Principle
Arabidopsis thaliana is a cruciferous plant, a biennial herb, 7-40 cm tall. It is widely used in research on plant genetics, developmental biology and molecular biology, and has become a typical model plant. A. thaliana has the following characteristics:
- The shape is small, strong vitality and large number of seeds.
- The growth cycle is fast, usually only 6-8 weeks from sowing to harvesting the seeds.
- The genome is small (2n=10), the smallest among known plant genomes, but it contains all the genes needed to complete complex plant growth and development.
- A. thaliana is a self-pollinated plant and homozygous mutants are easily obtained.
The Agrobacterium flower dip method is used to transform A. thaliana, which is a convenient and efficient plant transgenic method. It is mostly used to transform dicotyledonous plants. After improvement, it can also be used to transform monocotyledonous plants.
Procedures
1. Cultivation of A. thaliana
Arabidopsis seeds were sterilized with 2% sodium hypochlorite and rinsed with sterile water 4-5 times, and then inoculated on MS medium. After being treated at low temperature at 4°C and protected from light for 2 d, they were transferred to a light incubator and cultured at 22°C and 10 h of light for 1-2 weeks. After the sterile seedlings grow, they are transferred to soil with a ratio of vermiculite to nutrient soil of 1:3 to continue growing. The environment in which the plants were grown was maintained at a constant temperature of 22°C and 16 h of light/8 h of darkness.
2. Activation of Agrobacterium
- Pick the monoclonal positive Agrobacterium GV3101 strain containing the target gene plasmid into 5 mL of fresh YEB liquid medium (50 mg/L Kan, 125 mg/L Rif), and shake culture at 28°C for 24 h.
- Take 0.1 mL of the above bacterial solution and add it to 50 mL of fresh YEB liquid medium (50 mg/L Kan, 125 mg/L Rif), and culture it at 28°C and 220 r/min for 2-4 h until the OD600 value reaches about 0.8.
- Take 1 mL of the above bacterial solution in an EP tube, centrifuge at room temperature 22°C and 5,500 g for 15 min, discard the supernatant, and resuspend the pellet in transformation medium until the OD600 value reaches about 0.8.
3. Agrobacterium infects A. thaliana
- Lay the Arabidopsis plant to be transformed flat, insert the flower bud part into a 2 mL EP tube, add the above transformation solution, and soak for 5 min. Gently shake off the dip liquid and mark.
- Place the Arabidopsis seedlings in dark and humid conditions for 16 h and then resume normal culture.
Note:
- After transformation, the culture of A. thaliana should be restored to normal management. Once side knots appear again or branches appear on the main shoot, they should be cut off in time.
- After 5-6 weeks of transformation, in order to accelerate the maturation of A. thaliana, you can pour an appropriate amount of nutrient solution in small amounts. After individual A. thaliana siliques begin to wither and turn yellow, the siliques can be cut off and placed in a petri dish to dry. After most of the A. thaliana siliques have withered, all the seeds can be collected and stored in a 1.5 mL EP tube (punch a small hole in the lid for drying). After the seeds are completely dry, place them in a new 1.5 mL EP tube for short-term storage at 4°C. If necessary, it can be stored in a -20°C refrigerator for long-term storage.
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