pGreen-TRAIL

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	Transformation of N. tabacum cells was performed by co-cultivation technique (30) with some modifications. At first, 10 mL of freshly activated transformed A. tumefaciens (OD: 1, 600 nm) (Optical Density) was centrifuged (3000 rpm, 5 min, 25 oC) and the medium was replenished by 5 mL modified LS medium containing 100 μM Acetosyringon.

Protocol tips
Transformation of N. tabacum cells was performed by co-cultivation technique (30) with some modifications. At first, 10 mL of freshly activated transformed A. tumefaciens (OD: 1, 600 nm) (Optical Density) was centrifuged (3000 rpm, 5 min, 25 oC) and the medium was replenished by 5 mL modified LS medium containing 100 μM Acetosyringon.

Publication protocol

N. tabacum callus cells were grown at 25 oC in the darkness condition on solid LS medium (Linsmaier & Skoog Medium) (29) supplemented with Indole acetic acid 3 μg/mL, Naphthyl acetic acid 3 μg/mL, Kinetin 100 ng/mL, Myoinositol 100 μg/mL and Sucrose 30 mg/mL Since Zn2+ plays important role in proper assembly of the TRAIL, two fold of Zn2+ ion regular concentration was used for LS medium preparation. Transformation of N. tabacum cells was performed by co-cultivation technique (30) with some modifications. At first, 10 mL of freshly activated transformed A. tumefaciens (OD: 1, 600 nm) (Optical Density) was centrifuged (3000 rpm, 5 min, 25 oC) and the medium was replenished by 5 mL modified LS medium containing 100 μM Acetosyringon. Afterward, one gram of freshly grown N. tabacum cells was added to the bacterial culture and was incubated at 100 rpm about 2 h at 25 oC. The incubated cells were centrifuged (1000 rpm, 10 min, 25 oC) and the cells pellet was placed on the sterile filter paper for 5 min to wipe excess water. Inoculated plant cells were subsequently transferred to the solid modified LS medium containing 100 μM Acetosyringon and were kept for two days at 25 oC in the darkness condition. Remained Agrobacterium cells were eradicated by means of sub-culturing on medium containing Cefotaxime (200 μg/mL). Finally, transformed N. tabacum cells were screened through frequent sub-culturing on selection medium containing Hygromycin (30 μg/mL) and Cefotaxime (200 μg/mL) every two weeks. Selected N. Tabacum cells in the week 8th were used for the rest of the experiments.

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