Method for preparing and regenerating phomopasis asparagi protoplast

A technology of asparagus stem blight and protoplasts, which is applied in the field of asparagus stem blight protoplast preparation and regeneration, and can solve the molecular mechanism and research of unfavorable pathogenic bacteria

Inactive Publication Date: 2013-12-18
VEGETABLE & FLOWER INST JIANGXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no reports on the protoplast preparation system of Asparagus stem blight at home and ab...

Method used

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Examples

Experimental program
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Effect test

example 1

[0038] Example 1: Collect the conidia produced by the pycnidia on oat solid medium (OA), inoculate them in complete liquid medium (CM), and culture them on a shaker at 150r / min at 25°C for 36h to obtain fresh bacteria Silk, using a mixed enzyme of 1.5% lyase, 1.0% collapse enzyme, and 1.5% helicase: pH 6.00, enzymatically hydrolyze in a water bath at 30°C for 3.0h, and use 1.0mol / L MgSO 4 (10mmol / LPBS preparation, the volume ratio is MgSO 4 : PBS=3:1) as an osmotic pressure stabilizer; dilute the protoplast suspension 100 times with a 0.6mol / L sucrose solution, take 0.1mL of the diluted solution and spread it on a potato dextrose agar plate and a hyperosmotic potato dextrose agar plate Cultured at 25°C for 3-5 days, the number of protoplasts reached 1.7×10 7 cells / mL, the regeneration rate was 22.0%.

example 2

[0039] Example 2: Collect the conidia produced by the pycnidia on the oat solid medium (OA), and inoculate them in complete liquid medium (CM), and cultivate them on a shaker at 150r / min for 48 hours at 25°C to obtain fresh bacteria Silk, using a mixed enzyme of 1.5% lyase, 1.0% collapse enzyme and 1.5% helicase: pH 7.60, enzymolysis in 35°C water bath for 4.0h, with 1.0mol / L MgSO 4 (10mmol / LPBS preparation, the volume ratio is MgSO 4 : PBS=3:1) as an osmotic pressure stabilizer; dilute the protoplast suspension 100 times with a 0.6mol / L sucrose solution, take 0.1mL of the diluted solution and spread it on a potato dextrose agar plate and a hyperosmotic potato dextrose agar plate Cultured at 25°C for 3-5 days, the number of protoplasts reached 2.2×10 7 cells / mL, the regeneration rate was 22.5%.

example 3

[0040] Example 3: Collect the conidia produced by the pycnidia on the oat solid medium (OA), and inoculate them in complete liquid medium (CM), and cultivate them on a shaking table at 150r / min for 72h at 25°C to obtain fresh bacteria Silk, using a mixed enzyme of 1.5% lyase, 1.0% collapse enzyme and 1.5% helicase: pH 6.98, hydrolyze in a water bath at 33°C for 4.5h, and use 1.0mol / L MgSO 4 (10mmol / LPBS preparation, the volume ratio is MgSO 4 : PBS=3:1) as an osmotic pressure stabilizer; dilute the protoplast suspension 100 times with a 0.6mol / L sucrose solution, take 0.1mL of the diluted solution and spread it on a potato dextrose agar plate and a hyperosmotic potato dextrose agar plate Cultured at 25°C for 3-5 days, the number of protoplasts reached 2.8×10 7 cells / mL, the regeneration rate was 24.0%.

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PUM

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Abstract

The invention discloses a method for preparing and regenerating a phomopasis asparagi protoplast, which relates to the technical field of fungus protoplast preparation and regeneration in cell engineering. The method comprises the following detailed operation steps of: (1) preparing a phomopasis asparagi conidium suspension; (2) preparing a fresh mycelium; (3) performing enzymolysis on mycelium cell walls; (4) separating the protoplast; and (5) regenerating the protoplast. The method is easy to operate, low in requirements on equipment and short in enzymolysis time and regeneration period, and effectively enhances the efficiency of preparation and regeneration of the protoplast.

Description

technical field [0001] The invention relates to the technical field of preparation and regeneration of fungal protoplasts in cell engineering, in particular to a method for preparing and regenerating protoplasts of stem blight of asparagus. Background technique [0002] Asparagus (Asparagus officinalis L.), also known as Asparagus officinalis L., is a perennial herbaceous plant of Asparagus officinalis. It is a health-care vegetable with high nutritional value and enjoys the reputation of "king of vegetables" internationally reputation. Asparagus stem blight is an important worldwide fungal disease caused by Phomopsis asparagi, which has been reported in the United States, Europe and Australia. The disease mainly harms stems or young shoots (stems or spears), and can also infect branches and pseudo-leaves. It is initially water-soaked streaks, and then gradually becomes dark black spindles. Small black dot-shaped pycnidia are scattered on it, eventually causing the whole p...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12R1/645
Inventor 张岳平陈光宇罗绍春周劲松汤泳萍黄燕萍
Owner VEGETABLE & FLOWER INST JIANGXI ACADEMY OF AGRI SCI
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