Tomato
  Agrobacterium based
  A modification of Fillatti et al. Bio/Tech 5:726-730

Day 1 Germinate sterile tomato seed on 1/10 B5 medium for 5-7 days. Germination container should be tall enough to allow elongation of seedlings. Note: Preferred cultivars are UC82B (an OP commercial canning type), Better Boy (a Hybrid Home Garden type), and Micro-Tom (an OP research type).
Day 6 Start 2 ml liquid LB culture of Agrobacterium from frozen stock or single isolated colony in appropriate antibiotics. Culture overnight 27°C/200 rpm.
Day 7 Agrobacterium Culture Innoculate 50 mL liquid LB culture with 10 µL of previous night's culture. Again, incubate overnight 27°C/200 rpm. Tomato Pre-culture Excise sterile cotyledons. Cut each cotyledon longitudinally and place on LYT+AS media. Incubate overnight in the dark 27°C.
Day 8 Agrobacterium Culture Take OD600 reading of overnight culture. If greater than 1.0, dilute 1:10 and culture for 6 additional hours. If culture is between 0.2 and 0.8, then centrifuge and resuspend to an OD600 equal 0.25 in LTY+AS. Place on shaker until ready to use. Tomato Transformation Remove explants from dark incubation to fresh petri dish. Cover with Agrobacterium suspension and soak for 15 minutes. Remove explants from suspension and place on sterile filter paper placed on top of LYT+AS solid media. Co-culture 48 hours at 24°C.
Day 10 Transfer explants to LYC media plus antibiotics. A light halo of bacteria may be evident around each explant. Note: LYC media should be made fresh prior to use due to lability of Zeatin and some antibiotics.
Day 13 Sub-culture explants to LYS media plus antibiotics. No bacteria should be evident around explants.
Day 23 Sub-culture explants to LYS media plus antibiotics if warranted by bacterial overgrowth. Transgenic callous may be appearing around this time, but it is preferrable to see emerging shoots. Some regions of explants may be beginning to yellow or turn necrotic.
Days 30-45 Shoots may begin to elongate. Those shoots greater than 1 cm in length may be excised and transferred to rooting media.
Days 45-60 Excised shoot should form roots within 14 days. Rooted shoots may be hardened and transferred to the greenhouse.
Appendix Selective Agents Hygromycin 25 mg/Liter Kanamycin 100 mg/Liter Antibiotics Rifampicin 100 mg/Liter Tetracycline 12.5 mg/Liter Carbenicillin 100 mg/Liter Cefoxatime 500 mg/Liter Media (in mg/L) LYT LYC LYS Rooting MS Macro 100.0 - 100.0 100.0 MS Micro 1.0 - 1.0 1.0 Ca Stock 5.0 - 5.0 5.0 Fe Stock 10.0 - 10.0 10.0 B5 Vitamins 2.0 - 2.0 2.0 Thiamine HCI 0.2 - 0.2 0.2 IAA Stock 10.0 1.0 1.0 - KH2PO4 200.0 - 200.0 - Zeatin - 5.0 2.0 - Pyridoxine-HCI - - - 0.5 Nicotinic Acid - - - 0.5 Glycine - - - 2.0 Sucrose (g/L) 30.0 - 20.0 30.0 Phytagar 7.0 - 7.0 7.0 pH 5.7 - 5.8 7.0 Growth Regulator Stocks Thiamine 84 mg/L (4°C) Pyridoxine HCI 1 mg/mL (4°C) Nicotinic Acid 1 mg/mL (4°C) Zeatin 2 mg/mL (-20°C) IAA 100 mg/L (4°C) Glycine 1 mg/mL (4°C)