7. After about 2–3 months, fine photosynthetic cell suspension
cultures, consisting in very small cell aggregates, are obtained
(Fig. 1c). They contain chloroplasts as functional types of
plastids, as confirmed by confocal microscopy observations of
chlorophyll autofluorescence (Fig. 1f, g) and transmission electron microscopy (TEM) analyses (Fig. 1h–k). For TEM procedures, see [16].
3.2.5 Determination
of the Cell Suspension
Culture Growth Curve
To determine the growth curve (see Note 20), the fresh weight
(mg/mL) of the suspension-cultured cells can be measured at fixed
days after the onset of the subculture, using the following
procedure:
1. Weigh a 1.5 mL Eppendorf-like tube (see Note 21).
2. Open the Erlenmeyer flask containing the cell suspension culture and resuspend the flask content at least ten times, by using
the pipet-aid and a sterile disposable 10 mL pipette.
3. Transfer exactly 1 mL of the resuspended cell culture into the
tube by using a P1000 micropipette and a sterile tip with a wide
bore (see Note 22).
4. Centrifuge the tube containing the cell culture aliquot at maximum speed for 1 min at room temperature.
5. Remove most of the culture medium by using a 1 mL syringe
with a thin needle.
6. Centrifuge the tube again and remove all the remaining culture
medium as above.
7. Weigh the tube again and take note of the gross weight.
8. Subtract the tare to calculate the net weight and plot it against
time in order to visualize the growth curve of the cell suspension culture (Fig. 1l).
3.3 Establishment
and Maintenance
of Heterotrophic Cell
Suspension Cultures
3.3.1 Surface
Sterilization of Seeds,
Sowing on Petri Dishes,
and Growing of Seedlings
Surface sterilize the seeds as described in Subheading 3.2.1. Proceed with sowing as described in Subheadings 3.2.2, by transferring
the seeds on Petri dishes containing Solid Medium for Seed Germination (see Subheading 2.4). After 1 week the seeds will have
germinated and developed into small seedlings (Fig. 2a).
3.3.2 Induction
and Maintenance
of Heterotrophic Calli
1. In the laminar flow hood gently transfer the seedlings onto the
inner side of the Petri dish lid by using sterile forceps.
2. Remove the roots by using the sterile scalpel.
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Enrico Cortese et al.
cultures, consisting in very small cell aggregates, are obtained
(Fig. 1c). They contain chloroplasts as functional types of
plastids, as confirmed by confocal microscopy observations of
chlorophyll autofluorescence (Fig. 1f, g) and transmission electron microscopy (TEM) analyses (Fig. 1h–k). For TEM procedures, see [16].
3.2.5 Determination
of the Cell Suspension
Culture Growth Curve
To determine the growth curve (see Note 20), the fresh weight
(mg/mL) of the suspension-cultured cells can be measured at fixed
days after the onset of the subculture, using the following
procedure:
1. Weigh a 1.5 mL Eppendorf-like tube (see Note 21).
2. Open the Erlenmeyer flask containing the cell suspension culture and resuspend the flask content at least ten times, by using
the pipet-aid and a sterile disposable 10 mL pipette.
3. Transfer exactly 1 mL of the resuspended cell culture into the
tube by using a P1000 micropipette and a sterile tip with a wide
bore (see Note 22).
4. Centrifuge the tube containing the cell culture aliquot at maximum speed for 1 min at room temperature.
5. Remove most of the culture medium by using a 1 mL syringe
with a thin needle.
6. Centrifuge the tube again and remove all the remaining culture
medium as above.
7. Weigh the tube again and take note of the gross weight.
8. Subtract the tare to calculate the net weight and plot it against
time in order to visualize the growth curve of the cell suspension culture (Fig. 1l).
3.3 Establishment
and Maintenance
of Heterotrophic Cell
Suspension Cultures
3.3.1 Surface
Sterilization of Seeds,
Sowing on Petri Dishes,
and Growing of Seedlings
Surface sterilize the seeds as described in Subheading 3.2.1. Proceed with sowing as described in Subheadings 3.2.2, by transferring
the seeds on Petri dishes containing Solid Medium for Seed Germination (see Subheading 2.4). After 1 week the seeds will have
germinated and developed into small seedlings (Fig. 2a).
3.3.2 Induction
and Maintenance
of Heterotrophic Calli
1. In the laminar flow hood gently transfer the seedlings onto the
inner side of the Petri dish lid by using sterile forceps.
2. Remove the roots by using the sterile scalpel.
176
Enrico Cortese et al.