2 Materials
2.1 Growth Facilities
The establishment and maintenance of plant cell cultures requires
the availability of several in vitro growth facilities, such as a growth
chamber equipped with light/temperature control and a laminar
flow hood where all the procedures that require sterile conditions
are conducted. The growth chamber for cell cultures is set at a
temperature of 24
C and a photoperiod of 16 h light/8 h dark.
2.2 Preparation
of Phytohormones
1. 0.25 mg/mL 6-benzylaminopurine (BAP).
Slowly dissolve 25 mg of BAP in 5 mL of 0.1 N HCl, then
make up to 100 mL volume with distilled H 2 O. In the laminar
flow hood sterilize the solution by filtration through sterile
disposable 0.22 μm filter units and store at 4
C.
2. 0.5 mg/mL 2,4-dichlorophenoxyacetic acid (2,4-D).
Slowly dissolve 50 mg of 2,4-D in 5 mL of 95% (v/v)
ethanol, then make up to 100 mL volume with distilled
H 2 O. Sterilize and store as described above.
3. 2 mg/mL kinetin.
Weigh 30 mg kinetin in a 15 mL tube, dissolve in 3 mL
0.1 N KOH, then make up to 15 mL volume with distilled
H 2 O. In the laminar flow hood sterilize the solution by filtration through sterile disposable 0.22 μm filter units and store at
À20
C in 1 mL aliquots in sterile 1.5 mL Eppendorf-like
tubes.
2.3 Preparation
of Culture Media
for Photosynthetic
Cultures
Culture media are based on Murashige and Skoog (MS) basal
medium (micro- and macroelements including vitamins). Sterilize
them by autoclaving on the same day of the preparation, because
most of them contain sucrose. They can be stored at room temperature for several months (see Note 1).
1. Solid Medium for Green Callus Induction: full-strength 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.
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).
For Component A, dissolve 4.4 g of Murashige and Skoog
basal medium in a beaker containing about 800 mL of deionized H 2 O. Use a magnetic stirrer in order to ensure optimal
and rapid dissolution of the reagents. Add 30 g sucrose and stir
until completely dissolved. Adjust the pH to 5.5 using 1 N
KOH. Add deionized H 2 O and bring the volume up to 1 L in a
cylinder. Transfer the solution into a 1 L Duran bottle. Autoclave for 20 min at 121
C. After autoclaving and cooling down
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169
2.1 Growth Facilities
The establishment and maintenance of plant cell cultures requires
the availability of several in vitro growth facilities, such as a growth
chamber equipped with light/temperature control and a laminar
flow hood where all the procedures that require sterile conditions
are conducted. The growth chamber for cell cultures is set at a
temperature of 24
C and a photoperiod of 16 h light/8 h dark.
2.2 Preparation
of Phytohormones
1. 0.25 mg/mL 6-benzylaminopurine (BAP).
Slowly dissolve 25 mg of BAP in 5 mL of 0.1 N HCl, then
make up to 100 mL volume with distilled H 2 O. In the laminar
flow hood sterilize the solution by filtration through sterile
disposable 0.22 μm filter units and store at 4
C.
2. 0.5 mg/mL 2,4-dichlorophenoxyacetic acid (2,4-D).
Slowly dissolve 50 mg of 2,4-D in 5 mL of 95% (v/v)
ethanol, then make up to 100 mL volume with distilled
H 2 O. Sterilize and store as described above.
3. 2 mg/mL kinetin.
Weigh 30 mg kinetin in a 15 mL tube, dissolve in 3 mL
0.1 N KOH, then make up to 15 mL volume with distilled
H 2 O. In the laminar flow hood sterilize the solution by filtration through sterile disposable 0.22 μm filter units and store at
À20
C in 1 mL aliquots in sterile 1.5 mL Eppendorf-like
tubes.
2.3 Preparation
of Culture Media
for Photosynthetic
Cultures
Culture media are based on Murashige and Skoog (MS) basal
medium (micro- and macroelements including vitamins). Sterilize
them by autoclaving on the same day of the preparation, because
most of them contain sucrose. They can be stored at room temperature for several months (see Note 1).
1. Solid Medium for Green Callus Induction: full-strength 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.
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).
For Component A, dissolve 4.4 g of Murashige and Skoog
basal medium in a beaker containing about 800 mL of deionized H 2 O. Use a magnetic stirrer in order to ensure optimal
and rapid dissolution of the reagents. Add 30 g sucrose and stir
until completely dissolved. Adjust the pH to 5.5 using 1 N
KOH. Add deionized H 2 O and bring the volume up to 1 L in a
cylinder. Transfer the solution into a 1 L Duran bottle. Autoclave for 20 min at 121
C. After autoclaving and cooling down
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