the culture medium, in the laminar flow hood add 2 mL/L
stock solutions of BAP and 2,4-D (see Subheading 2.2) in order
to obtain a 2Â concentration. Gently shake the bottle (lid
closed) to ensure accurate mixing.
For Component B, proceed as described above for Component A. After bringing the solution to pH 5.5, divide it into
two 500 mL Duran bottles and add 8 g agar directly into each
bottle (2Â concentration of agar, 16 g/L) before autoclaving
(see Note 2).
The medium for callus induction is obtained by mixing
Component A and Component B in equal volume directly in
the Petri dishes, as explained in Subheading 2.5.
2. Liquid Media for Photosynthetic Cell Suspension Cultures:
full-strength MS, variable amount (w/v) of sucrose, 0.25 μg/
mL BAP, 0.5 μg/mL 2,4-D, pH 5.5.
To set up photosynthetic cell suspension cultures, culture
media containing progressively reduced sucrose concentrations
(2%, 1%, 0.5% (w/v)) are used. To prepare them, proceed as
described above, adding the proper amount of sucrose (20 g/
L, 10 g/L, 5 g/L) depending on the desired final concentration. Stir until dissolution, bring to pH and final volume,
autoclave, and cool them down before adding 1 mL/L of the
stock solution of BAP and 2,4-D (see Subheading 2.2).
2.4 Preparation
of Culture Media
for Heterotrophic
Cultures
Culture media are based on MS and Gamborg B5 basal medium
(micro- and macroelements including vitamins).
1. Solid Medium for Seed Germination: half-strength (½) MS,
1.5% (w/v) sucrose, 0.8% (w/v) agar, pH 5.5.
Dissolve 2.2 g/L MS basal medium and 15 g/L sucrose in
distilled H 2 O, bring to pH, make up to volume, add 8 g/L agar
directly into the bottle and autoclave. Store at room
temperature.
2. Solid Callus Induction Medium (CIM) for heterotrophic cultures: 3.2 g/L Gamborg B5 basal medium, 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES), 2% (w/v) sucrose,
0.05 μg/mL kinetin, 0.5 μg/mL 2,4-D, 0.8% (w/v) agar,
pH 5.7.
To obtain this medium, first prepare a liquid medium containing a twofold concentration of phytohormones (Component A’), and a solid medium containing a twofold
concentration of agar (Component B’), analogously to what
described in Subheading 2.3. The medium for callus induction
is obtained by mixing Component A’ and Component B’ in
equal volume (10 mL + 10 mL) directly in the Petri dishes, as
described in Subheading 2.6.
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Enrico Cortese et al.
stock solutions of BAP and 2,4-D (see Subheading 2.2) in order
to obtain a 2Â concentration. Gently shake the bottle (lid
closed) to ensure accurate mixing.
For Component B, proceed as described above for Component A. After bringing the solution to pH 5.5, divide it into
two 500 mL Duran bottles and add 8 g agar directly into each
bottle (2Â concentration of agar, 16 g/L) before autoclaving
(see Note 2).
The medium for callus induction is obtained by mixing
Component A and Component B in equal volume directly in
the Petri dishes, as explained in Subheading 2.5.
2. Liquid Media for Photosynthetic Cell Suspension Cultures:
full-strength MS, variable amount (w/v) of sucrose, 0.25 μg/
mL BAP, 0.5 μg/mL 2,4-D, pH 5.5.
To set up photosynthetic cell suspension cultures, culture
media containing progressively reduced sucrose concentrations
(2%, 1%, 0.5% (w/v)) are used. To prepare them, proceed as
described above, adding the proper amount of sucrose (20 g/
L, 10 g/L, 5 g/L) depending on the desired final concentration. Stir until dissolution, bring to pH and final volume,
autoclave, and cool them down before adding 1 mL/L of the
stock solution of BAP and 2,4-D (see Subheading 2.2).
2.4 Preparation
of Culture Media
for Heterotrophic
Cultures
Culture media are based on MS and Gamborg B5 basal medium
(micro- and macroelements including vitamins).
1. Solid Medium for Seed Germination: half-strength (½) MS,
1.5% (w/v) sucrose, 0.8% (w/v) agar, pH 5.5.
Dissolve 2.2 g/L MS basal medium and 15 g/L sucrose in
distilled H 2 O, bring to pH, make up to volume, add 8 g/L agar
directly into the bottle and autoclave. Store at room
temperature.
2. Solid Callus Induction Medium (CIM) for heterotrophic cultures: 3.2 g/L Gamborg B5 basal medium, 0.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES), 2% (w/v) sucrose,
0.05 μg/mL kinetin, 0.5 μg/mL 2,4-D, 0.8% (w/v) agar,
pH 5.7.
To obtain this medium, first prepare a liquid medium containing a twofold concentration of phytohormones (Component A’), and a solid medium containing a twofold
concentration of agar (Component B’), analogously to what
described in Subheading 2.3. The medium for callus induction
is obtained by mixing Component A’ and Component B’ in
equal volume (10 mL + 10 mL) directly in the Petri dishes, as
described in Subheading 2.6.
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Enrico Cortese et al.