3. Solid Medium for Heterotrophic Callus Maintenance: fullstrength MS, 3% (w/v) sucrose, 0.25 μg/mL BAP, 0.5 μg/
mL 2,4-D, 0.8% (w/v) agar, pH 5.5.
First prepare a liquid culture medium containing a twofold
concentration of BAP and 2,4-D (Component A’’) and a solid
medium containing a twofold concentration of agar (Component B’’), analogously to what described in Subheading 2.3.
Combine them in equal volumes (10 mL + 10 mL) when you
prepare the plates containing agarized medium, as described in
Subheading 2.6.
4. Liquid Medium for Heterotrophic Cell Suspension Cultures:
full-strength MS, 3% (w/v) sucrose, 0.25 μg/mL BAP, 0.5 μg/
mL 2,4-D, pH 5.5.
Proceed as explained for photosynthetic cell cultures in
Subheading 2.3 but adding 30 g/L sucrose.
2.5 Preparation
of Plates Containing
Agarized Medium
for Photosynthetic
Cultures
1. By using the microwave oven at low power, melt Component B
of the Solid Medium for Green Callus Induction (see Subheading 2.3) until it is fully melted (see Note 3).
2. Close the bottle and gently shake it in order to mix its content
to homogeneity.
3. Let the medium cool down until you can easily handle the
bottle in your hands without scorching (see Note 4).
4. By using the pipet-aid and a sterile disposable 10 mL pipette
inside the laminar flow hood, transfer in round sterile Petri
dishes for cell cultures (100 mm diameter  20 mm height)
10 mL Component A and 10 mL Component B (see Note 5).
5. Gently mix the content of the Petri dish by sliding the plate on
the surface of the bench under the laminar flow hood with a
cross-like motion for 3 times.
6. Partially close the dish with its lid, placing it on top slightly
inclined and let the medium solidify (it takes about 15 min).
2.6 Preparation
of Plates Containing
Agarized Medium
for Heterotrophic
Cultures
1. For seed germination: melt the Solid Medium for Seed Germination (see Subheading 2.4) in a microwave oven and then
transfer 20 mL in a Petri dish.
2. For callus induction: transfer 10 mL of Component A’ + 10 mL
Component B’ of CIM (see Subheading 2.4) in a Petri dish and
gently mix (see Subheading 2.5).
3. For callus maintenance: transfer 10 mL of Component
A’’ + 10 mL Component B’’ of Solid Medium for Heterotrophic Callus Maintenance (see Subheading 2.4) in a Petri dish
and gently mix (see Subheading 2.5).
Cell Suspension Cultures
171
mL 2,4-D, 0.8% (w/v) agar, pH 5.5.
First prepare a liquid culture medium containing a twofold
concentration of BAP and 2,4-D (Component A’’) and a solid
medium containing a twofold concentration of agar (Component B’’), analogously to what described in Subheading 2.3.
Combine them in equal volumes (10 mL + 10 mL) when you
prepare the plates containing agarized medium, as described in
Subheading 2.6.
4. Liquid Medium for Heterotrophic Cell Suspension Cultures:
full-strength MS, 3% (w/v) sucrose, 0.25 μg/mL BAP, 0.5 μg/
mL 2,4-D, pH 5.5.
Proceed as explained for photosynthetic cell cultures in
Subheading 2.3 but adding 30 g/L sucrose.
2.5 Preparation
of Plates Containing
Agarized Medium
for Photosynthetic
Cultures
1. By using the microwave oven at low power, melt Component B
of the Solid Medium for Green Callus Induction (see Subheading 2.3) until it is fully melted (see Note 3).
2. Close the bottle and gently shake it in order to mix its content
to homogeneity.
3. Let the medium cool down until you can easily handle the
bottle in your hands without scorching (see Note 4).
4. By using the pipet-aid and a sterile disposable 10 mL pipette
inside the laminar flow hood, transfer in round sterile Petri
dishes for cell cultures (100 mm diameter  20 mm height)
10 mL Component A and 10 mL Component B (see Note 5).
5. Gently mix the content of the Petri dish by sliding the plate on
the surface of the bench under the laminar flow hood with a
cross-like motion for 3 times.
6. Partially close the dish with its lid, placing it on top slightly
inclined and let the medium solidify (it takes about 15 min).
2.6 Preparation
of Plates Containing
Agarized Medium
for Heterotrophic
Cultures
1. For seed germination: melt the Solid Medium for Seed Germination (see Subheading 2.4) in a microwave oven and then
transfer 20 mL in a Petri dish.
2. For callus induction: transfer 10 mL of Component A’ + 10 mL
Component B’ of CIM (see Subheading 2.4) in a Petri dish and
gently mix (see Subheading 2.5).
3. For callus maintenance: transfer 10 mL of Component
A’’ + 10 mL Component B’’ of Solid Medium for Heterotrophic Callus Maintenance (see Subheading 2.4) in a Petri dish
and gently mix (see Subheading 2.5).
Cell Suspension Cultures
171