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Hydrangea macrophylla protoplast preparation and transient transformation method

A protoplast and instantaneous transformation technology, applied in the field of cell biology, can solve the problems of not being limited by time, limited operation, time limit of protoplast, etc., and achieve the effect of simple formula, increased stability and maintaining vitality

Pending Publication Date: 2021-03-05
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there have been researches on the method of using hydrangea sepals to isolate protoplasts, but the sepals used can only be sampled during the flowering period, and the isolation of protoplasts is limited by time and operation. Conversion, the operation will not be limited by time

Method used

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  • Hydrangea macrophylla protoplast preparation and transient transformation method
  • Hydrangea macrophylla protoplast preparation and transient transformation method
  • Hydrangea macrophylla protoplast preparation and transient transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the preparation of hydrangea leaf protoplast

[0047] In this embodiment, the enzymatic solution combination (including MES, mannitol, cellulase R-10, isolated enzyme R-10 and KH) is prepared for hydrangea leaf protoplasts 2 PO 4 ) for experiments, using five factors and four levels (L 16 (4 5 )) The method of orthogonal experimental design, concrete implementation is as follows:

[0048]S1. Select the leaves of hydrangea tissue culture seedlings, cut off the edge of the leaves with a blade, and cut the leaves into thin strips perpendicular to the main veins (about 1mm wide);

[0049] S2. Quickly put the leaves in step S1 into a petri dish filled with enzymatic hydrolysis solution, and culture in dark at 25-28° C. for 12-15 hours.

[0050] Wherein, the preparation method of the enzymatic hydrolyzate is as follows:

[0051] Add 5mM MES (pH 5.7), 0.8-1M mannitol, 1-1.5% (W / V) cellulase R-10 and 0.5-0.8% (W / V) isolated enzyme R-10 into a 50mL centrifuge ...

Embodiment 2

[0059] Embodiment 2: the preparation of hydrangea leaf protoplast

[0060] In this embodiment, the enzymatic hydrolysis time for hydrangea leaf protoplast preparation is tested, and 0, 6, 9, 12, 15, 16, 17, 18 hours are set, and the specific implementation is as follows:

[0061] S1. Select the 3-6th leaf under the terminal bud of the hydrangea plant, cut off the edge of the leaf with a blade, and cut the leaf into thin strips (about 1mm wide) perpendicular to the main vein;

[0062] S2. The leaves in step S1 are quickly put into enzymatic hydrolysis solution (5mM MES (pH 5.7), 0.8M mannitol, 1.5% (W / V) cellulase R-10, 0.8% (W / V) Isolase R-10, 50mM KH 2 PO 4 and 0.1% bovine serum albumin) in a culture dish at 25-28° C. for 12-15 hours in the dark.

[0063] S3. Slowly add an equal volume of W5 solution to the digested enzymolysis solution along the wall of the petri dish, mix gently to stop the digestion, and then filter the digested protoplasts into a centrifuge tube with a...

Embodiment 3

[0068] Embodiment 3: Hydrangea leaf protoplast preparation

[0069] In this example for KH 2 PO 4 The influence of the concentration on the protoplast viability of hydrangea leaves was tested, and the settings were 0, 20, 30, 40, 50, 60mM, and the specific implementation was as follows:

[0070] S1. Select the tender leaves at the top of the hydrangea tissue culture seedlings, cut off the leaf margins with a blade, and cut the leaves into thin strips perpendicular to the main veins (about 1mm wide);

[0071] S2. The leaves in step S1 are quickly put into enzymatic hydrolysis solution (5mM MES (pH 5.7), 0.8M mannitol, 1.5% (W / V) cellulase R-10, 0.8% (W / V) Isolated enzyme R-10, 0~60mM KH 2 PO 4 and 0.1% bovine serum albumin) in a culture dish at 25-28° C. for 12-15 hours in the dark.

[0072] S3. Slowly add an equal volume of W5 solution to the digested enzymolysis solution along the wall of the petri dish, mix gently to stop the digestion, and then filter the digested prot...

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Abstract

The invention relates to a hydrangea macrophylla protoplast preparation and transient transformation method, and belongs to the technical field of cell biology. The method comprises the following steps: selecting hydrangea macrophylla leaves, cutting off leaf margin, cutting the leaves into strips perpendicular to main leaf veins, and putting the strips into an enzymolysis liquid for enzymolysis;after enzymolysis, adding a W5 solution to terminate digestion, and then performing filtering to obtain hydrangea macrophylla protoplast; and performing transient transformation on the protoplast. Themethod uses the leaves to prepare the protoplast, is not limited by seasons, and is simple to operate and easy to implement; the used enzymolysis liquid is simple in formula, and the obtained protoplast is high in yield and strong in activity; KH2PO4 is added into the enzymolysis liquid, so that the stability of the plasma membrane of the protoplast can be remarkably improved, and the activity ofthe protoplast is effectively maintained; the corresponding transient transformation method is established for the hydrangea macrophylla leaf protoplast, the operation is simple and convenient, and the transformation efficiency is high; and the important technical support is provided for the basic and application research of hydrangea macrophylla, so that the development of the hydrangea macrophylla industry is promoted.

Description

technical field [0001] The invention relates to a hydrangea protoplast preparation and instantaneous transformation method, which belongs to the technical field of cell biology. Background technique [0002] Plant protoplasts are naked cells from which the cell wall has been removed. They are pluripotent and have the potential to redifferentiate into complete organisms. It has been widely used in the field of plant cell biology research, such as transient expression of genes, improvement of germplasm resources, subcellular localization of target genes, protein interaction, etc. Protoplasts have broad application prospects in plant transgenic and gene function verification. [0003] Hydrangea [Hydrangea macrophylla (Thunb.) Ser.], also known as hydrangea, hydrangea, hydrangea, etc., is an ornamental plant of the genus Hydrangea in the saxifrage family. It has the advantages of strong adaptability, strong stress resistance, and less pests and diseases. Moreover, the plants o...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8201
Inventor 陈双双邓衍明齐香玉冯景陈慧杰秦紫艺王华娣
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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