The preparation method of the protoplast of Gracilaria gracilaria in Chile

A technology for protoplasts and gracilis, which is applied in the field of preparation of gracilaria protoplasts in Chile, and can solve problems such as poor enzymatic hydrolysis effect and the like

Active Publication Date: 2020-04-07
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the present invention finds that the enzyme solution derived from the digestive tissue of abalone has a poor enzymatic effect on Gracilaria Chilean tissue

Method used

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  • The preparation method of the protoplast of Gracilaria gracilaria in Chile
  • The preparation method of the protoplast of Gracilaria gracilaria in Chile
  • The preparation method of the protoplast of Gracilaria gracilaria in Chile

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Preparation of tissue enzyme solution: homogenate it with seawater 0°C with 2 times the volume of sea cucumber intestine; extract at 4°C for 12 hours, then adjust the pH value of the extract to 7.6, refrigerate and centrifuge for 5 minutes, and take the supernatant. Sea cucumber enzyme solution; tissue enzyme solution is obtained by mixing sea cucumber enzyme solution, cellulase and seawater. The specific composition of the tissue enzyme solution is 2.4ml cellulase, 0.5ml sea cucumber enzyme solution and 17.1ml sterilized seawater.

[0017] Preparation of protoplasts: Take 2g of Chilean river fence material, blot dry with absorbent paper, chop the algae with a scalpel (the more broken the better), add the prepared tissue enzyme solution, and put it on a constant temperature shaker at 25°C Dark enzymolysis. Microscopically check once every 30 minutes, observe and take pictures. After 5 hours, filter with a 200-mesh sieve to remove undigested cells, cell clusters and de...

Embodiment 2

[0019] Detection of protoplasts:

[0020] Determination of Cell Density: Determination of Cell Density Using a Hemocytometer

[0021] The specific results are as follows:

[0022]

[0023]

Embodiment 3

[0033] The protoplasts prepared in Example 1 were collected and cultured under low light in hypertonic seawater with an illumination intensity of 100-200lx. After the protoplast regenerated the wall, the osmotic pressure of seawater was gradually reduced to normal seawater, and the illumination intensity was gradually increased to 3000lx for cultivation.

[0034] Such as figure 2 As shown, after 42 hours of culture, the protoplasts have not yet formed a complete cell wall, and after 4 days of culture, most of the protoplasts have regenerated their cell walls. After 7 days of culture, the cells have been divided into multiple cells, and the cells are arranged more regularly. After 12 days of culture, the number of cells has been increasing and they are arranged in different shapes.

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Abstract

The invention relates to a method for preparing gracilaria chilensis protoplast and belongs to the field of algae cell engineering. The preparation method comprises the following steps: preparing a tissue enzyme solution, and performing enzymolysis on gracilaria chilensis tissues, wherein the step of preparing the tissue enzyme solution comprises the following steps: homogenizing with seawater in an amount of 1-10 times that of the volume of sea cucumber intestines at a temperature of 0 DEG C; extracting at a temperature of 4 DEG C for 4-18 hours, regulating the pH value of the extracting solution to be 7.0-8.0, freezing and centrifuging for 5 minutes, and taking the supernatant so as to obtain a sea cucumber enzyme solution; and mixing the sea cucumber enzyme solution, cellulase and seawater so as to obtain the tissue enzyme solution.

Description

technical field [0001] The invention relates to a method for preparing protoplasts of Gracilaria gracilarius, belonging to the field of algae cell engineering. Background technique [0002] Plant protoplast refers to a viable cell whose cell wall is removed and surrounded by plasma membrane. Its structure includes cell membrane, cytoplasm (including various organelles, cytoskeleton system and cytomatrix) and nucleus and other parts. Protoplast technology refers to a series of technical operations based on the separation and cultivation of protoplasts, mainly including protoplast plant regeneration, protoplast fusion and cell hybridization, transgenic and other biotechnologies for cultivating new types of mutations. Protoplast culture and plant regeneration technology has the advantages of wide application range and strong feasibility, and is the basis of plant bioengineering. [0003] The preparation of plant protoplasts includes the selection and pretreatment of materials,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
CPCC12N5/04C12N2509/00
Inventor 刘涛金月梅
Owner OCEAN UNIV OF CHINA
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