Agrobacterium-mediated transformation in tomato
(Licopersicon esculetum)
1. Inoculate Agrobacterium tumefaciens LBA4404 or EHA105 containing the binary
vector in 5 ml YEP liquid medium at 28℃ overnight.(pCAMIBA2301-kanamycin
selection in plant, kanamycin in E. coli).
＊The Agrobacteria is from the de-freezer(-80℃).
2. Spread the bacteria to YEP+Kan solid medium, and incubate for two days at 28℃.
3. 25ml AB with 100 ppm Kan medium in 125 ml flask was inoculated with the
single colony for 2 days at 28℃.
4. Add AS (acetosyringone) and glucose to AB medium (step 3) to yield a final
concentration of 100 μM and 5%, respectively. Then incubate for 4 hr at 28℃.
5. Cut the cotyledons or hypocotyl and incubate in KCMS+AS (100μM) solid
medium (pH 5.6) for 1 day.
6. The Agrobacterium in step 4 are centrifuged at 5000 rpm for 5 min.
7. Discard the supernatant and resuspend the pellet with KCMS+AS (100μM)
+Glucose(5%), pH5.6 liquid medium. The suspension is diluted such that the final
A600 reading is 0.5-1.
8. Add 15 ml Agrobacterium tumefaciens to each petri dish in step 5. Co-culture for
1 hr (at room temperature).
9. Excess Agrobacterium is blotted from the explants on filter paper to avoid excess
growth of the bacteria during co-cultivation.
10. Transfer the infected explants to another KCMS+AS(100μM) +G(5%), pH5.6
solid medium for 48 hr at 26℃.
11. Wash the explants in KCMS liquid medium containing the 200mg/L Timentin
(Powder freshly prepare, filter sterile, and add before use). Repeatedly wash for
three times. (The first time is shaking at 220 rpm, and other times are shaking at
100 rpm, each time is for 30 min).
12. Dry the explants with Whatmann No. 41 or No. 1 filter paper. Transfer to MSZ
solid medium containing suitable antibiotic (100mg/L Kan) and 200 mg/L
Timentin at 26℃ with a 16 hr light/ 8 hr dark photoperiod at a light intensity of
100 to 200μEm2s-1.
13. Formation of the initial calli can be seen on the wounded site of explants after 3
weeks of transformation. Shoot will expectably respectably regenerate after 6
weeks the explants are cut form the calli and discarded.
14. The tomato shoots have regenerated from the calli and are ready to be transferred
to rooting media. This media lacks zeatin, but contains hygromycin for selection
of transformants.
Medium
AB medium
20X AB salt (/l)
20g NH4Cl
6g MgSO4•7H2O
3g KCl
0.2g CaCl2
50mg FeSO4•7H2O
20X AB buffer (/ L)
60g K2HPO4
23g NaH2PO4•H2O
Glucose solution(900ml): 5g glucose
Autoclave all three solutions separately and then mix 50 ml 20X AB salt and 50 ml
20X AB buffer, and 900 ml glucose solution to make 1L.
MSS medium (for tomato seedling), 1L (pH5.6)
MS salt
4.3 g
MS vitamin
1 ml
1 % Sucrose
10 g
Phytagel
3g
KCMS medium, 1L (pH5.6)
MS salt
4.3 g
MS vitamin
1 ml
KH2PO4
200 mg
Thiamine-HCl (0.9mg/L)
1 ml of 1000 x stock
2,4-D (0.2mg/L)
1 ml of 1000 x stock
Kinetine (0.2mg/L)
Phytagel
100 l of 10000 x stock
3g
MSZHT medium(for shooting), 1L (pH6.0)
MS salt
4.3 g
Nitch Vitamin
1 ml
3% Sucrose
30 g
Hygromycin
Zeatin
Timetin
200 l of 100 mg/ml stock
100 l of 20 mg/ml stock
200 mg
MSHT medium (for rooting), 1L (pH6.0)
MS salt
4.3 g
Nitch Vitamin
1 ml
3% Sucrose
30 g
Hygromycin
Timetin
200 l of 100 mg/ml stock
200 mg
1000 X MS vitamin stock, 100 ml (store at -20℃)
Thiamine-HCl
Pyridoxine-HCl
Nicotinit acid
Myo-inositol
100 mg
100 mg
1g
10 g
1000 X Nitch vitamin stock, 100 ml (store at -20℃)
Biotin
Folic asid
Glycine
Myo-inositol
Nicotinic acid
Pyridoxine-HCl
Thiamine-HCl
5 mg
50 mg
200 mg
10 g
0.5 g
50 mg
50 mg