Table 1.

Optimized steps, medium used and culture conditions in Agrobacterium-mediated transformation of Nicotiana alata.

Type of Medium	Medium content	Antibiotics applicaiton	Culture conditions	Culture time
Washing medium	MS basal medium (4.41 g/L), 6-Benzyladenine (6-BA) 1 mg/L, IAA 0.02 mg/l, 30 g/L sucrose, adjust the pH to 5.8 with 1 M KOH.	None	None	None
Co-cultivation	MS basal medium (4.41 g/L), 6-BA 1 mg/L, IAA 0.02 mg/l, 30 g/L sucrose, and 5 g/L Bacto agar, adjust the pH to 5.8 with 1 M KOH.	None	Dark, 25°C	3 days
Callus induction	MS basal medium (4.41 g/L), 6-BA 1 mg/L, IAA 0.02 mg/l, 30 g/L sucrose, and 5 g/L Bacto agar, adjust the pH to 5.8 with 1 M KOH.	Timentin (125 mg/L) for Agrobacterium inhibition and hygromycin (35 mg/L) for selectable antibiotic	Long-day condition (16-h light/8-h dark, 25 °C)	3-5 weeks
Shoot regeneration	MS basal medium (4.41 g/L), 6-BA 1 mg/L, 30 g/L sucrose, and 5 g/L Bacto agar, adjust the pH to 5.8 with 1 M KOH.	Timentin (125 mg/L) for Agrobacterium inhibition and hygromycin (35 mg/L) for selectable antibiotic	Long-day condition (16-h light/8-h dark, 25 °C)	3 weeks
Rooting	1/4 X MS basal medium (4.41 g/L), 0.8 mg/L 4-(3-Indolyl) butyric acid, 30 g/L sucrose, and 6 g/L Bacto agar, adjust the pH to 5.8 with 1 M KOH	Timentin (125 mg/L) for Agrobacterium inhibition	Long-day condition (16-h light/8-h dark, 25 °C)	3-5 weeks