5. Every 3 weeks, transfer the calli into new Petri dishes, containing fresh CIM medium.
6. After the third renewal in CIM, calli will consist in big, yellow,
friable clusters of completely dedifferentiated cells, almost
indistinguishable regardless of their origin from either cotyledons or hypocotyls (Fig. 2f).
7. Fractions of these calli can be subcultured monthly onto Solid
Medium for Heterotrophic Callus Maintenance (see Subheading 2.4) (see Note 23) or used to set up heterotrophic cell
suspension cultures.
3.3.3 Initiation
and Subculturing
of Heterotrophic Cell
Suspension Cultures
1. Proceed as described in Subheading 3.2.3, using as starting
material heterotrophic calli (instead of the green ones), and the
Liquid Medium for Heterotrophic Cell Suspension Cultures,
containing 3% (w/v) sucrose (instead of progressively reduced
sucrose concentrations) (see Subheading 2.4) (Fig. 2g).
2. Place the flask on an orbital shaker set at 80 rpm with continuous motion in the growth chamber under a light intensity of
50 Æ 10 μmol photons m
À2 s
À1 .
3. Do the subculturing once a week, by transferring 1 mL PCV
into 20 mL fresh medium, containing the same percentage of
sucrose.
4. After about 2–3 months, fine heterotrophic cell suspension
cultures are obtained (Fig. 2h). They are maintained in the
darkness by wrapping the flasks in aluminum foil. They contain
amyloplasts as functional types of plastids, as confirmed by the
absence of chlorophyll autofluorescence (Fig. 2i, j), Lugol
staining of starch granules (Fig. 2k, l), and TEM analyses
(Fig. 2m–p).
3.4 Interconversion
of Photosynthetic
and Heterotrophic Cell
Suspension Cultures
Photosynthetic cell suspension cultures (see Subheading 3.2) and
heterotrophic cell suspension cultures (see Subheading 3.3) are, at a
certain degree, interconvertible. Indeed, the gradual transfer of
photosynthetic cultures to liquid culture media containing stepwise
higher sucrose concentration allows, together with the gradual
transition from a 16 h light/8 h dark photoperiod to a condition
of constant darkness, for the obtainment of heterotrophic cell
suspension cultures [6] (Fig. 3) (see Note 24). Even the transition
in the other direction, i.e., from heterotrophic to autotrophic cell
suspension cultures, has been obtained and described [5, 8].
3.4.1 Pulse Amplitude
Modulation (PAM)
Fluorometry
The photosynthetic efficiency of the cell suspension cultures growing in the presence of different sucrose concentrations can be
measured by Pulse Amplitude Modulation (PAM) analyses.
1. In the laminar flow hood transfer 3 mL of 6-day-old suspension
cell culture into a sterile disposable 6-well plate.
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Enrico Cortese et al.
6. After the third renewal in CIM, calli will consist in big, yellow,
friable clusters of completely dedifferentiated cells, almost
indistinguishable regardless of their origin from either cotyledons or hypocotyls (Fig. 2f).
7. Fractions of these calli can be subcultured monthly onto Solid
Medium for Heterotrophic Callus Maintenance (see Subheading 2.4) (see Note 23) or used to set up heterotrophic cell
suspension cultures.
3.3.3 Initiation
and Subculturing
of Heterotrophic Cell
Suspension Cultures
1. Proceed as described in Subheading 3.2.3, using as starting
material heterotrophic calli (instead of the green ones), and the
Liquid Medium for Heterotrophic Cell Suspension Cultures,
containing 3% (w/v) sucrose (instead of progressively reduced
sucrose concentrations) (see Subheading 2.4) (Fig. 2g).
2. Place the flask on an orbital shaker set at 80 rpm with continuous motion in the growth chamber under a light intensity of
50 Æ 10 μmol photons m
À2 s
À1 .
3. Do the subculturing once a week, by transferring 1 mL PCV
into 20 mL fresh medium, containing the same percentage of
sucrose.
4. After about 2–3 months, fine heterotrophic cell suspension
cultures are obtained (Fig. 2h). They are maintained in the
darkness by wrapping the flasks in aluminum foil. They contain
amyloplasts as functional types of plastids, as confirmed by the
absence of chlorophyll autofluorescence (Fig. 2i, j), Lugol
staining of starch granules (Fig. 2k, l), and TEM analyses
(Fig. 2m–p).
3.4 Interconversion
of Photosynthetic
and Heterotrophic Cell
Suspension Cultures
Photosynthetic cell suspension cultures (see Subheading 3.2) and
heterotrophic cell suspension cultures (see Subheading 3.3) are, at a
certain degree, interconvertible. Indeed, the gradual transfer of
photosynthetic cultures to liquid culture media containing stepwise
higher sucrose concentration allows, together with the gradual
transition from a 16 h light/8 h dark photoperiod to a condition
of constant darkness, for the obtainment of heterotrophic cell
suspension cultures [6] (Fig. 3) (see Note 24). Even the transition
in the other direction, i.e., from heterotrophic to autotrophic cell
suspension cultures, has been obtained and described [5, 8].
3.4.1 Pulse Amplitude
Modulation (PAM)
Fluorometry
The photosynthetic efficiency of the cell suspension cultures growing in the presence of different sucrose concentrations can be
measured by Pulse Amplitude Modulation (PAM) analyses.
1. In the laminar flow hood transfer 3 mL of 6-day-old suspension
cell culture into a sterile disposable 6-well plate.
178
Enrico Cortese et al.