it in 10ml of medium, culture at 30°C, shake at 100rpm / min in the dark, and the composition of the medium is : 20% sucrose + 0.015% CaCl 2 +0.01%H 3 BO 3 ;

[0024] 2) Fix: Wash the cultured apple pollen obtained in step 1) twice with 4% paraformaldehyde (prepared with 50mM PIPES buffer with a pH value of 6.9), fix for 1.5h, and vacuumize at the same time Gas for 10-15min, get fixed pollen;

[0025] 3) Enzyme treatment: wash the immobilized pollen obtained in step 2) three times with 50 mM PIPES buffer, and then place the washed pollen in a solution containing 1% pectinase and 1% by mass Incubate in 50mM PIPES buffer solution of cellulase at 37°C for 15-20min to obtain enzyme-treated pollen;

[0026] 4) The enzyme-treated polle...

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Abstract

The invention discloses a labeling method of an apple pollen tube microfilament. The labeling method comprises the following steps: cultivating apple pollen, curing the apple pollen, performing enzyme treatment on the apple pollen, treating the apple pollen with a surfactant, performing dye labeling, and performing scanning and imaging. The labeling method has the beneficial effect that the labeling method of gymnosperm pollen tube microfilament is referred and improved on the basis of the labeling method of pear pollen tube microfilament, dyes enter cells easier through curing, enzymolysis of cell walls and the surfactant, the interference of cell outer wall autofluorescence due to insufficient enzymolysis as TRITC-Phalloidin is applied are avoided when FITC-Phalloidin labeling is applied, so that relatively complete apple pollen tube microfilament can be stored; moreover, the labeling method is efficient and quick, can be used for obtaining relatively clear and bright pictures, thus filling up the blank of the apple pollen tube microfilament labeling, providing reference for labeling microfilament in rosaceae and angiosperm pollen tube, and achieving a high application value.

Description

technical field [0001] The invention relates to a method for marking apple pollen tube filaments. Background technique [0002] At present, there are almost no reports on the labeling of microfilaments in apple pollen tubes at home and abroad. The microfilament labeling technology in pollen mainly includes chemical staining methods: labeling phalloidin with fluorescent dyes, followed by freezing fixation combined with microinjection and using gene recombination technology to transfer the green fluorescent protein gene into living pollen tubes However, at present, the method of chemical staining is more simple and efficient. In recent years, there are relatively many studies on microfilaments in pollen of gymnosperms Pinaceae, and most of them are labeled with Rhodamine-Phalloidin. In angiosperm Rosaceae, the labeling of pear pollen tube microfilaments has been reported. During the labeling process, the steps of enzymatic hydrolysis of pollen tube wall and Triton decontamin...

Claims

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Application Information

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IPC IPC(8): C12Q1/00
Inventor 房克凤秦岭曹庆芹郝敬虹杨瑞王建立张卿杨柳
Owner BEIJING UNIV OF AGRI
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