3. When these cells start to fall apart from the mother explant,
change to MIII plates and incubate them at 24
C with a
photoperiod of 16/8 h. In this media, the somatic embryos
will start to differentiate into a plantlet, process that takes
around 3 months (Fig. 3). Procure to transfer the globular
somatic embryos to new MIII each month, handling them
with care as this type of cells are very fragile and can be damaged very easily through manipulation with the tweezers.
4. When leaves and roots are developed (Fig. 4a), transfer the
seedlings to Magenta GA-7 Vessels with 30 mL of MIII to
allow shoot extension and plant development (Fig. 4b).
5. When plants are big enough to manipulate (3 cm long), change
them to long pots with sterilized soil. Very carefully, remove
the plant from the culture media and remove any traces of the
solid media that could be on the root.
Fig. 2 Somatic embryo development from preembryonic cells. (a) Explants after 30 days in MII plates. (b)
Detail of immature embryo development that emerges from the mother explant. (c) Detail of globular embryos.
White arrowheads indicate somatic embryos; red arrowhead indicates the mother explant
Fig. 3 Somatic embryos develop into seedlings. (a) In MIII, the somatic embryos will develop until they produce
a new seedling. (b) Amplification of an explant that produces several embryos that are developing into a
complete seedling. Line: 1 cm
Carrot Nuclear Transformation
317
change to MIII plates and incubate them at 24
C with a
photoperiod of 16/8 h. In this media, the somatic embryos
will start to differentiate into a plantlet, process that takes
around 3 months (Fig. 3). Procure to transfer the globular
somatic embryos to new MIII each month, handling them
with care as this type of cells are very fragile and can be damaged very easily through manipulation with the tweezers.
4. When leaves and roots are developed (Fig. 4a), transfer the
seedlings to Magenta GA-7 Vessels with 30 mL of MIII to
allow shoot extension and plant development (Fig. 4b).
5. When plants are big enough to manipulate (3 cm long), change
them to long pots with sterilized soil. Very carefully, remove
the plant from the culture media and remove any traces of the
solid media that could be on the root.
Fig. 2 Somatic embryo development from preembryonic cells. (a) Explants after 30 days in MII plates. (b)
Detail of immature embryo development that emerges from the mother explant. (c) Detail of globular embryos.
White arrowheads indicate somatic embryos; red arrowhead indicates the mother explant
Fig. 3 Somatic embryos develop into seedlings. (a) In MIII, the somatic embryos will develop until they produce
a new seedling. (b) Amplification of an explant that produces several embryos that are developing into a
complete seedling. Line: 1 cm
Carrot Nuclear Transformation
317