Preparing method of carnation leaf protoplast

A protoplast and carnation technology, applied in the field of plant tissue and cell culture, can solve the problems of low yield, achieve high yield, shorten enzymatic hydrolysis time, and improve genetic traits

Active Publication Date: 2019-04-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although predecessors have reported the carnation protoplast culture system, its yield is generally low, and the original carnation protoplast culture system is in urgent need of improvement and improvement. It will be necessary to improve the protoplast activity while increasing the protoplast output. Use protoplasts to lay a solid foundation for research on gene function identification, subcellular localization, and protein interaction, etc.

Method used

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  • Preparing method of carnation leaf protoplast
  • Preparing method of carnation leaf protoplast
  • Preparing method of carnation leaf protoplast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The preparation method of embodiment 1 carnation leaf protoplast

[0040] The present embodiment provides the method for the separation and purification of carnation leaf protoplasts, said method comprising the following steps:

[0041] A preparation method for carnation leaf protoplasts, comprising the following steps:

[0042] (1) Preparation of plant material: In the sunny and dry morning, shoots of carnation plants grown in the field were collected. Properly cut off the leaves, wash them with washing powder water first, then rinse them under running water for about 1 hour, and then carry out disinfection treatment on the ultra-clean workbench. The process is as follows: first, soak the tender shoots in a jar of 2% sodium hypochlorite solution for 30 minutes, shake it properly for 5-6 times during the soaking, and wash it with sterile water for 3 times after soaking; then use 75% sodium hypochlorite solution Soak in ethanol for 30s, rinse with sterile water 2-3 tim...

Embodiment 2

[0052] The influence of embodiment 2 different enzymolysis time on carnation leaf protoplast preparation

[0053] For the enzymatic hydrolysis treatment in Example 1, the present invention sets 5 gradient enzymatic hydrolysis times, namely: 0.5h, 1h, 1.5h, 3h, and 4h. The enzymolysis mixture treated by enzymolysis was subjected to microscopic examination to observe the state of release of protoplasts (see Table 1). The results showed that the protoplasts were released in large quantities after 3 hours of enzymatic hydrolysis, and part of the protoplasts began to be damaged at 4 hours. Therefore, the suitable time for enzymatic hydrolysis of protoplasts was 3h-4h, and should not exceed 4h as far as possible. 3.5h enzymatic hydrolysis time is the best enzymatic hydrolysis time (see Table 1).

[0054] Table 1 The release status of protoplasts from carnation leaves after different enzymatic hydrolysis time treatments

[0055]

[0056]

Embodiment 3

[0057] The influence of embodiment 3 different enzyme concentration ratios on carnation leaf protoplast preparation

[0058] For the enzymolysis treatment in embodiment 1, the present invention sets the concentration test of the cellulase of four kinds of levels, is respectively 0.5%, 1.0%, 1.5%, 2.0%, the test of the pectinase concentration level of three levels, They are 0.1%, 0.2%, and 0.5%, respectively. According to the test conditions of each group, the enzymatic hydrolysis was carried out, and the yield and activity of protoplasts were counted. The results are shown in Table 2. It can be seen from Table 2 that in combination 8 under comprehensive conditions, that is, when the enzyme concentration ratio is 1.5% cellulase + 0.2% pectinase, the yield of protoplasts is the highest, up to 183 × 10 5 Each / gFW is about, and the activity of protoplasts is also higher than 90%, reaching 94.26% (see Table 2).

[0059] Table 2 Effects of different enzyme concentration combinati...

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Abstract

The invention belongs to the technical field of plant tissue and cell culture, and particularly relates to a preparing method of carnation leaf protoplast. With a carnation tissue culture seedling asthe test material, an efficient protoplast preparing method for purification through a low-speed oscillation enzymolysis-sedimentation method is established. The method is convenient to operate and can be implemented all year round, the yield of the protoplast subjected to low-speed oscillation enzymolysis separation and density gradient purification is high and can reach 183*105 p / gFW, the cell integrity is high, and it is ensured that the activity is 75% or above. By means of the method, a foundation is laid for the research on the aspects of gene function authentication, sub-cellular localization, protein interaction and the like of a carnation leaf instantaneous converting system.

Description

technical field [0001] The invention belongs to the technical field of plant tissue and cell culture, in particular to a method for preparing carnation leaf protoplasts. Background technique [0002] Carnation (Dianthus caryophyllus), a plant of the genus Caryophyllum in the family Caryophyllaceae, also known as carnation, is one of the "four major cut flowers" in the world and has extremely high ornamental and economic value. In conventional breeding work, carnation is usually not fruitful due to its high double petals, which brings great difficulties to carnation hybrid breeding. Due to the loss of the cell wall coating, plant protoplasts are more receptive to exogenous DNA and other substances. The combination of protoplast transient expression system or protoplast regeneration technology can provide a new technical route for plant breeding. In addition, protoplast fusion technology is useful for cultivating new varieties. It is also of great significance. At present, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/0025C12N5/04
Inventor 傅小鹏林胜男姜敏包满珠冯晗
Owner HUAZHONG AGRI UNIV
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