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Preparation method of protoplast of Tilletia glabrata

A technique for the production of smut and protoplasts, which is applied in the field of preparation of protoplasts of smut of wheat, and can solve the problems of less research on the pathogenic mechanism of pathogens and the like.

Active Publication Date: 2021-02-26
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At home and abroad, the research on wheat light smut mainly focuses on the biological characteristics of the pathogen and molecular marker technology, and there are few studies on the pathogenic mechanism of the pathogen

Method used

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  • Preparation method of protoplast of Tilletia glabrata
  • Preparation method of protoplast of Tilletia glabrata
  • Preparation method of protoplast of Tilletia glabrata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Optimization of Tilletia glabra protoplast preparation method

[0053] Cultivate the teliospores of Tilletia tritici in 2% water agar medium at a temperature of 16°C and a relative humidity of 80%. Select the cells of different culture days and wash them down with sterilized distilled water, collect the bacterial liquid and filter it , centrifuge at 5000r / min for 10min to remove the supernatant, take 1g of mycelium, add 20ml of different types of compound enzyme solution, 28°C, 180r / min oscillating enzymolysis, observe the protoplast yield with a hemocytometer every half hour, and screen out The most suitable bacterial age, enzymatic hydrolysis time, enzymatic hydrolysis system, and osmotic pressure stabilizer for protoplast preparation.

[0054] 1. Bacteria culture time

[0055] Cultivate the teliospores of Tilletia tritici on the water agar medium, the temperature is 16 ℃, and the relative humidity is 80%. After 4 days, the first mycelium begins to germ...

Embodiment 2

[0073] Embodiment 2: Adopt optimized method to prepare Tilletia glabra protoplasts

[0074] 1.2 mol / L potassium chloride was used to prepare a composite enzyme solution with a mass concentration of 1.5% collapsing enzyme, 1.5% lysozyme and 1.5% helicase. Then prepare protoplasts according to the following steps:

[0075] (1) Mycelia culture: Cultivate the teliospores of Tilletia tritici on 2% water agar medium for 7 days, the culture condition is 16 ℃ of temperature, 80% relative humidity, then the teliospores of germination are rinsed with sterilized distilled water After coming down, collect the bacterial liquid.

[0076] (2) Enzymatic hydrolysis of the mycelial cell wall: filter the collected bacterial liquid, then centrifuge at 5000r / min for 10min to remove the supernatant, take 1g of mycelium, add 20mL compound enzyme solution, 28°C, 180r / min, oscillating for 2h, and Filtrate the enzymatic solution and centrifuge at 5000r / min for 10min to obtain protoplasts of Tilletia ...

Embodiment 3

[0079] Embodiment 3: the regeneration of Tilletia tritici protoplast

[0080] The protoplasts prepared in Examples 1 and 2 were resuspended with 1.2 mol / L potassium chloride, respectively, and spread on PDA and TB3 solid medium, cultured at 16° C. for 4 days, and observed single colonies grown.

[0081] The result is as Figure 6 Shown, the protoplast of Tilletia tritici grows more single bacterium colony on TB3 medium, and no single bacterium colony grows on PDA medium, therefore, TB3 medium is more suitable for protoplast regeneration than PDA medium, It can be used for regeneration culture of protoplast of Tilletia tritici.

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Abstract

The invention relates to a preparation method of tilletia foetida protoplasts. The preparation method comprises the following steps: (1) mycelium culture: culturing teleutospores of tilletia foetida in a water agar culture medium for 6-9 days, flushing the teleutospores off from the water agar culture medium by virtue of sterile distilled water, collecting bacteria liquid, and carrying out centrifugation to remove supernatant, so as to obtain mycelia; and (2) cell wall cracking: preparing mixed enzyme liquid by taking a potassium chloride solution as an osmotic pressure stabilizer, adding the mixed enzyme liquid into the mycelia, and carrying out vibration enzymolysis at 28 DEG C for 1.5-2.5 hours, so as to obtain the tilletia foetida protoplasts, wherein the mixed enzyme liquid contains the following components in percentage by mass: 1.5% of driselase, 1.5% of lyticase and 1.5% of helicase. The method is used for preparing the tilletia foetida protoplasts, the yield of the tilletia foetida protoplasts reaches up to 17.0*10<5>unit / mL-18.0*10<5>unit / mL, and the released protoplasts are uniformly distributed in a scattered manner and are not aggregated into a pile.

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 protoplasts of Tilletia tritici. Background technique [0002] Common bunt of wheat (common bunt of wheat) caused by Tilletia foetida Walle.Lindr. is a worldwide disease, which has the characteristics of wide distribution, strong prevalence, and serious damage. It seriously affects Production of wheat. Susceptible plants usually have no obvious symptoms in appearance. In the later stage of the disease, the glumes open slightly, and the whole ear becomes a diseased ear. There is a layer of gray film outside the diseased grain, and the inside is full of black powder, which is the teliospore of the pathogenic fungus. , Teliospores have a fishy smell, causing a reduction in wheat yield and quality. Tilletia wheat belongs to Basidiomycotina, Teliomycetes, Ustilaginales, Tilletiaceae, Tilletia. Tel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12R1/645
CPCC12N1/14
Inventor 高利沈慧敏陈万权刘太国刘博
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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