Transformation of Tomato

Tomato is a plant of the Solanaceae family and is an annual or perennial herbaceous plant. Tomato is one of the most cultivated vegetable crops in the world. Because it has the advantages of self-pollination, ease of cultivation, and its genome has been sequenced, it is one of the model plants in biological research.

The genetic transformation of tomatoes mainly uses Agrobacterium-mediated genetic transformation . This method is simple, fast, mostly single-copy insertion and has good genetic stability.

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Procedures

1. Sowing on MS medium

The seeds were soaked (5 min), disinfected (soaked in 2% sodium hypochlorite for 10 min), and rinsed (4-5 times with sterile water). Dot the seeds evenly on the MS in a clean bench, culture them in the dark for 3-4 d, then move them to a sunlight incubator and culture them at 25°C to the cotyledon plateau stage (a total of 6-7 d).

2. Activation of Agrobacterium

First, shake Agrobacterium with YEP overnight, then shake the bacteria with 100 mg/L Kan+60 mg/L Rif for 7-8 h until the OD value is 0.6-0.8. Collect the bacterial cells and resuspend Agrobacterium in MS medium 1: (3-4) to an OD value of 0.2.

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3. Obtaining explants

Take the young leaves of the sterile seedlings, cut them into 0.5 cm² leaf discs, and infect them with resuspended Agrobacterium for 5 min, while shaking occasionally.

4. Co-culture

Use sterile filter paper to absorb the bacterial solution on the surface of the cotyledons, inoculate the cotyledons on MS+2 mg/L ZT medium (the surface is covered with a layer of filter paper), and co-culture at 25°C in the dark for 2 d.

5. Recovery culture

Rinse the co-cultured cotyledons in carbenzyl water for several minutes (to inhibit Agrobacterium and reduce browning), blot dry with filter paper, inoculate on MS+2 mg/L ZT+500 mg/L Carb medium, and culture for 2-3 d.

6. Screening and culture

The cotyledons after recovery were rinsed in carboxylic water for 5 min, dried with filter paper, and then inoculated on MS+2 mg/L ZT+25 mg/L Kan+500 mg/L Carb medium. And subcultured on the same culture medium, the culture conditions were 25°C and light 14 h/d.

7. Rooting culture

Cut off the induced shoots (with some callus tissue) and inoculate them in MS+0.2 mg/L NAA medium to induce rooting.

8. Obtain complete redifferentiated plants

Note:

• Genetic transformation operations and culture processes are all conducted under sterile conditions.
• Appropriate selection pressure should be selected when selecting for culture.

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