Sugarcane tip rot pathogenic bacterium protoplast preparation and regeneration method

A technology for sugarcane tip rot and protoplasts, which is applied in the field of preparation and regeneration of fungal protoplasts, to achieve the effects of improved preparation efficiency, short enzymatic hydrolysis time, and easy operation

Inactive Publication Date: 2019-01-04
SOUTH ASIAN TROPICAL AGRI SCI RES INST OF GUANGXI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on the preparation of protoplasts of the pathogenic bacteria of sugarcane tip rot. In order to screen the pathogenicity variant strains of the pathogenic bacteria of sugarcane tip rot, strengthen the research on the pathogenic mechanism of the pathogen, and provide a theoretical basis for the breeding of sugarcane disease resistance, it is necessary to produce sugarcane tip rot. Study on the preparation and regeneration of protoplasts of rot pathogen

Method used

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  • Sugarcane tip rot pathogenic bacterium protoplast preparation and regeneration method

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Embodiment 1

[0034] A method for preparing and regenerating protoplasts of sugarcane tip rot pathogen, comprising the following steps,

[0035] (1) Preparation of sugarcane tip rot pathogen spore suspension: inoculate the pathogenic bacteria in the potato dextrose agar PDA medium and activate it for 3 days, then pick the mycelium on the edge of the colony and inoculate it in the potato dextrose water PDW medium, at a speed of 220rpm , cultivated in a constant temperature shaker at 28°C for 3 days, collected the bacteria by centrifugation, and prepared a spore suspension with sterile water;

[0036] (2) Preparation of fresh mycelium: Take 1ml of the prepared spore suspension and inoculate it in 100ml of potato dextrose water PDW medium, cultivate it in a constant temperature shaker with a rotation speed of 220rpm and a temperature of 28°C for 20h, and wipe it with bacteria. Obtain fresh mycelium by filter with lens paper, and fully wash with pre-cooled 0.8mol / L NaCl solution;

[0037] (3) ...

Embodiment 2

[0045] A method for preparing and regenerating protoplasts of sugarcane tip rot pathogen, comprising the following steps,

[0046] (1) Preparation of sugarcane tip rot pathogen spore suspension: inoculate the pathogenic bacteria in the potato dextrose agar PDA medium and activate it for 3 days, then pick the mycelium on the edge of the colony and inoculate it in the potato dextrose water PDW medium, at a speed of 220rpm , cultivated in a constant temperature shaker at 28°C for 3 days, collected the bacteria by centrifugation, and prepared a spore suspension with sterile water;

[0047] (2) Preparation of fresh mycelium: Take 1ml of the prepared spore suspension and inoculate it in 100ml of potato dextrose water PDW medium, cultivate it in a constant temperature shaker with a rotation speed of 220rpm and a temperature of 28°C for 18h, and wipe it with bacteria Obtain fresh mycelium by filter with lens paper, and fully wash with pre-cooled 0.8mol / L NaCl solution;

[0048] (3) C...

Embodiment 3

[0056] A method for preparing and regenerating protoplasts of sugarcane tip rot pathogen, comprising the following steps,

[0057] (1) Preparation of sugarcane tip rot pathogen spore suspension: inoculate the pathogen in the potato dextrose agar PDA medium and activate it for 4 days, then pick the mycelia on the edge of the colony and inoculate it in the potato dextrose water PDW medium, at a speed of 220rpm , cultivated in a constant temperature shaker at a temperature of 28°C for 4 days, collected the bacteria by centrifugation, and prepared a spore suspension with sterile water;

[0058] (2) Preparation of fresh mycelium: Take 1ml of the prepared spore suspension and inoculate it in 100ml of potato dextrose water PDW medium, cultivate it in a constant temperature shaker with a rotation speed of 220rpm and a temperature of 28°C for 18h, and wipe it with bacteria Obtain fresh mycelium by filter with lens paper, and fully wash with pre-cooled 0.8mol / L NaCl solution;

[0059] ...

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Abstract

The invention discloses a sugarcane tip rot pathogenic bacterium protoplast preparation and regeneration method which specifically comprises the following steps: (1) preparing a sugarcane tip rot pathogenic bacterium spore suspension; (2) preparing fresh mycelia; (3) preparing lyticase; (4) carrying out enzymolysis of hypha cell walls; (5) collecting protoplast; (6) regenerating the protoplast; (7) investigating regeneration situations. The method is low in cost, simple to operate, low in equipment requirement, short in enzymolysis time and regeneration cycle, capable of greatly improving sugarcane tip rot pathogenic bacterium protoplast preparation efficiency and well maintaining activity of the protoplast, good in regeneration capability and beneficial to genetic transformation of laterpathogenic bacteria and screening of transformants.

Description

technical field [0001] The invention relates to the technical field of preparation and regeneration of fungal protoplasts in molecular plant pathology, in particular to a method for preparing and regenerating protoplasts of pathogens of sugarcane tip rot. Background technique [0002] Sugarcane tip rot (Pokkah Boeng) is a fungal disease caused by Fusarium (Gibberella fujikuroi Saw.). There are many kinds of Fusarium fungi that cause sugarcane tip rot, including Fusarium moniliforme, Fusarium proliferatum, Fusarium verticillioides, etc. The dominant bacteria of Fusarium are different in different regions. The pathogenic bacteria of sugarcane tip rot found in my country is mainly Fusarium verticillioides Mainly. The pathogen of sugarcane tip rot mainly spreads to the heart leaves of sugarcane through airflow to infect and damage the leaves, causing leaf death and stunted plant growth. In severe cases, it can cause plant death, resulting in a 5%-20% reduction in sugarcane yield...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12R1/645
CPCC12N1/14
Inventor 郭强马文清唐利球陈海生秦昌鲜彭崇闭德金施泽升何洪良刘连军廖韦卫江清梅罗晟昇蒋亚琴韦海球
Owner SOUTH ASIAN TROPICAL AGRI SCI RES INST OF GUANGXI
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