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Method for sorting 2n pollen from pollen

A pollen and sorting technology, which is applied in the analysis of materials, individual particle analysis, particle and sedimentation analysis, etc., can solve the problems of high cost, complicated operation, and low 2n pollen acquisition rate, and achieves reduction of sorting errors and efficient sorting. Effect

Active Publication Date: 2019-12-06
SOUTH CHINA AGRI UNIV
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  • Description
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AI Technical Summary

Problems solved by technology

The acquisition rate of 2n pollen is generally low. In order to improve the efficiency of creating polyploids, 2n pollen must be separated from normal pollen, and artificial pollination must be carried out after reaching a higher ratio.
According to reports, the diameter of unreduced 2n pollen is 1.28 to 1.30 times that of normal pollen. Therefore, according to the pollen diameter, the methods for separating 2n pollen mainly include: screening method, velocity sedimentation method and electrostatic pollen sorting method, but these methods are not It has the disadvantages of low screening efficiency, high cost, complicated operation, etc.

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  • Method for sorting 2n pollen from pollen
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  • Method for sorting 2n pollen from pollen

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This embodiment provides a method for sorting 2n pollen from pollen. The specific operation process is as follows:

[0057] 1 Materials and methods

[0058] 1.1 Test materials

[0059] The material used for analysis and sorting is the longan hybrid pollen induced by the applicant through 4 treatments in the spring of 2019. The variety is a hybrid of longan 'Shixia' as the female parent and litchi 'Zi Niangxi' as the male parent The intergeneric hybrid 'Shizi 19' was planted in the orchard of South China Agricultural University.

[0060] 1.2 Flow cytometer software operation

[0061] Adjust the ambient temperature of the flow cytometer to 20°C and the humidity to 48%.

[0062] The instrument is started, and the C&ST quality control (testing the status of the instrument to ensure that the lasers of the instrument are operating normally) and the DropDelay process (to ensure accurate sorting of the instrument) are carried out.

[0063] (1) Create a new experiment, sele...

Embodiment 2

[0113] This embodiment is a modification example of Embodiment 1, and the changes relative to Embodiment 1 include:

[0114] (1) The ambient temperature for sorting 2n pollen by flow cytometry is 18° C., and the humidity is 46%.

[0115] (2) The solvent used is an aqueous sucrose solution with a mass percentage of 0.6%.

[0116] (3) The parameter values ​​of the flow cytometry protocol are set as:

[0117] Threshold (ThreShold): 130; GFP voltage (PMT): 24.62; SSC voltage (PMT): 24.20; FSC voltage (PMT): 0.50.

[0118] See Table 7 for the results. According to Table 7, there is no significant difference between the results obtained in this example and the traditional sorting method. Moreover, the activity of the 2n pollen obtained in this example is very good, and the germination rate reaches 71.88%.

[0119] Table 7. Using three different methods to count the 2n pollen rate of each treatment pollen

[0120]

Embodiment 3

[0122] This embodiment is a modification example of Embodiment 1, and the changes relative to Embodiment 1 include:

[0123] (1) The ambient temperature for sorting 2n pollen by flow cytometry is 22° C., and the humidity is 50%.

[0124] (2) The mass percent of the sucrose aqueous solution adopted is 1.5%.

[0125] (3) The parameter values ​​of the flow cytometry protocol are set as:

[0126] Threshold (ThreShold): 3372; GFP voltage (PMT): 50.86; SSC voltage (PMT): 29.07; FSC voltage (PMT): 8.5.

[0127] See Table 8 for the results. According to Table 8, there is no significant difference between the results obtained in this example and the traditional sorting method. And the activity of the 2n pollen obtained in this example is very good, and the germination rate reaches 75.14%.

[0128] Table 8. Using three different methods to count the 2n pollen rate of each treatment pollen

[0129]

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Abstract

The invention relates to a method for sorting 2n pollen from pollen. The method comprises the steps of 1, taking a pollen sample to prepare pollen suspension; and 2, sampling the pollen suspension toa flow cytometry for sorting. According to the method, the flow cytometry is applied into the 2n pollen, so that the problem that the 2n pollen sorting is low in efficiency and complicated in operation is ingeniously solved. Furthermore, in cooperation with the setting of the sorting environment temperature, humidity and parameter value of the flow cytometry, the sorting error of the flow cytometry can be reduced.

Description

technical field [0001] The invention relates to the technical field of biological breeding, in particular to a method for sorting 2n pollen from pollen. Background technique [0002] Flow cytometry (flow cytometry, FCM) refers to the rapid quantitative analysis of the physical and chemical characteristics of cell populations using flow cytometry and the precise sorting of these physical and chemical characteristics. It is mainly divided into flow cytometry and flow cytometry. It is widely used in various fields of basic medicine and biological research. The application of flow cytometry in plants started relatively late, mainly focusing on flow analysis, including nuclear DNA content analysis, ploidy analysis, chromosome structure analysis, cell cycle analysis, etc. In terms of flow sorting, there are many applications for sorting chromosomes and protoplasts and then combining other analysis methods such as genome, transcriptome and proteome. At present, there are no relev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
CPCG01N15/1404G01N15/1434
Inventor 傅嘉欣刘成明熊海胡桂兵甘吉昌毛晓丹
Owner SOUTH CHINA AGRI UNIV
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