2.7 Surface
Sterilization of Seeds
and Sowing on Petri
Dishes
1. Seeds of Arabidopsis thaliana, Columbia 0 (Col-0) ecotype (see
Note 6).
2. Seed Sterilization Solution 1 (70% (v/v) ethanol, 0.05%
(v/v) Triton X-100).
3. Seed Sterilization Solution 2 (100% (v/v) ethanol).
4. Petri dishes containing Solid Medium for Green Callus Induction (see Subheading 2.3) or Solid Medium for Seed Germination (see Subheading 2.4).
2.8 Preparation
of Enzymatic Solution
and Protoplast
Resuspension Buffer
1. Enzymatic Solution: 1.5% (w/v) cellulase “Onozuka” R-10,
0.5% (w/v) Macerozyme R-10, 0.55 M mannitol in Liquid
Culture Medium (see Subheadings 2.3 and 2.4) without phytohormones. Dissolve mannitol and the enzymes in the liquid
medium and make up to volume. In the laminar flow hood
sterilize the solution through sterile disposable vacuum filtration units. Store at À20
C in 10 mL aliquots in sterile 15 mL
tubes, wrapped in aluminum foil.
2. Protoplast Resuspension Buffer: 0.55 M mannitol in Liquid
Culture Medium (see Subheadings 2.3 and 2.4) without phytohormones. Sterilize the solution in autoclave and store at
À20
C in sterile 15 mL tubes.
3 Methods
3.1 Sterility
Conditions
The maintenance of sterility at all times is a conditio sine qua non for
the establishment of in vitro cultures.
1. All appropriate manipulations must be carried out in a laminar
flow hood, using sterile disposable plasticware (pipettes, tips,
tubes, Petri dishes), sterilized glassware (Erlenmeyer flasks,
beakers, bottles), and sterilized metalware (forceps, scalpels)
(see Note 7).
2. Wash your hands and clean them with denatured alcohol before
working in the laminar flow hood, or wear gloves.
3. Wipe the bench under the laminar flow hood with denatured
alcohol before and after use.
4. Never pass over open sterile labware with your hands or arms,
when you are working in the laminar flow hood. Avoid to
speak, cough, sneeze.
5. Once a week, sterilize the environment under the laminar flow
cabinet by using the UV lamp according to manufacturer’s
instructions.
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Sterilization of Seeds
and Sowing on Petri
Dishes
1. Seeds of Arabidopsis thaliana, Columbia 0 (Col-0) ecotype (see
Note 6).
2. Seed Sterilization Solution 1 (70% (v/v) ethanol, 0.05%
(v/v) Triton X-100).
3. Seed Sterilization Solution 2 (100% (v/v) ethanol).
4. Petri dishes containing Solid Medium for Green Callus Induction (see Subheading 2.3) or Solid Medium for Seed Germination (see Subheading 2.4).
2.8 Preparation
of Enzymatic Solution
and Protoplast
Resuspension Buffer
1. Enzymatic Solution: 1.5% (w/v) cellulase “Onozuka” R-10,
0.5% (w/v) Macerozyme R-10, 0.55 M mannitol in Liquid
Culture Medium (see Subheadings 2.3 and 2.4) without phytohormones. Dissolve mannitol and the enzymes in the liquid
medium and make up to volume. In the laminar flow hood
sterilize the solution through sterile disposable vacuum filtration units. Store at À20
C in 10 mL aliquots in sterile 15 mL
tubes, wrapped in aluminum foil.
2. Protoplast Resuspension Buffer: 0.55 M mannitol in Liquid
Culture Medium (see Subheadings 2.3 and 2.4) without phytohormones. Sterilize the solution in autoclave and store at
À20
C in sterile 15 mL tubes.
3 Methods
3.1 Sterility
Conditions
The maintenance of sterility at all times is a conditio sine qua non for
the establishment of in vitro cultures.
1. All appropriate manipulations must be carried out in a laminar
flow hood, using sterile disposable plasticware (pipettes, tips,
tubes, Petri dishes), sterilized glassware (Erlenmeyer flasks,
beakers, bottles), and sterilized metalware (forceps, scalpels)
(see Note 7).
2. Wash your hands and clean them with denatured alcohol before
working in the laminar flow hood, or wear gloves.
3. Wipe the bench under the laminar flow hood with denatured
alcohol before and after use.
4. Never pass over open sterile labware with your hands or arms,
when you are working in the laminar flow hood. Avoid to
speak, cough, sneeze.
5. Once a week, sterilize the environment under the laminar flow
cabinet by using the UV lamp according to manufacturer’s
instructions.
172
Enrico Cortese et al.