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Method for establishing ecdysone-enriched ajuga multiflora bunge suspension cell line

A technology of multiflora and suspension cells, which is applied in the biological field, can solve the problems of sensitive culture conditions and suspension culture of Labiatae plants, and achieve the effect of uniform cell size, good dispersion and rapid growth

Inactive Publication Date: 2015-04-08
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is suspension culture of alfalfa anthers. In the culture method, alfalfa belongs to the perennial herbaceous plant of leguminous family, while Sinus multiflora is a plant of the genus Muscaria of Lamiaceae. There is a certain gap between the two in genetic characters, and the young leaves In order to induce materials to be relatively more sensitive to external culture conditions, the culture method of alfalfa callus obviously cannot be used for suspension culture of Labiatae plants, and the culture system needs to be re-established
But so far, there is no relevant report on the establishment of suspension cell lines of S. sinensis

Method used

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  • Method for establishing ecdysone-enriched ajuga multiflora bunge suspension cell line
  • Method for establishing ecdysone-enriched ajuga multiflora bunge suspension cell line
  • Method for establishing ecdysone-enriched ajuga multiflora bunge suspension cell line

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Induction of callus tissue from young leaves of S. multiflora

[0032] C. multiflora used in this experiment was collected from Fuling Forest Park, Shenyang, Liaoning Province, and transplanted to the laboratory of Forestry College of Northeast Forestry University for cultivation. Pick its young leaves in early April as explants. First, rinse the explants under running water for 30 minutes, then disinfect the explants with alcohol with a mass concentration of 75% in an ultra-clean workbench for 30 seconds, and then wash them with sterile water. After rinsing 3 times, place it in an appropriate amount of 2.5% NaClO solution, and shake it on a shaker at 100 rpm for 8 minutes. After removing the NaClO solution, rinse the remaining NaClO solution thoroughly with sterile water for about 5-6 times. Finally, use sterile filter paper to absorb the moisture on the surface of the explants. Use a scalpel to cut off the edge of the leaf, then cut it into 1cm 2 size, the leaves ar...

Embodiment 2

[0035] Obtaining Stable Suspension Cells

[0036] 1. Induction of suspension cells

[0037] In the last subculture, after the callus was cultured for 14 days, the granular embryogenic callus with loose shape, yellow-green color, and rapid growth was selected, and the callus was gently crushed with tweezers, and inoculated in a culture medium containing 50 mL of Suspension shaking culture was carried out in a 100mL Erlenmeyer flask containing liquid. Culture conditions: the temperature is (25±1)°C, the light-dark cycle is 16h / 8 hours, the light intensity is 2000lx, and the shaker rotates at 120-140 rpm.

[0038] 2. Obtaining stable suspension cells

[0039] After the cells in 1 were suspended and shaken for 24 hours, filter to remove larger and tighter cell clusters and continue to culture. After culturing under the above conditions for 8-12 days, obtain a cell suspension, and then add an equal volume of the suspension to the obtained suspension. Suspend the culture medium a...

Embodiment 3

[0043] Determination of Factors Affecting Suspension Cell Growth

[0044] 1. The effect of inoculum amount on the length of suspended cell dust of C. multiflora

[0045] Callus II was inoculated into the suspension medium according to different inoculum amounts (5%, 10%, 15%, 20%, 25%, 30%) of the cells cultured for 8 days after suspension culture of callus tissue 2, and after culturing for 12 days The PCV, fresh weight and dry weight were determined respectively. Take the inoculation amount as the abscissa, and take the fresh weight proliferation number as the ordinate to make the influence curve of the inoculum amount on the growth of the suspension cells of S. multiflora, and the results are as follows figure 1 shown. When the initial inoculum amount was 10% in the suspension culture of S. sinensis, that is, when the inoculum amount was 100g / L, the PCV, fresh weight and dry weight of the suspension cells had the highest multiplication multiples, and the multiplication mul...

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Abstract

The invention discloses a method for establishing an ecdysone-enriched ajuga multiflora bunge suspension cell line. The method comprises the following steps: 1, placing an ajuga multiflora bunge explant on an induction culture medium for induction culture to obtain a first callus; 2, placing the first callus on a subculture medium for carrying out subculture for more than 5 times to obtain a second callus; 3, placing the second callus on a suspension culture medium for suspension shake culture; and 4, harvesting cells and extracting ecdysone. By the method disclosed by the invention, the ajuga multiflora bunge suspension cell line is firstly established and haploid plants having a full set of parental genes can be quickly and efficiently obtained. The method is conductive to deeply analyzing the mechanism for synthesizing ecdysone from ajuga multiflora bunge from the perspective of cytology and lays the technical foundation for the genetic transformation and genetic modification of ajuga multiflora bunge.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for establishing a suspension cell line of C. multiflora rich in ecdysone. Background technique [0002] With the improvement of people's awareness of environmental protection, the development of pesticides has tended to be less toxic, safer and more environmentally friendly. Environmentally friendly botanical insecticides that are easy to decompose and whose decomposed products do not damage the environment have become research hotspots for plant protectors in various countries. Therefore, the demand for ecdysterone will become larger and larger, making its wild resources unable to meet the market demand. The production of secondary metabolites by plant cell suspension culture has remarkable characteristics such as increasing yield, shortening cycle, regulating its production process, and improving product quality. [0003] Internationally, there have been many studies on th...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12R1/91
Inventor 赵佳佳迟德富宇佳
Owner NORTHEAST FORESTRY UNIVERSITY
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