Iris ensata thunb mesophyll protoplast and preparation method thereof

A technology of protoplast and horsetail, which is applied in the field of cell biology and biology, to achieve high-efficiency dissociation and overcome technical bottlenecks

Inactive Publication Date: 2019-02-22
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the isolation of protoplasts from S.

Method used

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  • Iris ensata thunb mesophyll protoplast and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Select young leaves of Sperus japonica with a healthy seedling age of 30, 45, and 60 days as treatment materials, wash them with tap water, put them into an ultra-clean workbench, sterilize with 70vt% absolute ethanol for 1min, sterilize with 5wt% sodium hypochlorite for 10-15min, and aseptically Rinse with water and set aside;

[0036] (2) Put the material obtained in step (1) on the sterile and dry filter paper in the ultra-clean workbench, and use sterile scissors or a scalpel to cut the leaves into 1-2 mm wide thin slices along the direction of the leaf veins. leaf shreds;

[0037] (3) In the ultra-clean workbench, put the cut fine leaf shreds into the beaker containing the sterile plasmolysis liquid at a ratio of 1g:30ml, and then wrap the beaker with sterile aluminum foil to avoid light for plasmolysis. Separation treatment 2h;

[0038] (4) Take the above-mentioned thin leaf silks that have undergone plasmolysis treatment, put them into pre-prepared beakers ...

Embodiment 2

[0043] (1) Select young leaves of Sperus japonica with a healthy seedling age of 30, 45, and 60 days as treatment materials, wash them with tap water, put them into an ultra-clean workbench, sterilize with 70vt% absolute ethanol for 1min, sterilize with 5wt% sodium hypochlorite for 10-15min, and aseptically Rinse with water and set aside;

[0044] (2) Put the material obtained in step (1) on the sterile and dry filter paper in the ultra-clean workbench, and use sterile scissors or a scalpel to cut the leaves into 1-2 mm wide thin slices along the direction of the leaf veins. leaf shreds;

[0045] (3) In the ultra-clean workbench, put the cut fine leaf shreds into the beaker containing the sterile plasmolysis liquid at a ratio of 1g:30ml, and then wrap the beaker with sterile aluminum foil to avoid light for plasmolysis. Separation treatment 2h;

[0046] (4) Take the above-mentioned fine leaf silks that have undergone plasmolysis treatment, put them into pre-prepared beakers ...

Embodiment 3

[0051] (1) Select young leaves of Sperus japonica with a healthy seedling age of 30, 45, and 60 days as treatment materials, wash them with tap water, put them into an ultra-clean workbench, sterilize with 70vt% absolute ethanol for 1min, sterilize with 5wt% sodium hypochlorite for 10-15min, and aseptically Rinse with water and set aside;

[0052] (2) Put the material obtained in step (1) on the sterile and dry filter paper in the ultra-clean workbench, and use sterile scissors or a scalpel to cut the leaves into 1-2 mm wide thin slices along the direction of the leaf veins. leaf shreds;

[0053] (3) In the ultra-clean workbench, put the cut fine leaf shreds into the beaker containing the sterile plasmolysis liquid at a ratio of 1g:30ml, and then wrap the beaker with sterile aluminum foil to avoid light for plasmolysis. Separation treatment for 3h;

[0054] (4) Take the above-mentioned thin leaf silks that have undergone plasmolysis treatment, put them into pre-prepared beak...

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Abstract

The invention discloses an iris ensata thunb mesophyll protoplast and a preparation method thereof. Through pretreatment and plasmolysis of an iris ensata thunb material, and through combined enzymolysis of an iris ensata thunb mesophyll tissue by using cellulase, macerozyme and pectinase, preparation of the iris ensata thunb protoplast is effectively achieved, a technical bottleneck that the irisensata thunb protoplast is difficultly dissociated is overcome, and the efficient preparation method of the iris ensata thunb mesophyll protoplast is effectively established. An effective operating platform is provided for achieving rapid genetic transformation of the iris ensata thumb and transient gene expression research, and a technical support is provided for iris ensata thunb protoplast culture, protoplast fusion, protogenetic genetic transformation, transient gene expression research and the like for establishment of an iris ensata thunb cell engineering technological system.

Description

technical field [0001] The invention belongs to the field of cell biology and biotechnology, and in particular relates to the protoplast of the eel mesophyll and its preparation method. Background technique [0002] Because of the lack of cell walls, plant protoplasts have unique advantages compared to tissue, organ or individual levels: First, protoplasts can get rid of the constraints of the cytoskeleton and the interaction between cells, and can study the function and pluripotency of a cell alone; secondly , Protoplast is a "cell" population with good uniformity, and a large number of consistent research objects can be obtained in a short time. At the same time, protoplasts can be used for different levels of cytology and molecular biology research. Protoplasts are an ideal single-cell system to study cell wall regeneration, cell division and differentiation, uptake into organelles, virus infection mechanism, membrane permeability and ions. Basic theoretical research suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 唐君常有宏李建斌于利王神云余方伟
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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