Culture method for aquilaria sinensis callus cell suspension system
A callus and cell suspension technology, which is applied in tissue culture, cell culture medium, plant cells, etc., can solve the problems of difficulty in culture methods, and achieve the effect of maintaining sustainable development and utilization, and strong cell vitality
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Embodiment 1
[0023] Example 1 Suspension culture of callus cells of Akiras sinensis
[0024] (1) Induction culture of callus: take 0.8cm of young stems of aseptic seedlings of Akira sinensis and inoculate them on MS+1.0mg / L6-BA2.5mg / L 2,4-D+5g / L agar+30g / The callus was induced in the solid medium of L sucrose, the induction conditions were: temperature 25°C, light intensity 2000lx, light time 12h / d, callus was induced on the 35th day, transferred to the same medium for 45 days, This is the first generation callus, continue to subculture in the same medium for 40 days, this is the second generation callus, culture to the third generation in turn, the subculture time is 120 days, you can get loose light yellow white callus;
[0025] (2) Cell suspension culture: Get 4.0 g of the loose light yellow-white callus obtained in step (1), divide it into mung bean size with sterilized tweezers, inoculate it into a 100 ml Erlenmeyer flask containing 40 ml of liquid medium, Suspension culture was ca...
Embodiment 2
[0027] Example 2 Determination of Growth Curve of Akiras sinensis Suspension Cells
[0028] The first-generation subcultured cells obtained in Example 1 were filtered with a 40-mesh cell mesh, and the filtrate was continued to be filtered with a 200-mesh cell mesh; Shaped flasks, numbered sequentially, placed in a constant temperature shaker at 25°C, carried out suspension culture at 135rpm, 12h light and 12h dark conditions, and the cells were measured every 0d, 2d, 4d, 6d, 8d, 10d, 12d, 14d The fresh weight and dry weight of the cells were recorded, each group was repeated three times, and the growth curve of the cells was drawn.
[0029] The result is as figure 1 As shown, the growth curve of the suspension cells of A. sinensis is in line with the growth of general suspension cells, showing an "S"-shaped curve. 0-4d is the adaptation period of the cells, and the average growth rate is 0.041g / d; 4-8d is the logarithmic phase , the growth rate of the cells is fast, and the ...
Embodiment 3
[0030] Example 3 Detection of Vitality of Akiras sinensis Suspension Cells
[0031] The first-generation subcultured cells of Akiras sinensis obtained in Example 1 were subcultured to the third generation under the same conditions, and 0.25 g of cells were weighed at 0d, 2d, 4d, 6d, 8d, 10d, 12d, and 14d, respectively. In a 10ml EP tube, add 2.5mL of 0.5% TTC solution and 2.5ml of PBS buffer solution with a pH of 8.0, cover the tube, and place it in the dark for 12h. Take out, filter out the solution, wash with distilled water for 3 times, add 5mL of 95% ethanol, bathe in 60°C water for 30 minutes, and shake it two or three times in the middle, so that the dyed substance is fully dissolved in ethanol, and stop the water bath after all the dyed red parts disappear. Filter, collect the filtrate, add 95% ethanol to make up to 5mL, detect OD (485nm) by ultraviolet light, and use ethanol for control.
[0032] The result is as figure 2 As shown, the cell viability in the suspensi...
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