7. Quickly pass the aluminum foil lid, as well as the flask neck,
over the Bunsen burner flame (paying attention not to burn
your hands) and close the flask by wrapping the aluminum foil
around the top of the flask (Fig. 1b).
8. Place the flask on an orbital shaker set at 80 rpm with continuous motion in the growth chamber under a light intensity of
130 Æ 20 μmol photons m
À2 s
À1 (to stimulate photosynthetic
activity).
9. After a couple of days, gently resuspend the culture about ten
times, using the pipet-aid and a sterile disposable 10 mL pipette
deprived of the tip (see Note 17) to favor the fragmentation of
calli.
10. After 1 week, by using the pipet-aid and a sterile disposable
10 mL pipette without the tip, transfer all the calli in a 100 mL
sterile Erlenmeyer flask, containing 20 mL fresh medium with
the same concentration (2%, w/v) of sucrose and phytohormones (see Note 13).
3.2.4 Setup
and Subculturing
of Photosynthetic Cell
Suspension Cultures
Cell suspension cultures are renewed weekly by transferring 1 mL
packed cell volume (PCV) of late-exponential phase (7-day-old) (see
Subheading 3.2.5) cells into 20 mL fresh culture medium. Sucrose
concentration in the culture medium is gradually reduced from 2 to
1% and then to 0.5% (w/v) every 2–3 weeks, in order to stimulate
the photosynthetic activity of the cells.
1. Transfer 20 mL fresh Liquid Medium for Photosynthetic Cell
Cultures (containing stepwise 50% less sucrose content every
2–3 weeks) (see Subheading 2.3) into a sterile 100 mL Erlenmeyer flask.
2. Resuspend the content of the 7-day-old cell suspension culture
at least ten times, by using the pipet-aid and a sterile disposable
10 mL pipette, taking care not to form air bubbles in the flask.
3. Suck in the pipette about 5 mL of the culture, then position the
pipette tip against the inner bottom of the flask, slowly forcing
out only the liquid medium (the cells will remain inside the
tip). Repeat this step until you have amassed 1 mL PCV inside
the tip (see Note 18).
4. Start the new subculture by transferring 1 mL PCV in the
100 mL Erlenmeyer flask containing 20 mL fresh culture
medium prepared above (see Note 19).
5. Pipette up and down at least ten times in order to accurately
resuspend the new subculture and fully release all the cells
sticking inside the pipette.
6. Close the flask with the aluminum foil lid and place it on the
orbital shaker in the growth chamber.
Cell Suspension Cultures
175
over the Bunsen burner flame (paying attention not to burn
your hands) and close the flask by wrapping the aluminum foil
around the top of the flask (Fig. 1b).
8. Place the flask on an orbital shaker set at 80 rpm with continuous motion in the growth chamber under a light intensity of
130 Æ 20 μmol photons m
À2 s
À1 (to stimulate photosynthetic
activity).
9. After a couple of days, gently resuspend the culture about ten
times, using the pipet-aid and a sterile disposable 10 mL pipette
deprived of the tip (see Note 17) to favor the fragmentation of
calli.
10. After 1 week, by using the pipet-aid and a sterile disposable
10 mL pipette without the tip, transfer all the calli in a 100 mL
sterile Erlenmeyer flask, containing 20 mL fresh medium with
the same concentration (2%, w/v) of sucrose and phytohormones (see Note 13).
3.2.4 Setup
and Subculturing
of Photosynthetic Cell
Suspension Cultures
Cell suspension cultures are renewed weekly by transferring 1 mL
packed cell volume (PCV) of late-exponential phase (7-day-old) (see
Subheading 3.2.5) cells into 20 mL fresh culture medium. Sucrose
concentration in the culture medium is gradually reduced from 2 to
1% and then to 0.5% (w/v) every 2–3 weeks, in order to stimulate
the photosynthetic activity of the cells.
1. Transfer 20 mL fresh Liquid Medium for Photosynthetic Cell
Cultures (containing stepwise 50% less sucrose content every
2–3 weeks) (see Subheading 2.3) into a sterile 100 mL Erlenmeyer flask.
2. Resuspend the content of the 7-day-old cell suspension culture
at least ten times, by using the pipet-aid and a sterile disposable
10 mL pipette, taking care not to form air bubbles in the flask.
3. Suck in the pipette about 5 mL of the culture, then position the
pipette tip against the inner bottom of the flask, slowly forcing
out only the liquid medium (the cells will remain inside the
tip). Repeat this step until you have amassed 1 mL PCV inside
the tip (see Note 18).
4. Start the new subculture by transferring 1 mL PCV in the
100 mL Erlenmeyer flask containing 20 mL fresh culture
medium prepared above (see Note 19).
5. Pipette up and down at least ten times in order to accurately
resuspend the new subculture and fully release all the cells
sticking inside the pipette.
6. Close the flask with the aluminum foil lid and place it on the
orbital shaker in the growth chamber.
Cell Suspension Cultures
175