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Preparation method of litsea cubeba mesophyll cell protoplast

A technology of protoplasts and litsea cubeba leaves, which is applied in the field of plant cell protoplast preparation, can solve the problems of litsea cubeba protoplast separation failure and poisoning of protoplasts, shorten the time required for enzymatic hydrolysis, promote development, and maintain normal morphology Effect

Active Publication Date: 2019-10-22
HUNAN NUOZ BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far, there is no relevant content that can efficiently obtain the protoplasts of Litsea cubeba mesophyll cells; in addition, compared with plants that successfully obtained protoplasts, Litsea cubeba leaf tissue contains a large number of secondary metabolites, which can be obtained in the future. When the cell wall is enzymatically hydrolyzed, the secondary metabolites released by the damaged leaf tissue are oxidized due to long-term exposure to the air, and converted into a large amount of browning substances that are poisonous to the protoplasts, which easily leads to the failure of the separation of litsea cubeba protoplasts; therefore, how to use An efficient and economical method to prepare litsea cubeba protoplasts is a difficult research and development problem

Method used

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  • Preparation method of litsea cubeba mesophyll cell protoplast
  • Preparation method of litsea cubeba mesophyll cell protoplast
  • Preparation method of litsea cubeba mesophyll cell protoplast

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Experimental program
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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 litsea cubeba mesophyll cell protoplast

[0028] The preparation method of litsea cubeba mesophyll cell protoplast comprises the following steps:

[0029] Step 1. Material selection: In mid-May 2019, mature leaves of litsea cubeba with good growth and no pests and diseases were collected in Shending Mountain, Miluo City, Hunan Province, and the collected leaves were sterilized with 0.1% mercury chloride solution for 3 minutes to obtain no Litsea cubeba leaves, set aside;

[0030] Step 2. Leaf enzymatic hydrolysis

[0031] Use a sterile scalpel to cut the sterilized litsea cubeba leaves into strips with a width of 1mm, take 0.1g of the pieces and place them in 3ml of enzymatic hydrolysis solution, and let them stand in the dark Enzymolysis for 6 hours; the enzymolysis solution contains the following final concentrations of each component: 10g / l cellulase R-10, 10g / l isolated enzyme R-10, 91g / l mannitol, 0.1g / l sodium dihydrogen phosphate...

Embodiment 2

[0044] The preparation of embodiment 2 litsea cubeba mesophyll cell protoplast

[0045] The preparation method of litsea cubeba mesophyll cell protoplast comprises the following steps:

[0046] Step 1, material selection: tissue culture test-tube plantlets have the advantage of being sterile and can be obtained throughout the year. Therefore, the present embodiment uses the young leaves of litsea cubeba aseptic test-tube plantlets with a seedling height of about 3cm as material to obtain aseptic litsea cubeba. Cotyledons, to be used;

[0047] Step 2. Leaf enzymatic hydrolysis

[0048] Use a sterile scalpel to cut the sterilized leaves of Litsea Cubeba into strips with a width of 2 mm, take 0.3 g of the leaves and place them in 5 ml of enzymatic hydrolysis solution, and let them stand in the dark Enzymolysis for 12 hours; the enzymolysis solution contains the following final concentrations of each component: 20g / l cellulase R-10, 20g / l isolated enzyme R-10, 128g / l mannitol, 0...

Embodiment 3

[0056] Influence of different enzymatic hydrolysis solutions on the separation effect of protoplasts when preparing litsea cubeba mesophyll cell protoplasts in embodiment 3

[0057] 1. Effects of different concentrations of vitamin C, Tween 80 and PEG 4000 on the separation of protoplasts from litsea cubeba mesophyll cells

[0058] In step 2 of Example 1, vitamin C, Tween 80 and PEG 4000 were adjusted in different concentrations to obtain protoplast cells. The morphology of protoplast cells was observed and counted under a microscope. The results are shown in Table 1.

[0059] Table 1 Effects of different concentrations of vitamin C, Tween 80 and PEG 4000 on protoplast production of litsea cubeba mesophyll cells

[0060]

[0061] As can be seen from the above table 1, the number of protoplasts prepared from test group 1 to test group 3 was significantly lower than that of test group 4 to test group 8, which indicated that a certain proportion of vitamin C and PEG 4000 were ...

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Abstract

The invention discloses a preparation method of a litsea cubeba mesophyll cell protoplast. The method comprises the following steps that sterile litsea cubeba leaves are obtained; the sterile litsea cubeba leaves are cut into strips and placed in enzymatic hydrolysate, and under the dark condition, standing and enzymolysis are conducted for 6-12 hours, wherein the mass-volume ratio of the strips to the enzymatic hydrolysate is (1-3):(30-50); the enzymatic hydrolysate obtained after enzymolysis is filtered, filtrate is taken and subjected to centrifugal treatment, supernatant is discarded, andthe protoplast is obtained; a protoplast washing solution A is added into the obtained protoplast, and the protoplast is resuspended; then a protoplast washing solution B is added, after centrifugation, protoplast cells at the liquid level boundary are collected, and the litsea cubeba mesophyll cell protoplast is obtained. According to the preparation method, the litsea cubeba mesophyll cell protoplast can be efficiently obtained, the obtained protoplast is normal in form and free of damage, (8.69+ / -0.57)*10<6> protoplasts can be obtained from each gram of fresh leaf tissue, and important technical support is provided for basic research and application research of the litsea cubeba.

Description

technical field [0001] The invention relates to the technical field of plant cell protoplast preparation, more specifically, to a method for preparing litsea cubeba mesophyll cell protoplast. Background technique [0002] Litsea cubeba (Litsea cubeba) is a unique industrial raw material and spice tree species in southern my country. The essential oil extracted from its fruit has a high content of citral and is widely used in medicine, food, cosmetics and other fields. However, the current basic research related to Litsea Cubeba is relatively weak, which seriously restricts the development of Litsea Cubeba industry. Plant cell protoplasts play an important role in basic research and genetic breeding, mainly in the following aspects: 1. Compared with intact cells, protoplasts remove the cell wall, which is more conducive to cell membranes, organelles and Observation of protein subcellular localization; 2. Protoplast culture is an important way for plant asexual propagation; 3...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 陈昊王阳张付豪
Owner HUNAN NUOZ BIOLOGICAL TECH CO LTD
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