Experimental material Tobacco cells: BY-2
Experimental procedure
1. BY-2 cell culture
The suspension of BY-2 cells was cultured on a shaker at a temperature of 260 C and a rotation speed of 130 rpm. Every 7 days, 1 ml of cells were transferred to 20 ml of fresh liquid medium to continue the culture. BY -2 callus was grown on solid medium and subcultured every 3-4 weeks. The transgenic suspension cells and callus are grown in a medium containing the corresponding antibiotic.
2. Preparation of dry transformed Agrobacterium GV3101
(1) Inoculation of Agrobacterium single colonies into 2 ml of fresh YEB liquid medium, 28" culture overnight;
(2) Add 200 ul of culture to 10 ml of new medium and continue to culture for 3-4 hours:
(3) '5000 rpm, centrifuge for 5 min, and resuspend the cells with BY-2 liquid medium. The OD is about 0.5, and it is ready for use.
3. Foreign gene transformation of BY-2 suspension cells mediated by Agrobacterium
(1) Take 4 ml of BY-2 cells grown to the 3rd or 4th day, add 100ul of the above-mentioned spare Agrobacterium liquid, and co-culture for 3 days;
(2) Centrifuge at 500 g for 2 min, harvest the cells and wash 3 times with BY-2 liquid medium (Amp 500 mg/L):
(3) The cells were diluted and plated on a plate of BY-2 solid medium (Kan 100 mg/L, Amp 500 mg / L), and cultured in the dark at 26 °C.
(4) After four weeks, the well-grown callus pieces were taken out, dispersed, and placed in a liquid medium suspension culture.
4. Agrobacterium-mediated transformation of exogenous genes in BY-2 callus
(1) A small piece of tobacco callus cultured for 2-3 weeks was immersed in Agrobacterium liquid for 20 min, taken out and dried with a sterile filter paper, and placed on a BY-2 medium plate (without antibiotics) for 2 days;
(2) remove the callus pieces, wash 4 times with sterile water, and then soak 30-60m in sterile water containing antibiotics (Amp 500 mg / L);
(3) The callus pieces were taken out, blotted dry with a sterile filter paper, and placed on a plate for culturing BY-2 (Amp 500 mg/L, Kan 100 mg/).
(4) After four weeks of culture, pick up the growing callus and transfer to a new resistant plate (Kan 100 mg/L)
Continue to grow;
(5) Take out the well-grown callus pieces, disperse them, put them into liquid medium, and incubate them in a dark place on a shaker at 26 ° C, 130 rpm;
(6) Subsequent every 7 days.
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