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-80 °C freezer
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Autoclave (any model that allows a temperature of 121 °C)
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Plant growth chamber (any model with temperature and light control)
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Incubator shaker (e.g., New Brunswick Scientific, model: Excella E25)
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pH meter (e.g., ThermoFisher, Orion Star A111)
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Laminar flow hood (e.g., LABCONCO, model: 36125)
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Pipetman® 20, 200,1000 µl (e.g., RANIN, Pipet-Lite XLS)
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Spectrophotometer (e.g., Eppendorf, 6131 23550)
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Orbital shaker (e.g., Heidolph Rotamax 120 Orbital Platform Shaker)
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Plant tissue culture room with controlled light and temperature
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Kanamycin (50 mg/ml)
Dissolve 500 mg kanamycin sulfate in 10 ml MilliQ water
Filter sterilize using a 0.2 µm syringe filter
Store at -20 °C in 1 ml aliquots
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Rifampicin (25 mg/ml)
Dissolve 250 mg rifampicin in 10 ml DMSO
Store at -20 °C in 1 ml aliquots wrapped in aluminium foil to protect from light
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Timentin (200 mg/ml)
Dissolve 2 g timentin in 10 ml MilliQ water
Filter sterilize using a 0.2 µm syringe filter
Store at -20 °C in 1 ml aliquots
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Thidiazuron (TDZ) (1 mg/ml)
Dissolve 50 mg of TDZ in 1 ml 1 N NaOH and gradually dilute with sterile MilliQ water to make a final volume of 50 ml
Store at 4 °C and is good to use for several months
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NAA (0.5 mg/ml)
Dissolve 25 mg NAA in 1 ml 1 N NaOH. Bring the volume to 50 ml using sterile MilliQ water by constantly stirring the solution
Store at 4 °C and is good to use for several months
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Acetosyringone (AS) (0.2 M)
Dissolve 40 mg acetosyringone in 1 ml DMSO by vortexing to dissolve it completely
Wrap the tube in aluminium foil; Make fresh on the day of the experiment
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12% (v/v) Clorox Regular Bleach Disinfectant solution (300 ml)
Clorox Regular bleach 36 ml
MilliQ water 264 ml
Mix well
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LB agar supplemented with kanamycin (50 µg/ml) and rifampicin (15 µg/ml), 250 ml
LB Broth, Miller 6.25 g
Agar 3.75 g
Make up the volume to 250 ml using MilliQ water
Autoclave and once media cools to 50 °C, add 250 μl kanamycin (50 mg/ml) and 150 µl lrifampicin (25 mg/ml), swirl the media to mix well and pour in 100 x 15 mm Petri dishes
Store the plates at 4 °C and use within one month
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Agrobacterium Inoculation media (IN) [4.3 g/L Murashige and Skoog basal medium (MS), 3% sucrose, 16.8 μM thidiazuron (TDZ), pH 5.7, supplemented with 200 μM acetosyringone (AS)], 100 ml
MS basal medium 0.43 g
Sucrose 3 g
TDZ (1 mg/ml) 400 µl
Dissolve above in 80 ml MilliQ water, adjust pH to 5.7 with 0.5 N NaOH, and make up the volume to 100 ml using MilliQ water
Autoclave, add 100 μl AS (0.2 M) when it cools down to room temperature, cover with aluminium foil to avoid light.
Always make fresh on the day of the experiment
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Callus and shoot induction (SI) media (4.3 g/L MS, 3% sucrose, 0.8% phytoagar, 16.8 μM TDZ, pH 5.7) supplemented with 200 μM AS, 500 ml
MS basal medium 2.15 g
Sucrose 15 g
TDZ (1 mg/ml) 2 ml
Dissolve above in 400 ml MilliQ water, adjust pH to 5.7 with 0.5 N NaOH, and make up the volume to 500 ml using MilliQ water
Add 4 g Phytoagar
Autoclave, add 500 μl AS (0.2 M) when it cools down to about 50 °C, mix well and pour in cell culture dishes/Petri-plates, cover with foil to avoid light
Always make fresh on the day of the experiment
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Callus and shoot induction (SI) media supplemented with timentin (200 µg/ml) and kanamycin (30 µg/ml), 500 ml
Prepare SI media as above (9)
After autoclaving when the media cools down to 50 °C, add 500 µl timentin (200 mg/ml) and 300 µl kanamycin (50 mg/ml), swirl the media and pour in cell culture dishes
Media plates can be stored at 4 °C for two weeks
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Root induction (RI) media [4.3 g/L MS, 3% sucrose, 0.8% phytoagar, 0.054 μM NAA, pH 5.7] supplemented with timentin (200 µg/ml) and kanamycin (30 µg/ml), 500 ml
MS 2.15 g
Sucrose (3%) 15 g
NAA (0.5 mg/ml) 10 μl
Dissolve above in 400 ml MilliQ water, adjust pH to 5.7 with 0.5 N NaOH, and make up volume to 500 ml using MilliQ water
Add phytoagar 4 g
Autoclave, once media cools down to 50 °C, add 500 µl timentin (200 mg/ml) and 300 µl kanamycin (50 mg/ml), swirl the media and pour in plant tissue culture containers
Media containers can be stored at 4 °C for two weeks
This protocol was derived from the original research paper by Navet and Tian (2020). It was modified based on the methods developed for sweet basil regeneration described by Phippen and Simon (2000) and Agrobacterium-mediated transformation of sweet basil previously reported by Deschamps and Simon (2002). This work was supported by NIFA HATCH (Accession No. 1003536 and 1020611) and the USDA-ARS Agreement No. 58~5320~4~020, entitled, “Control of Pests and Diseases and Adding Value to Specialty Crops”, managed by the College of Tropical Agriculture and Human Resource, University of Hawaii at Manoa.
The authors declare no conflict of interest.