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Myricaria laxiflora callus and suspension cell granule culturing method

A technique for thinning cypress branches and callus, which is applied in the field of plant tissue or cell culture and achieves the effects of good uniformity, reduced production cost and fast growth rate.

Active Publication Date: 2015-02-18
上海揽微赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on Cypress spp. mainly focuses on its ecological environment, physiological changes, cutting and seed propagation directions, which cannot solve the current situation of the scarcity of Cypress genus resources.
No scholars have reported the callus induction and establishment of suspension cell culture system of S.

Method used

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  • Myricaria laxiflora callus and suspension cell granule culturing method
  • Myricaria laxiflora callus and suspension cell granule culturing method
  • Myricaria laxiflora callus and suspension cell granule culturing method

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Experimental program
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Effect test

Embodiment 1

[0034]2. The callus obtained in the above step 1 was inoculated in the following liquid medium without adding agar and without antioxidant for shaking culture: MS basic medium + sucrose 30g / L + 6-BA 0.5mg / L + 2, 4-D 0.2mg / L (the pH value of the medium is 5.9-6.1, sterilized at 121°C for 20 minutes); the rotation speed of the rotary shaker is 120r / min. On the 7th day of cultivation, the solution of suspended particles began to appear clear and transparent, and on the 28th day, it began to become cloudy. The culture conditions mentioned were that the temperature was 25°C, and the light intensity was controlled in sections as in step 1. The light cycle was 16h / d; take the culture solution Sonicate for 1 hour, centrifuge at 5000r / min, centrifuge for 10min, collect the suspended particle water, weigh the fresh weight and dry weight; use the Folin’s method to determine the polyphenol content to be 6wt% of the dry weight of the suspended cells; use T-AOC The total antioxidant capacit...

Embodiment 2

[0037] 2. Measure the number of cells with a hemocytometer, draw a growth curve, and measure the cell size and cell structure with a microscope. Since the cells cultured in suspension do not appear as free single cells, it is difficult to perform reliable cell recording by directly sampling in a culture flask. The cell mass was treated with 0.25% pectinase, and the diameter of the cells obtained from the stems of Cypress cypress branch was 7.25-13.75 μm, and the cell structure was as follows: Figure 1-3 , cells in mitosis can be seen under a 100X microscope. The cell growth curve of the branches and stems of Cypress cypress Figure 4 , we can see that with 1×10 6 cells / ml inoculated at the initial concentration, the cell division speed reached the maximum on the 4th day of culture.

[0038] Example 3

Embodiment 3

[0040] 2. Cell clusters or cell lines obtained from single cell division and propagation can be considered to have the same genes and characteristics, and large-scale cell culture with this cell line can obtain relatively uniform cell clusters and metabolites. Based on the cell concentration measured by the hemocytometer, adjust the suspension cell concentration to 1×10 4 , inoculated in fresh solid MS medium at 50°C, mixed evenly, placed horizontally, and controlled the thickness of the medium within 1mm, so as to facilitate observation with a microscope; 3. The single cells in the inoculation medium were shaken in the following liquid medium : MS basic medium + sucrose 30g / L + 6-BA 0.5mg / L + 2,4-D 0.2mg / L (the pH value of the medium is 5.8-6.1, sterilized at 121°C for 20min); rotary shaker The bed rotation speed was 120r / min, and cultured for 15 days to obtain a single-cell suspension system.

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Abstract

The invention relates to a plant tissue and cell culturing method, in particular to a method for producing calluses and high-quality suspension cell granules in a large scale by taking stems of a specific rare or endangered plant myricaria laxiflora in the Three Gorge basin as explants, belongs to the field of plant tissue or cell culture, and discloses a callus and suspension cell granule culturing method. The method comprises the following steps: 1, obtaining aseptic myricaria laxiflora plant body organs, and performing comparison to obtain parts with the most prominent overall performance in roots, stems and leaves of the system; 2, inoculating the aseptic stems into a callus inducing medium to induce and subculture calluses to obtain the calluses using myricaria laxiflora tissues as raw materials; 3, inoculating the calluses to a corresponding liquid medium, and performing shaking culturing to obtain stable-character suspension cell granules originated from the myricaria laxiflora plant body organs. The calluses are stable in character, the obtained suspension cell granules are high in growth speed and good in dispersity, cell clusters are smaller, and a solution is clear and transparent.

Description

technical field [0001] The present invention relates to a kind of cultivation method of plant tissue or cell, involves using Shuibai branch ( Myricaria laxiflora The invention relates to a method for large-scale production of callus originating from plant organs and excellent suspended cell particles, and belongs to the field of plant tissue or cell culture. Background technique [0002] Tamaraceae (Tamaraceae) Tamaricaceae ) Cypress ( Myricaria ) is a perennial shrub that is restrictedly distributed in the middle and lower sandy beaches of the natural drawdown zone on both sides of the Yangtze River in the Three Gorges Reservoir area, with an altitude of about 80-155 m. It is the only plant species listed as a rare and endangered plant in China due to the Three Gorges Project. The plants of the genus Shuibai mainly have the functions of dispelling the wind, relieving the exterior, dispelling the wind and dredging the collaterals. It is mainly used to treat impermeable m...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 刘士平曾文薛艳红孔玉珊蒋维秦王格格
Owner 上海揽微赛尔生物科技有限公司
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