of 1 mg/mL 2.4-D). Mix the media very gently to avoid
generating bubbles, and deposit 25 mL of media in each plastic, sterile petri dish and let solidify. 90 mm Petri dishes are
recommended.
4. Sterile absorbent paper: In an autoclavable flask, deposit
squares of absorbent paper of approximately 4 cm
2 . Sterilize
by autoclave and then dry in an oven at 60
C by 2 days
minimum.
5. Sterile quirurgic and glass equipment: use, preferably, a 773A
tweezers, as it is better for handling the small and thin carrot
explants. A small-blade scalpel is recommended as well. The
equipment should be sterilized at 150
C for 15 min and dried
in an oven for 2 days minimum before use.
2.2 Somatic
Embryogenesis
Induction
1. Media I: Solid MS 1Â media supplemented with 0.5 mg/L
2.4-D.
2. Media II: Solid MS 1Â media supplemented with 0.25 mg/L
2.4-D.
3. Media III plates: Solid MS 1Â media without hormones.
Melt the solid MS 1Â media and let it cool, then measure
30 mL of media inside a 50 mL, sterile plastic tube. When correspond, add 2.4-D to a final concentration of 0.5 mg/L or
0.25 mg/L. Mix the media very gently to avoid generating bubbles, and deposit the media in a plastic, sterile petri dish and let
solidify.
3 Methods
3.1 Carrot Explant
Preparation
1. Cut 2 cm-long carrot epicotyls from 3 weeks old in vitrogerminated plants and deposit in the coculture plates.
2. Incubate the explants for 3 days in dark at 24
C.
3.2 Agrobacterium
tumefaciens
Suspension
Preparation
1. Inoculate 30 mL of liquid Luria–Bertani broth supplemented
with the required selection agents of the A. tumefaciens strain
being used (see Note 3) and let the culture grown in agitation
at 28
C overnight.
2. Centrifuge the cells at 4000 Â g for 15 min at 4
C. Discard the
media and add Liquid MS 1Â until an Optical Density at
600 nm of 0.3 is reached.
3. Resuspend the pellet very gently by pipetting (see Note 4).
4. Add acetosyringone to a final concentration of 1 μM and
incubate the suspension at room temperature for 20 min (see
Note 5).
Carrot Nuclear Transformation
315
generating bubbles, and deposit 25 mL of media in each plastic, sterile petri dish and let solidify. 90 mm Petri dishes are
recommended.
4. Sterile absorbent paper: In an autoclavable flask, deposit
squares of absorbent paper of approximately 4 cm
2 . Sterilize
by autoclave and then dry in an oven at 60
C by 2 days
minimum.
5. Sterile quirurgic and glass equipment: use, preferably, a 773A
tweezers, as it is better for handling the small and thin carrot
explants. A small-blade scalpel is recommended as well. The
equipment should be sterilized at 150
C for 15 min and dried
in an oven for 2 days minimum before use.
2.2 Somatic
Embryogenesis
Induction
1. Media I: Solid MS 1Â media supplemented with 0.5 mg/L
2.4-D.
2. Media II: Solid MS 1Â media supplemented with 0.25 mg/L
2.4-D.
3. Media III plates: Solid MS 1Â media without hormones.
Melt the solid MS 1Â media and let it cool, then measure
30 mL of media inside a 50 mL, sterile plastic tube. When correspond, add 2.4-D to a final concentration of 0.5 mg/L or
0.25 mg/L. Mix the media very gently to avoid generating bubbles, and deposit the media in a plastic, sterile petri dish and let
solidify.
3 Methods
3.1 Carrot Explant
Preparation
1. Cut 2 cm-long carrot epicotyls from 3 weeks old in vitrogerminated plants and deposit in the coculture plates.
2. Incubate the explants for 3 days in dark at 24
C.
3.2 Agrobacterium
tumefaciens
Suspension
Preparation
1. Inoculate 30 mL of liquid Luria–Bertani broth supplemented
with the required selection agents of the A. tumefaciens strain
being used (see Note 3) and let the culture grown in agitation
at 28
C overnight.
2. Centrifuge the cells at 4000 Â g for 15 min at 4
C. Discard the
media and add Liquid MS 1Â until an Optical Density at
600 nm of 0.3 is reached.
3. Resuspend the pellet very gently by pipetting (see Note 4).
4. Add acetosyringone to a final concentration of 1 μM and
incubate the suspension at room temperature for 20 min (see
Note 5).
Carrot Nuclear Transformation
315