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Preparation method of catalpa fargesii leaf blade protoplasm

A technology of protoplasts and leaves, which is applied in the field of preparation of protoplasts from leaves of Ash catalpa to achieve the effect of a simple and efficient preparation method

Active Publication Date: 2017-05-31
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, most of the researches on Ash Catalpa are focused on the evaluation of genetic diversity, the breeding of fine new varieties and the research on high-efficiency breeding technology, etc., and the research on the separation and preparation of the protoplasts of Ash Catalpa has not been reported yet.

Method used

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  • Preparation method of catalpa fargesii leaf blade protoplasm
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  • Preparation method of catalpa fargesii leaf blade protoplasm

Examples

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Effect test

Embodiment 1

[0038] A preparation method of ash catalpa leaf protoplasts, comprising the following steps:

[0039] (1) Sample screening: Take the 3-4th young leaves from the top to bottom of the tissue-cultured seedlings of Catalpa chinensis with a height of 6-8cm.

[0040] (2) Cut the selected young leaves into strips of 1mm×0.1mm with a blade, and place the leaf strips in the enzymatic hydrolysis according to the ratio of enzymatic hydrolysis solution volume (mL): leaf mass (g) = 10:1 solution, the temperature is 28°C, the rotation speed is 50rpm, and the enzyme is hydrolyzed for 10h under dark conditions. The composition of the enzymolysis solution is: 0.01M CaCl 2 , 0.02M KCl, 0.02M MES, 0.1% BSA, 2% cellulase Onozuka RS, 0.5% resolase Macerozyme R-10, 0.4M mannitol.

[0041] (3) The enzymatic hydrolyzate was filtered through a 300-mesh filter, centrifuged at 600 rpm for 5 minutes, the supernatant was sucked off, the precipitate was resuspended in buffer, and the above centrifugation...

Embodiment 2

[0045] A preparation method of ash catalpa leaf protoplasts, comprising the following steps:

[0046] (1) Sample screening: Take the 3-4th young leaves from the top to bottom of the tissue-cultured seedlings of Catalpa chinensis with a height of 6-8cm.

[0047] (2) Cut the selected young leaves into strips of 1mm×0.1mm with a blade, and place the leaf strips in the enzymatic hydrolysis according to the ratio of enzymatic hydrolysis solution volume (mL): leaf mass (g) = 10:1 solution, the temperature is 25°C, the rotation speed is 50rpm, and the enzyme is hydrolyzed for 12h under dark conditions. The composition of the enzymolysis solution is: 0.01M CaCl 2, 0.02M KCl, 0.02M MES, 0.1% BSA, 2% cellulase Onozuka RS, 2% resolase Macerozyme R-10, 0.3M mannitol.

[0048] (3) The enzymatic hydrolyzate was filtered through a 300-mesh filter, centrifuged at 600 rpm for 5 minutes, the supernatant was sucked off, the precipitate was resuspended in buffer, and the above centrifugation wa...

Embodiment 3

[0052] A preparation method of ash catalpa leaf protoplasts, comprising the following steps:

[0053] (1) Sample screening: Take the 5-6th young leaves from the top to bottom of the tissue-cultured seedlings of Catalpa chinensis with a height of 6-8cm.

[0054] (2) Cut the selected young leaves into strips of 1mm×0.1mm with a blade, and place the leaf strips in the enzymatic hydrolysis according to the ratio of enzymatic hydrolysis solution volume (mL): leaf mass (g) = 10:1 solution, the temperature is 30°C, the rotation speed is 50rpm, and the enzyme is hydrolyzed for 8h under dark conditions. The composition of the enzymolysis solution is: 0.01M CaCl 2 , 0.02M KCl, 0.02M MES, 0.1% BSA, 3% cellulase Onozuka RS, 1% resolase Macerozyme R-10, 0.6M mannitol.

[0055] (3) The enzymatic hydrolyzate was filtered through a 300-mesh filter, centrifuged at 600 rpm for 5 minutes, the supernatant was sucked off, the precipitate was resuspended in buffer, and the above centrifugation wa...

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Abstract

The invention relates to a preparation method of catalpa fargesii leaf blade protoplasts. The catalpa fargesii leaf blade protoplasts are obtained by controlling the ratios and the concentrations of cellulase Onozuka RS and Macerozyme R-10, the concentration of mannitol in enzymatic hydrolysate, enzymolysis temperature, enzymolysis time and other factors. Free protoplasts obtained by the method disclosed by the invention are high in quantity and vitality, and can lay a foundation for researching catalpa fargesii protoplast culture, plant regeneration, genetic transformation, somatic hybridization, protoplast fusion breeding and the like.

Description

technical field [0001] The invention relates to a method for preparing protoplasts of ash catalpa leaves, belonging to the field of biotechnology. Background technique [0002] Ash catalpa (Catalpa fargesii) is a tall deciduous tree belonging to Bignoniaceae (Bignoniaceae) and is a high-quality and precious timber and garden ornamental tree species traditionally cultivated in my country. Ash catalpa has a well-developed root system, fast growth, strong wind resistance, strong soil fixation ability, cold and drought resistance, straight texture, tough and dense texture, and is a high-quality fast-growing timber tree species. [0003] Protoplast is the product of plant cells after the cell wall is removed. It can be applied to the fusion between distant parents, easy to introduce foreign genes, and is also a good tool for studying the function of proteins on the cell membrane (such as ion channels, aquaporins, signal receptors, etc.). Materials, and can provide technical supp...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04C12N2509/00
Inventor 麻文俊王军辉易飞
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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