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Method for separating and purifying amomum villosum mesophyll protoplasts

A protoplast, separation and purification technology, applied in plant cells and other directions, can solve the problem of unclear protoplast yield and vitality factors, and achieve the effects of simple operation, broad application prospects, high yield and vitality

Active Publication Date: 2019-04-02
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The successful isolation and cultivation of protoplasts is the premise of somatic cell fusion. So far, no research has reported on the separation and purification of Yangchunsha protoplasts. The types and concentrations of enzymes involved in the separation and purification process, enzymatic hydrolysis time, osmotic pressure, etc. Factors affecting protoplast yield and viability are also unclear

Method used

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  • Method for separating and purifying amomum villosum mesophyll protoplasts
  • Method for separating and purifying amomum villosum mesophyll protoplasts
  • Method for separating and purifying amomum villosum mesophyll protoplasts

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Embodiment 1

[0045] Embodiment 1 A kind of method for separation and purification of mesophyll protoplasts of S. vulgaris

[0046] The present embodiment provides a method for isolating and purifying mesophyll protoplasts of S. solani, comprising the following specific steps:

[0047] S1. Material preparation and pretreatment: The shoot tips of rhizomes of the long-fruit cultivation type were inoculated on MS solid medium to induce sterile seedlings. Robust and lush sterile seedlings, select healthy leaves with a leaf length of 30-40 mm for the preparation of protoplasts; remove the veins of the sterile seedling leaves with a sterilized blade, and cut them into about 0.5 mm in the vertical direction of the veins. The thin strips were pretreated by adding a CPW solution containing 0.5M mannitol in a mass-to-volume ratio of 1g:20mL, and suspended in the dark, 28°C, and 120r / min for 1h to complete the preplasma separation;

[0048] S2. Isolation of protoplasts: add enzymatic hydrolysis solut...

Embodiment 2

[0054] Example 2 The effect of pretreatment on protoplast isolation

[0055] 1. Experimental group: Take the sterile leaves of S. solani, tear off the lower epidermis with tweezers or cut into 0.5mm thin slices, weigh 1 g and place it in a triangular flask containing 20 mL of CPW solution containing 13% mannitol, and place it in a dark place. , 28 ℃, 120r / min suspension culture for 1h, to complete the separation of the pre-plasma wall. Control group: no pretreatment. The mesophyll cells of P. solani before and after preplasma separation treatment were as follows: figure 1 shown.

[0056] 2. Put the experimental group and the control group in step 1 into 15 mL of enzymatic hydrolysis solution respectively for enzymatic hydrolysis. After 10 hours of enzymatic hydrolysis, the yield and activity of protoplasts were observed under a microscope. The result is as figure 2 The results showed that there was no significant difference in the protoplast yield between the two groups...

Embodiment 3

[0057] Example 3 Effects of different enzymolysis solutions on the yield and activity of protoplasts

[0058] Cut the sterile leaves of Cordyceps sinensis into 0.5mm and put them into each combination of enzymolysis solution (Table 1), and shake the enzyme solution at low speed for 10h in the dark. Observe the separation of protoplasts under a microscope to determine the best combination of enzyme concentrations. . The results are shown in Table 1 and image 3 shown. The results showed that protoplasts could be obtained by the combination of different concentrations of cellulase and isolated enzymes. When the concentration of isolated enzyme is 0.5%, the yield and activity of protoplasts have concentration effects on cellulase. When the concentration of cellulase is 1.5%, the yield and activity of protoplasts are the highest at 6.12×10 6 Pieces / g·Fw and 81.21%. When the concentration of isolated enzyme was increased to 1.0%, the yield and activity of protoplasts also had t...

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Abstract

The invention discloses a method for separating and purifying amomum villosum mesophyll protoplasts. The method comprises the following steps: preparing and pre-treating materials, separating protoplasts by an enzymolysis method, and purifying the protoplasts by combining a sedimentation method and a drifting method. By studying the factors affecting the yield and vitality of the protoplasts, suchas material pretreatment or no material pretreatment, enzyme types, enzyme concentration, enzymolysis time and concentration of an osmotic pressure regulator and by studying the influence of different purification methods on the purification effect of the mesophyll cell protoplasts, the invention optimizes the experimental conditions for the separation and purification of the amomum villosum mesophyll protoplasts so as to obtain the high-yield and high-vitality amomum villosum protoplasts, and lays a foundation for the cultivation of new varieties of amomum villosum by somatic cell fusion.

Description

technical field [0001] The present invention relates to the technical field of separation and purification of protoplasts, in particular to a method for separation and purification of mesophyll protoplasts of Pseudomonas vulgaris. Background technique [0002] Amomum glutinosa is one of the famous "Four Southern Medicines" in my country, and the genuine product is the ginger plant Amomum vulgaris Amomum villosum Lour., green shell sand Amomum Villosum Lour. var. xanthioides T. L. Wu et Senjen, Hainan Sand Amomumlongiligulare Dried ripe fruit of T.L.Wu. Among them, the fruit of Yangchunsha is the mainstream variety. Its properties are pungent and warm, and it returns to the spleen, stomach and kidney meridians. It has a long history of medicinal and clinical use in my country. [0003] In recent years, due to the development and expansion of the amomum processing industry, the supply of spring amomum is in short supply in the market, and the price is rising year by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 何国振杨锐培苏晓欣李明晓何卓航
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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