Method for separation of Chinese cabbage microspores at different development stages

A developmental stage, microspore technology, applied in the field of plant biology, can solve problems such as the difficulty of consistent specific standards, the difficulty of building a Chinese cabbage regeneration system, and the difficulty of ensuring repeatability.

Inactive Publication Date: 2016-05-04

SHENYANG AGRI UNIV

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AI-Extracted Technical Summary

Problems solved by technology

[0002] As the free microspore culture technology of cruciferous crops is becoming more and more mature, the research on the mechanism of microspore embryogenesis is becoming a hot spot, but the method of isolating microspores at specific developmental stages has not been successfully solved so far; at present, with the development of functional genomics , the construction of a genetic transformation system is becoming more and more important, and it is difficult to construct a Chinese cabbage regeneration system. At present, researchers are beginning to use microspore culture and cell-penetrating peptides combined with a gene gun for trans...

Abstract

The invention discloses a method for separation of Chinese cabbage microspores at different development stages. The method includes: preparing a percoll separation liquid, placing a Chinese cabbage microspore crude extracted solution to the top, carrying out 150rpm low speed centrifugation to separate microspore at different development stages. Microspores at different development stages have close relation with the formation of vacuoles and other organelles, i.e. usually showing the difference of relative density. Therefore, separation of microspores at different development stages can be achieved by percoll density gradient centrifugation. The method provided by the invention can efficiently and stably separate microspores at different development stages, overcomes the disadvantages of difficult grasp of petal length/anther length ratio, flower bud length and the like in experiments utilizing traditional morphological indices, solves the problem of in-vitro preparation of microspore cells at similar development stages, and can provide materials for the mechanism study of Chinese cabbage microspore culture and provide receptors for gene gun mediated Chinese cabbage genetic transformation.

Application Domain

Plant cells

Technology Topic

BudChemistry +15

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Examples

Experimental program(1)

Example Embodiment

[0015] In the following examples, Chinese cabbage microspores were collected from the experimental field of the College of Horticulture, Shenyang Agricultural University; the stock solution of percoll was purchased from sigma reagent company.

[0016] Example 1

[0017] Sample preparation

[0018] 1. Accelerate the germination of Chinese cabbage seeds. After they are exposed to whiteness, treat them at a low temperature of 2 to 4°C for 20 days. Sow the germinated seeds on the plugs in the greenhouse and take the materials during the flowering period.

[0019] 2. Preparation of crude microspore extract

[0020] Select flower buds with petal length/anther length between 0.5-0.75, rinse with running water for 30 minutes, put them in a small beaker of sterilization, disinfect the surface with 70% ethanol solution for 30 seconds, and use 0.1% (m/v) HgCl 2 Disinfect the solution for 8 minutes, wash with sterile water 3 times, 5 minutes each time. Add 8-10mL B5 liquid medium containing 13% (m/v) sucrose, squeeze the flower buds with a sterile pestle to squeeze out the microspores. The suspension containing the microspores was filtered with a 400-mesh nylon mesh, and the filtrate was collected in a 10 mL centrifuge tube, and centrifuged at 1000 rpm for 3 min.

[0021] 3. Separation of microspores in different periods

[0022] (1) Take the stock solution of percoll and prepare the mother liquor of percoll containing 13% (m/v) sucrose, and then mix the mother liquor of percoll and the sucrose solution with a concentration of 13% (m/v) to 28% (V/v) according to the volume ratio. V), 50% (V/V)) percoll separation liquid.

[0023] (2) Take the 50% (V/V) percoll separation solution of step (1) 750uL and place it at the bottom of the 2ml centrifuge tube ( figure 1 Number 2 in A), and then carefully add 28%(V/V)percoll separation solution 750uL ( figure 1 The label in A 1).

[0024] (3) The percoll separation solution prepared in step (2) is allowed to stand at 4°C for 3 hours to form a stable separation working solution ( figure 1 The label in B 3).

[0025] (4) 200uL of crude microspore extract corresponding to about 20 buds ( figure 2 1) Add the top layer of the percoll separation working solution of step (3), centrifuge at low speed for 40 minutes, and take out the sample tube after centrifugation. The developmental stages of microspores distributed in 50% (V/V) percoll separation solution and 28% (V/V) percoll separation solution are the metaphase of mononuclear ( figure 2 Mark 2 in B) and early single-core ( figure 2 The label in B 3).

[0026] (5) Carefully extract the microspores obtained in step (4), suspend them in 13% (m/v) NLN liquid medium, and quickly centrifuge at 4°C for 25 minutes with a centrifuge revolution of 150 rpm. The resulting pellet is purified After removing percoll's Chinese cabbage microspores, it can be used as a recipient for genetic transformation experiments.

PUM

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