pressure. Do not completely remove the lid, otherwise sterility
will be lost.
4. This is important to preserve phytohormone activity, once
Component A and B have been mixed. On the other hand,
avoid excessive cooling down, otherwise the medium will solidify again in the bottle or cause wrinkled plates.
5. If in vitro cultures need to be set up from a transgenic line, add
the proper concentration of antibiotic/herbicide in the
medium.
6. Arabidopsis seeds are kept in 1.5 mL or 2 mL Eppendorf-like
tubes, which must to be properly labeled (especially if there are
many different transgenic lines in the laboratory). For collection and preservation of seeds, see [17].
7. Erlenmeyer flasks and beakers, covered with thick aluminum
foil, are sterilized in the oven at 180
C for 4 h. Bottles, tip
boxes, cell culture media, and all other solutions (except ethanol) are sterilized in autoclave at 121
C for 20 min. Before
sterilization, apply autoclave tape on all glassware and tip
boxes: it will become black after autoclaving. Forceps and
scalpels can be wrapped in aluminum foil and sterilized in
the oven.
8. Seeds can be poured from the storage tube onto a clean filter
paper. This step is particularly useful if you need to precisely
count the seeds. To facilitate the transfer into the Eppendorflike tube, fold the piece of filter paper like a chute or bend it in a
funnel-shape manner. Alternatively, if only very few seeds are
needed, you can simply make them stick to your index finger
and then carefully release them into the Eppendorf-like tube.
Do not fill the tube with too many seeds (<0.5 cm
3 final
volume), otherwise the sterilization process may be less efficient. If a large amount of seeds needs to be surface-sterilized,
subdivide them in multiple Eppendorf-like tubes. The number
of seeds required for a single round Petri dish is usually small
(20–50 maximum).
9. Do not incubate seeds in either Seed Sterilization Solutions for
more than 5 min. Exceeding this time interval may severely
impair seed germination.
10. Try to suck the seeds in about 0.5 mL of ethanol. In this way
the seeds will not remain in ethanol for too long because
ethanol will dry out quickly.
11. The described procedure is, in our hands, the fastest and most
straightforward way to sow the seeds on Petri dishes. Less
experienced researchers may prefer to transfer the seeds individually by using a sterile toothpick, or a sterilized glass Pasteur
pipette (previously bent and sealed at the tip on a Bunsen
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Enrico Cortese et al.
will be lost.
4. This is important to preserve phytohormone activity, once
Component A and B have been mixed. On the other hand,
avoid excessive cooling down, otherwise the medium will solidify again in the bottle or cause wrinkled plates.
5. If in vitro cultures need to be set up from a transgenic line, add
the proper concentration of antibiotic/herbicide in the
medium.
6. Arabidopsis seeds are kept in 1.5 mL or 2 mL Eppendorf-like
tubes, which must to be properly labeled (especially if there are
many different transgenic lines in the laboratory). For collection and preservation of seeds, see [17].
7. Erlenmeyer flasks and beakers, covered with thick aluminum
foil, are sterilized in the oven at 180
C for 4 h. Bottles, tip
boxes, cell culture media, and all other solutions (except ethanol) are sterilized in autoclave at 121
C for 20 min. Before
sterilization, apply autoclave tape on all glassware and tip
boxes: it will become black after autoclaving. Forceps and
scalpels can be wrapped in aluminum foil and sterilized in
the oven.
8. Seeds can be poured from the storage tube onto a clean filter
paper. This step is particularly useful if you need to precisely
count the seeds. To facilitate the transfer into the Eppendorflike tube, fold the piece of filter paper like a chute or bend it in a
funnel-shape manner. Alternatively, if only very few seeds are
needed, you can simply make them stick to your index finger
and then carefully release them into the Eppendorf-like tube.
Do not fill the tube with too many seeds (<0.5 cm
3 final
volume), otherwise the sterilization process may be less efficient. If a large amount of seeds needs to be surface-sterilized,
subdivide them in multiple Eppendorf-like tubes. The number
of seeds required for a single round Petri dish is usually small
(20–50 maximum).
9. Do not incubate seeds in either Seed Sterilization Solutions for
more than 5 min. Exceeding this time interval may severely
impair seed germination.
10. Try to suck the seeds in about 0.5 mL of ethanol. In this way
the seeds will not remain in ethanol for too long because
ethanol will dry out quickly.
11. The described procedure is, in our hands, the fastest and most
straightforward way to sow the seeds on Petri dishes. Less
experienced researchers may prefer to transfer the seeds individually by using a sterile toothpick, or a sterilized glass Pasteur
pipette (previously bent and sealed at the tip on a Bunsen
182
Enrico Cortese et al.