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A method for evaluating the effect of transgenic plants on their endophytic microorganisms

A microorganism and plant technology, which is applied in the application field of evaluating the influence of transgenic plants on endophytic microorganisms, can solve the problems of influence, immersion in plant tissues, and death of endophytic bacteria, and achieve the effect of obvious enrichment effect.

Active Publication Date: 2021-05-11
INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the experiments of inserting endophytes reported in the literature, the concentration of endophytes used has affected the detection of bacteria content, and the isolation methods of plant endophytic cultivable bacteria in the prior art usually use cleaning-shearing segmentation-segmentation Segmental disinfection - tissue smashing - tissue fluid coating culture, during a long disinfection process, the disinfectant can easily penetrate into the plant tissue through the cut surface, resulting in the death of some endophytic bacteria; or excessively cut the edge of the plant segment by secondary shearing The disinfection part is removed, and the juicer is used to crush the plant tissue under pre-cooling conditions. Although the reduction of endophytic bacteria sources can be avoided to a certain extent, it is suitable for extracting a large amount of plant tissue, and it is not suitable for a small amount of plant samples. Determination of plant endophytic bacteria

Method used

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  • A method for evaluating the effect of transgenic plants on their endophytic microorganisms
  • A method for evaluating the effect of transgenic plants on their endophytic microorganisms
  • A method for evaluating the effect of transgenic plants on their endophytic microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Soaking Cotton Seeds with Different Concentrations of Indicating Endophytic Bacteria

[0044] 1. Soil sterilization

[0045] Weigh 1000g of natural air-dried loam, put it in a PE bag that can be sterilized by high temperature and high pressure, and sterilize it three times in an autoclave (LDZX-40BI, Shanghai Shen’an) at a temperature of 121°C and a pressure of 0.105MPa. , sterilize for 3 hours each time, and sterilize once every 24 hours.

[0046] 2. Culture of endogenous indicator bacteria

[0047] Activate the endogenous indicator bacteria B916-gfp on the LB plate medium containing 5 μg / mL chloramphenicol, and place it into 200mL / 500mL LB liquid medium containing 5 μg / mL chloramphenicol after a single colony grows , 30°C, 200rpm shaker culture until sporulation, 10000rpm centrifugal concentration.

[0048] 3. Seed disinfection

[0049] Cotton seeds were first acidified with 0.1 wt% HgCl 2 Aqueous solution (25mL 36wt% hydrochloric acid in 1L aqueous solution) was s...

Embodiment 2

[0065] Different concentrations indicate that endophytic bacteria irrigate cotton plants

[0066] Soil sterilization, endophytic bacteria culture and seed disinfection treatment are equal to embodiment 1.

[0067] The difference is:

[0068] After soaking the seeds in step 3 with sterile distilled water for 24 hours, raise seedlings with sterilized vermiculite on the seedling tray. After the cotton seeds germinate and grow to the cotyledon fully unfolded, move it to a plastic pot (selected plastic box Same as step 1)), put 1200g of sterilized dry soil in the plastic box, and water the soil surface with a concentration of 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 Spores / mL B916-gfp bacteria solution 150mL, so that the soil is fully soaked, and then use sterile water for watering management. The seedlings were collected on the 1st, 7th, 14th, 21st, 28th and 35th day after root irrigation and inoculation to measure the amount of live bacteria of B916-g...

Embodiment 3

[0075] Comparison of Different Disinfection Methods for Cotton Tissue

[0076] The cotton plants treated on the 7th day in Example 1 were selected, and three different disinfection methods were used to compare the differences in the bacteria content measured in different tissues.

[0077] Method 3.1: Same as Example 1, that is, the whole plant is sterilized.

[0078] Method 3.2: Washing the whole plant with running water - cutting into segments - segmental disinfection - tissue smashing - tissue fluid coating and cultivation, the disinfectant and other operations used are the same as in Example 1.

[0079] Method 3.3: Washing the whole plant with running water - cutting into segments - segmental disinfection - cutting off the edges of the segment - mashing the tissue - coating and culturing the tissue fluid, the disinfectant and other operations used are the same as in Example 1.

[0080] The results are shown in Table 3.

[0081] Table 3 Comparison of bacteria content in di...

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Abstract

The present invention provides a method for evaluating the effect of transgenic plants on their endophytic microorganisms. Wherein, the method for isolating plant endophytic microorganisms comprises the following steps: 1) collecting the plant of the target size, immersing the whole plant of the plant in the first disinfectant solution for overall disinfection to obtain a sterilized plant; 2) obtaining the sterilized plant from the Obtain the tissue at the site of the endophyte to be separated from the sterilized plant; 3) add a buffer solution containing alkylphenol polyoxyethylene ether sulfosuccinate to the container containing the tissue, and grind to obtain a tissue grinding solution ; 4) culturing the microorganisms in the tissue grinding fluid for subsequent analysis. The method of the present invention can be used to evaluate the influence of transgenic plants on their endophytic microorganisms, especially for evaluating the influence of transgenic plants such as transgenic cotton on their endophytic microorganisms at the seedling stage.

Description

technical field [0001] The invention provides a method for isolating plant endophytic microorganisms, especially its application in evaluating the impact of transgenic plants on endophytic microorganisms. Background technique [0002] Between 1996 and 2016, biotech crops were planted on 18.15 billion hectares globally (ISAAA, 2017). Genetically modified crops have largely succeeded in mitigating many major agricultural challenges, bringing numerous benefits to growers around the world. Although GM crops have brought enormous economic benefits to agricultural production, GM crops are still controversial in terms of biosafety and ecological compatibility, including their impact on plant endophytic microorganisms. [0003] Some microbial communities in the soil may enter the roots, stems and leaves of plants through the rhizosphere and become endophytic microorganisms. Plant endophytic bacteria can colonize and live in healthy plant tissues and organs, but do not cause substa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/02C12Q1/06C12Q1/04
CPCC12N1/02C12Q1/04C12Q1/06
Inventor 曹伟平杜立新宋健郭庆港鹿秀云
Owner INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI