Materials and reagents

Biological materials

1. Immature male inflorescences at least 10 from field plants

Reagents

1. Murashige and Skoog (MS) basal medium (Sigma, catalog number: M5524; contains MS macro and micro salts without vitamins)

2. Murashige and Skoog (MS) basal medium (Sigma, catalog number: M5519; contains MS macro, micro, and vitamins)

3. Biotin powder (Austratec, catalog number: B140)

4. 6-Benzylaminopurine (BAP) powder (Austratec, catalog number: B800)

5. 1-Naphthaleneacetic acid (NAA) powder (Austratec, catalog number: N600)

6. Indole-3-acetic acid (IAA) powder (Austratec, catalog number: I885)

7. Morel & Wetmore vitamin solution (100×) (Austratec, catalog number: M592)

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

9. Phytagel (Sigma, catalog number: P8169)

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

11. Sterile distilled or Milli-Q water

12. 1 M NaOH (Sigma, catalog number: 1091371000)

Solutions

1. Biotin stock (1 mg/mL) (see Recipes)

2. BAP stock (1 mg/mL) (see Recipes)

3. NAA stock (1 mg/mL) (see Recipes)

4. IAA stock (1 mg/mL) (see Recipes)

5. 2× Meristem formation media (see Recipes)

6. 2× Meristem proliferation media (see Recipes)

7. 2× Shoot differentiation media (see Recipes)

8. 2× Plant rooting and development media (see Recipes)

Recipes

1. Biotin stock (1 mg/mL)

Note: Biotin is only sparingly soluble in cold water. Warm the water to 40–50 °C before adding biotin to aid dissolution. Do not boil. Filter-sterilize the solution using a 0.22 μm filter. Dispense 1,000 μL aliquots into sterile 1.5 mL microfuge tubes. Store at -20 °C.

2. BAP stock (1 mg/mL)

Note: Dissolve BAP in a few drops of 1 N NaOH or ethanol before adding distilled water to make up 20 mL. Filter-sterilize using a 0.22 μm filter. Dispense 1,000 μL aliquots into sterile 1.5 mL microfuge tubes. Store at -20 °C.

3. NAA stock (1 mg/mL)

Note: Dissolve NAA in 14 mL of absolute ethanol, then add 6 mL of distilled water to reach 20 mL (final ethanol concentration: 70%). Filter sterilize (0.22 μm), aliquot at 1,000 μL, and store at -20 °C.

4. IAA stock (1 mg/mL)

Note: Dissolve IAA in 14 mL of absolute ethanol, then add 6 mL of distilled water to reach 20 mL (final ethanol concentration: 70%). Filter sterilize (0.22 μm), protect from light, aliquot at 1,000 μL, and store at -20 °C.

5. 2× Meristem formation media (1 L)

Note: When working with a new cultivar, it is recommended to try both 5 and 7 mg/L BAP concentrations to determine which is more effective for initiating meristem formation. Cytokinin responsiveness can vary between genotypes.

a. Dissolve MS powder (M5519) in 700 mL of sterile distilled water.

b. Add sucrose and mix well.

c. Add 1 mL of biotin (1 mg/mL stock) to achieve 1 mg/L final concentration.

d. Prepare two separate batches of media:

i. Add 5 mL of BAP stock (1 mg/mL) to one (for a 5 mg/L final concentration).

ii. Add 7 mL of BAP stock (1 mg/mL) to the second (for a 7 mg/L final concentration).

e. Adjust pH to 5.7 using 1 M NaOH (or HCl if needed).

f. Make up the volume to 1 L with distilled water.

g. Add 3 g of phytagel.

h. Autoclave at 121 °C, 15 psi for 15 min.

i. Cool to 50–60 °C and pour 25–30 mL per sterile 90 mm deep Petri plate under laminar airflow.

6. 2× Meristem proliferation media (1 L)

Note: As with meristem formation, when working with a new cultivar, it is recommended to try both 5 and 7 mg/L BAP concentrations, now in combination with 1 mg/L NAA, to determine the optimal hormonal balance for meristem proliferation. Auxin–cytokinin synergy can significantly influence shoot multiplication rates.

a. Dissolve MS powder (M5519) in ~700 mL of sterile distilled water.

b. Add sucrose and mix well.

c. Add 1 mL of biotin stock (1 mg/mL).

d. Prepare two separate media batches:

e. Add 5 mL of BAP stock (1 mg/mL) to one batch (for 5 mg/L BAP).

f. Add 7 mL of BAP stock (1 mg/mL) to the second batch (for 7 mg/L BAP).

g. Add 1 mL of NAA stock (1 mg/mL) to each batch.

h. Adjust pH to 5.8 using 1 M NaOH.

i. Bring volume to 1,000 mL with distilled water.

j. Add 3 g of phytagel per liter.

k. Autoclave at 121 °C, 15 psi, for 15 min.

l. Cool to 50–60 °C and pour 25–30 mL per sterile 90 mm deep Petri plate under laminar airflow.

7. 2× Shoot differentiation media (1 L)

Notes:

1. After successful meristem initiation and proliferation, the concentration of BAP is reduced from 5 or 7 mg/L to 2 mg/L in the shoot differentiation stage to encourage organized shoot development rather than excessive callus or shoot clustering.

2. High cytokinin levels (like 5–7 mg/L BAP) are beneficial for stimulating shoot bud formation, but prolonged exposure may inhibit elongation or result in vitrification. Reducing BAP supports shoot elongation and differentiation into distinct, transplantable shoots, particularly important for downstream rooting and acclimatization stages.

a. Dissolve 4.43 g of MS powder (M5519) in approximately 700 mL of sterile distilled water.

b. Add 30 g of sucrose and stir until fully dissolved.

c. Add 1 mL of biotin stock (1 mg/mL) to achieve a final concentration of 1 mg/L.

d. Add 2 mL of BAP stock (1 mg/mL) to reach a final concentration of 2 mg/L.

e. Adjust the pH of the solution to 5.8 using 1 M NaOH (or HCl if needed).

f. Make up the final volume to 1,000 mL with sterile distilled water.

g. Add 3 g of phytagel and mix well.

h. Autoclave the media at 121 °C, 15 psi, for 15 min.

i. Allow to cool to 50–60 °C.

j. Pour 25–30 mL of the media into sterile 90 mm deep Petri plates under laminar airflow.

8. 2× Plant rooting and development media (1 L)

Notes:

1. Root induction requires a hormonal environment with minimal cytokinin and low auxin, mimicking the natural physiological conditions needed for root initiation.

2. In this medium, BAP is reduced to 0.045 mg/L, and IAA is included at 0.2 mg/L, providing a hormonal balance conducive to healthy root development without excessive callusing or shoot regeneration.

3. The inclusion of Morel & Wetmore vitamins supports root formation by supplying essential cofactors. Notably, this mix contains calcium pantothenate (Vitamin B5), which enhances root development by promoting coenzyme A biosynthesis—a vital pathway for energy metabolism and cell division in developing root tissues.

4. Since IAA (Indole-3-acetic acid) is heat labile, it should indeed be added after autoclaving, once the medium has cooled to around 60 °C under sterile conditions. This helps preserve its biological activity and ensures consistent rooting results.

a. Dissolve MS powder and sucrose in approximately 700 mL of distilled water.

b. Add Morel & Wetmore Vitamin solution and BAP stock. Mix thoroughly.

c. Adjust the pH to 5.8 using 1 M NaOH or HCl.

d. Bring the volume up to 1,000 mL with distilled water.

e. Add phytagel and stir to disperse evenly.

f. Autoclave the media at 121 °C, 15 psi, for 15 min.

g. Allow the media to cool to 50–60 °C.

h. Under sterile conditions in a laminar airflow hood, add IAA stock (200 μL of 1 mg/mL) to the cooled media.

i. Mix gently and pour 70 mL per sterile 250 mL culture container under laminar airflow.

Laboratory supplies

1. Beakers (250 mL, 500 mL, 1 L)

2. Measuring cylinders (10 mL to 1 L)

3. Magnetic stirrer and stir bars (or glass rod for manual stirring)

4. Pipettes (adjustable micropipettes: 10–1,000 μL) and sterile tips

5. Syringe and 0.22 μm syringe filters (for sterilizing hormone stock solutions)

6. Sterile 250 mL culture containers or jars (with lids or breathable covers)

7. Forceps and scalpels (sterile, for explant transfer)

8. Glass or plastic media bottles (autoclavable, 1 L capacity)

9. Parafilm or sealing tape (for sealing culture vessels)

10. Lab marker pens (for labeling media and containers)

11. Autoclave-safe gloves and lab coat

12. Waste containers for biological and chemical waste

Equipment

1. Analytical balance for accurate weighing of media components

2. pH meter for adjusting medium pH

3. Autoclave for sterilizing media and reusable supplies

4. Laminar airflow hood (LAF cabinet) for aseptic operations

5. Hot plate with magnetic stirrer for dissolving components and mixing medium

6. Refrigerator/freezer (-20 °C) for storing stock solutions

7. Growth chamber or culture room for incubating cultures under controlled temperature and photoperiod

8. Water bath (optional) for gently warming stock solutions

9. Distilled or MilliQ water system for preparing media

10. Light meter (lux or PAR) (optional) to monitor light levels in culture room

Procedure