3.2 Establishment
and Maintenance
of Photosynthetic Cell
Suspension Cultures
3.2.1 Surface
Sterilization of Seeds
1. Transfer the seeds into a clean 1.5 or 2 mL Eppendorf-like
tube. This step can be performed on a bench, in the laboratory
(see Note 8).
2. In the laminar flow hood, slowly add 1 mL of Seed Sterilization
Solution 1 (see Subheading 2.7) to the Eppendorf-like tube
containing the seeds. Close the tube and shake it vigorously for
1 min by vortexing (see Note 9).
3. Remove the solution by using a P200 micropipette and a sterile
tip. Make sure you do not suck the seeds into the tip.
4. Add 1 mL of Seed Sterilization Solution 2 (see Subheading
2.7). Close the tube and shake it vigorously for 1 min as
above (see Note 9). Immediately proceed with sowing on
Petri dishes.
3.2.2 Sowing on Petri
Dishes and Induction
of Green Calli
1. In the laminar flow hood open a Petri dish containing Solid
Medium for Green Callus Induction (see Subheading 2.3) and
put the lid upside down.
2. Using a P1000 micropipette suck the seeds into a sterile tip (see
Note 10) and transfer them onto the inner side of the Petri
dish lid.
3. By using the same sterile tip, distribute the seeds so that they
are homogenously scattered inside the Petri dish lid. Let the
ethanol evaporate for a few min.
4. Reverse the Petri dish, containing the solid medium, upside
down over the lid, containing the seeds. Overturn the closed
Petri dish, in order to let the seeds fall onto the solid medium.
If some of them remain on the lid, gently flick the lid with your
fingers from outside to help their fall (see Note 11). If a large
amount of seeds needs to be sown, use more than one
Petri dish.
5. Seal the Petri dish with parafilm and place it horizontally, lid
side up, in the growth chamber (see Note 12). After exactly
3 weeks, well-developed green calli will have formed at the
hypocotyl level (Fig. 1a, d, e).
3.2.3 Initiation
of Photosynthetic Cell
Suspension Cultures
1. In the laminar flow hood carefully open a sterile 50 mL Erlenmeyer flask, and place the aluminum foil lid upside down.
2. Transfer in the flask, by using the pipet-aid and a sterile disposable pipette, 10 mL Liquid Medium for Cell Suspension Cultures containing 2% sucrose (see Subheading 2.3) (see Notes 5
and 13).
3. Using sterile forceps, transfer the green calli (Fig. 1a, d, e) one
by one inside the Petri dish lid (placed upside down on the
surface of the bench in the laminar flow hood).
Cell Suspension Cultures
173
and Maintenance
of Photosynthetic Cell
Suspension Cultures
3.2.1 Surface
Sterilization of Seeds
1. Transfer the seeds into a clean 1.5 or 2 mL Eppendorf-like
tube. This step can be performed on a bench, in the laboratory
(see Note 8).
2. In the laminar flow hood, slowly add 1 mL of Seed Sterilization
Solution 1 (see Subheading 2.7) to the Eppendorf-like tube
containing the seeds. Close the tube and shake it vigorously for
1 min by vortexing (see Note 9).
3. Remove the solution by using a P200 micropipette and a sterile
tip. Make sure you do not suck the seeds into the tip.
4. Add 1 mL of Seed Sterilization Solution 2 (see Subheading
2.7). Close the tube and shake it vigorously for 1 min as
above (see Note 9). Immediately proceed with sowing on
Petri dishes.
3.2.2 Sowing on Petri
Dishes and Induction
of Green Calli
1. In the laminar flow hood open a Petri dish containing Solid
Medium for Green Callus Induction (see Subheading 2.3) and
put the lid upside down.
2. Using a P1000 micropipette suck the seeds into a sterile tip (see
Note 10) and transfer them onto the inner side of the Petri
dish lid.
3. By using the same sterile tip, distribute the seeds so that they
are homogenously scattered inside the Petri dish lid. Let the
ethanol evaporate for a few min.
4. Reverse the Petri dish, containing the solid medium, upside
down over the lid, containing the seeds. Overturn the closed
Petri dish, in order to let the seeds fall onto the solid medium.
If some of them remain on the lid, gently flick the lid with your
fingers from outside to help their fall (see Note 11). If a large
amount of seeds needs to be sown, use more than one
Petri dish.
5. Seal the Petri dish with parafilm and place it horizontally, lid
side up, in the growth chamber (see Note 12). After exactly
3 weeks, well-developed green calli will have formed at the
hypocotyl level (Fig. 1a, d, e).
3.2.3 Initiation
of Photosynthetic Cell
Suspension Cultures
1. In the laminar flow hood carefully open a sterile 50 mL Erlenmeyer flask, and place the aluminum foil lid upside down.
2. Transfer in the flask, by using the pipet-aid and a sterile disposable pipette, 10 mL Liquid Medium for Cell Suspension Cultures containing 2% sucrose (see Subheading 2.3) (see Notes 5
and 13).
3. Using sterile forceps, transfer the green calli (Fig. 1a, d, e) one
by one inside the Petri dish lid (placed upside down on the
surface of the bench in the laminar flow hood).
Cell Suspension Cultures
173