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Separation method for plant endophyte

A technology for plant endophytes and isolation methods, which is applied in the field of separation of plant endophytes, can solve the problems of insufficient representation of quantity and species, uneven distribution of endophytes, limited number and types of endophytes, etc., to achieve The effect of ensuring accuracy and improving work efficiency

Inactive Publication Date: 2013-05-29
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods are simple and easy to implement and reduce pollution, they also make the number and types of endophytes isolated and obtained very limited.
This is due to the inhomogeneous distribution of endophytes in plants, resulting in the isolation results not being able to fully represent the number and types of endophytes in the whole plant.

Method used

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  • Separation method for plant endophyte
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Examples

Experimental program
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Effect test

Embodiment 1

[0031] —— Surface disinfection experiment of various plant tissues

[0032] Experimental materials and methods: The roots, stems and leaves of K326 tobacco were used as experimental materials, and healthy plants with good growth characteristics and no disease symptoms were selected for sampling. The root, stem, and leaf samples of K326 tobacco were separated from the plants, rinsed, numbered 1, 2, 3, 4, and 5, respectively, and packed in sealed bags, and kept fresh at 4°C for later use.

[0033] Randomly weigh 0.4 g of the cleaned root, stem and leaf samples of each tobacco, first shake and soak in 70% ethanol solution for 60 seconds, then shake and soak in sodium hypochlorite solution with 1% available chlorine content for 40~320 seconds, and then use non- Rinse with bacterial water 4 times. Coat the NA plate with the last rinse water, record the number of colonies after incubating at 28°C for 48 hours, calculate the surface sterilization rate to test the sterilization effe...

Embodiment 2

[0041] —— Isolation of Tobacco Endophytes

[0042] Experimental materials and methods: The roots, stems and leaves of K326 tobacco were used as experimental materials, and healthy plants with good growth characteristics and no disease symptoms were selected for sampling. The root, stem, and leaf samples of K326 tobacco were separated from the plant, rinsed, packed in sealed bags, and kept fresh at 4°C for later use.

[0043]Randomly weigh 0.4g of the cleaned tobacco roots, stems, and leaves, first shake and soak in 70% ethanol solution for 60s, then shake and soak the roots, stems, and leaves in sodium hypochlorite solution with 1% available chlorine 60s, 180s, 300s, then rinse with sterile water 4 times. Coat the NA plate with the last rinse water and incubate it at 28°C for 48 hours as a control for surface disinfection. If no colonies are formed on the control plate, it proves that the surface is fully disinfected, and all the isolates obtained in the subsequent steps are...

Embodiment 3

[0046] —— Isolation of Tobacco Endophytes

[0047] It is basically the same as Example 2, except that: the root, stem, and leaf of Yunyan 85 tobacco are used as experimental materials; the root, stem, and leaf samples of tobacco are soaked in a sodium hypochlorite solution with an available chlorine content of 1% for 40s, 170s, and 40s respectively. 310s; the grinding time is 240s, the frequency is 20r / s; the sizes of the sterilized steel balls are 3mm and 1mm respectively.

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Abstract

The invention discloses a separation method for plant endophyte. The separation method comprises the steps of: sampling, surface sterilization, tissue grinding, separation and pure cultivation, and particularly comprises sampling health plants, storing in classification, soaking in an ethanol solution, soaking again in an sodium hypochlorite solution, washing by sterile water, weighing 0.3-0.3 g, adding the sterile water and grinding. The separation method further comprises the steps of: diluting the ground slurry in gradients, plate cultivating after coating, repeating each processing for 3-5 times, selecting single colonies according to eight phenotypic characteristics of bacterial colonies, consisting of shape, size, humidity, color, texture, surface, edge and transparency, for pure cultivation, and moving the pure cultivated single colonies into the test tube slants for preservation. Through the application of the separation method for plant endophyte, the diversity separation result of the plant endophyte is effectively prevented from being manually enlarged or lost; and the separation method for plant endophyte plays a very important role in researching the composition characteristics of plant endophyte colonies and enriching the gene resources of the endophyte.

Description

technical field [0001] The invention belongs to the technical field of biology, further belongs to the technical field of microorganisms, and in particular relates to a method for isolating endophytic bacteria in plants. Background technique [0002] Plant endophytes refer to fungi or bacteria that live in various tissues and organs of healthy plants. During their life, the host plant does not show external symptoms. Plants and their endophytes are in a mutualistic symbiotic relationship. Plants provide photosynthetic products and minerals for endophytes, and the metabolites of endophytes can stimulate plant growth and development, and improve the resistance of host plants to biotic and abiotic stresses. Due to the unique advantages of plant endophytes as biocontrol factors: they are distributed in different tissues of plants, protected by plant tissues, have a stable ecological environment, and are easier to play a role in biocontrol compared with epiphytic bacteria, so , ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/14C12N1/02
Inventor 夏振远陈泽斌吴玉萍雷丽萍汪安云晋艳
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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