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Preparation method of chimonanthus praecox protoplast

A technology of protoplasts and wintersweet, applied in the field of preparation of wintersweet protoplasts, can solve problems such as long growth cycle and complex plant structure, and achieve the effect of genetic trait improvement

Active Publication Date: 2021-09-17
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a woody plant, wintersweet has a long growth cycle and complex plant structure. There is no related research on the preparation of wintersweet protoplasts, and it is still in its infancy

Method used

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  • Preparation method of chimonanthus praecox protoplast
  • Preparation method of chimonanthus praecox protoplast
  • Preparation method of chimonanthus praecox protoplast

Examples

Experimental program
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Embodiment 1

[0035] The preparation of Wintersweet protoplast, according to the following steps:

[0036] (1) Design an orthogonal experiment with 4 factors and 3 levels Table 1 is as follows:

[0037] Orthogonal experiment design (L 9 4 3 )

[0038]

[0039] Note: These genotypes were collected at Huazhong Agricultural University.

[0040] (2) Prepare 5mL enzymolysis solution: turn on the water bath, set it to 55°C for standby, weigh appropriate amount of cellulase and isolated enzyme according to Table 1, add 2.5mL 0.8M mannitol, 0.1mL 1M potassium chloride, 0.5 mL of 0.2M MES. After mixing evenly, incubate in a water bath for 10 minutes. After cooling to room temperature, add 0.1 mL of 1M calcium chloride, 0.5 mL of 10% BSA (bovine serum albumin), distilled water to 5 mL, and filter through a 0.45 μm filter into a conical In the bottle, ready to use.

[0041] (3) Weigh 0.2 g callus induced by 'Red Heart', 'Su Xin', and 'Wrinkle Petal' wintersweet anthers that have been subcultu...

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Abstract

The invention discloses a preparation method of chimonanthus praecox protoplast. The preparation method comprises the following steps of: culturing the anther of the chimonanthus praecox under a dark condition, and carrying out inducing to generate a callus; placing the callus in an enzymatic hydrolysis solution, and carrying out dark culture in a shake incubator to obtain the enzymatic hydrolysis solution containing the protoplast, wherein the enzymatic hydrolysis solution comprises cellulase and macerozyme; adding a proper quantity of W5 solutions into the enzymatic hydrolysis solution to carry out dilution, and carrying out filtering by a cell filter, wherein the W5 solution is a mixed solution of sodium chloride, calcium chloride, potassium chloride, 2-(N-morpholinyl) ethanesulfonic acid and water; and centrifuging the obtained filtrate, and discarding supernate to obtain a solution containing the chimonanthus praecox protoplast. According to the method, the chimonanthus praecox callus is used as a material to prepare the protoplast, the enzymatic hydrolysis solution combination of the cellulase and the macerozyme is adopted and is not influenced by material taking time and quantity, the 1.11 * 10 < 6 > / g average yield of the chimonanthus praecox protoplast can be obtained through enzymatic hydrolysis, and the method is efficient and rapid.

Description

technical field [0001] The invention relates to a preparation method of protoplasts, in particular to a preparation method of wintersweet protoplasts. Background technique [0002] Wintersweet (Chimonanthus praecox) is a rare flower and tree of the genus Chimonanthus in the family Cesaceae, and it is a precious winter flowering tree species. In recent years, people's demand for wintersweet has been increasing day by day, and the application and market of wintersweet have continued to expand. The trait inheritance and improvement of wintersweet has become the focus of scientific research and industry. As a woody plant, wintersweet is difficult to establish a genetic transformation system. Establishing wintersweet The genetic transformation system is of great significance to the gene functions of wintersweet flower color, fragrance, stress resistance, etc., and protoplast extraction is just a basic technology for transient expression and genetic trait improvement. [0003] Pl...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 赵凯歌李雅迪杨楠赵荣张倩向林陈龙清
Owner HUAZHONG AGRI UNIV
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