Materials and reagents

Biological materials

1. N. benthamiana seeds (lab stock; originally obtained from the Centre for Tropical Crops and Biocommodities, now Centre for Agriculture and Bioeconomy, Queensland University of Technology). Researchers interested in acquiring seeds are advised to contact laboratories or research groups working with N. benthamiana for seed sharing, as commercial suppliers are limited

2. In vitro–cultured N. benthamiana plants (generated from lab stock seeds as described in the protocol)

Reagents

1. Murashige and Skoog (MS) basal medium powder (Sigma, catalog number: M5519)

2. Kinetin powder (Sigma, catalog number: K3378)

3. Indole-3-butyric acid (IBA) powder (Sigma, catalog number: I5386)

4. Sucrose (PhytoTech Labs, catalog number: S391)

5. Agar (or alternative gelling agent like phytagel) (Sigma, catalog number: A5054)

6. Commercial bleach (e.g., White King^TM 4% sodium hypochlorite) (from local supermarket)

7. Tween-20 (10%) (Sigma, catalog number: P9416)

8. Ethanol (70% and absolute) (Sigma, catalog number: 459844)

9. 1 M NaOH (Sigma, catalog number: 1091371000)

9. Sterile Milli-Q water for seed sterilization

10. Milli-Q water for hormone and media preparation

Solutions

1. IBA stock solution (see Recipes)

2. Kinetin stock solution (see Recipes)

3. MS basal medium without hormones (hormone-free MS) (see Recipes)

4. MS basal medium with hormones (MS1) (see Recipes)

5. Seed sterilization solution (see Recipes)

Recipes

Note: Be precise about the ingredients (e.g., buffer or media), quantities, and conditions established for your experiments. Note that the omission of minor details from recipes (e.g., type of water used or storage conditions) might lead to the failure of the experiment.

1. IBA stock solution (1 mg/mL)

Weigh 20 mg of IBA powder and transfer to a 50 mL Falcon tube. Dissolve in 14 mL of absolute ethanol. Make up the final volume to 20 mL with Milli-Q water. The final concentration of ethanol will be 70%. Filter sterilize using a 0.2 μm syringe filter. Aliquot 1 mL into sterile 1.5 mL Eppendorf tubes. Store at -20 °C. Use 100 μL per 1 L of MS medium for a final IBA concentration of 0.1 mg/L.

2. Kinetin stock solution (1 mg/mL)

Weigh 20 mg of kinetin powder and transfer to a 50 mL Falcon tube. Dissolve in a few drops of 1 M NaOH and make up the final volume to 20 mL with Milli-Q water. Filter sterilize using a 0.2 μm syringe filter. Aliquot 1 mL into sterile 1.5 mL Eppendorf tubes. Store at -20 °C. Use 500 μL per 1 L of MS medium for a 0.5 mg/L final concentration.

3. MS basal medium without hormones (hormone-free MS)

Dissolve MS powder in 700 mL Milli-Q water. Add sucrose and mix well. Adjust pH to 5.8 using 1 M NaOH. Make up the volume to 1 L. Add 7 g of agar. Autoclave at 121 °C at 15 psi for 15 min. Cool to 40–50 °C and pour 70 mL per sterile 250 mL culture container in a laminar airflow hood.

Storage tip: Autoclaved hormone-free MS medium can be stored in the dark at room temperature (23–24 °C) for up to 3–4 weeks without compromising effectiveness.

4. MS basal medium with hormones (MS1)

Follow the same steps as for the hormone-free MS solution. After autoclaving and cooling to 40–50 °C, add 500 μL of kinetin stock (1 mg/mL) and 100 μL of IBA stock (1 mg/mL). Mix gently and pour 20 mL per sterile 120 mL culture container in a laminar airflow hood.

Storage tip: Hormone-supplemented MS medium is best used fresh or within 1–2 weeks when stored at room temperature (23–24 °C), as prolonged storage may reduce hormone activity.

Time-saving tip: Prepare hormone-free MS medium, autoclave, cool, and store at room temperature (23–24 °C) in the dark for 3–4 weeks. When needed, the stored hormone-free MS medium can be reheated in a microwave to melt the agar and poured into culture vessels. If hormone supplementation is required (e.g., kinetin or IBA), the appropriate stock solutions can be added to the melted medium once it cools slightly (to approximately 50 °C) before pouring. This approach saves time by eliminating the need to prepare fresh medium from scratch.

5. Seed sterilization solution

Prepare fresh. Add 1.25 mL of 4% bleach to 3.75 mL of sterile Milli-Q water. Add 25 μL of 10% Tween-20 to the bleach solution and mix well. This solution contains 1% sodium hypochlorite (from 4% commercial bleach) with 0.05% Tween-20, suitable for mild seed surface sterilization. Use for the 5-min treatment of seeds during sterilization.

Laboratory supplies

1. Sterile forceps

2. Scalpel

3. Petri dishes 94 mm × 16 mm (Sigma, catalog number: BR452000)

4. 1.5 mL sterile Eppendorf tubes (Sigma, catalog number: HS4323)

5. Sterile Whatman filter paper (90 mm diameter) (Sigma, catalog number: WHA1004090)

6. Wide-bore pipette tips (P1000); this can be prepared by cutting the ends of standard P1000 tips to create a larger orifice, then autoclaving for sterilization (cost-effective alternative)

7. Plant culture vessels (e.g., jars, tubes)

8. 50 mL Falcon tube Corning^® (Sigma, catalog number: CLS430829)

9. 0.2 μm syringe filter Corning^® (Sigma, catalog number: CLS431224)

Equipment

1. Laminar flow hood

2. Growth chamber or culture room with controlled temperature (27 ± 1 °C) and photoperiod (16 h light/8 h dark)

3. Light source (cool white fluorescent or LED providing 30–50 μmol·m^-2·s^-1)

4. Orbital shaker (for seed sterilization)

5. Autoclave

6. pH meter

Procedure