m
2
s) with a day/night temperature regime of 25/20
C for
6–8 weeks.
5. Check the cultures regularly and discard contaminated (see
Note 6) and hyperhydric plantlets (see Note 9).
6. Duration of a subculture depends on the genotype, and some
types will need longer time. Continue propagation until the
required number of plants for cryopreservation is achieved (see
Note 7).
7. Prior cryopreservation, in vitro plants are exposed to cold
hardening for a 16 h photoperiod (60 μmol/m
2 s) at
25/À1
C for 4 weeks.
3.5 Shoot Tip
Preparation
and Pretreatment
for Cryopreservation
3.5.1 In Vitro Plantlets
1. For potato and mint, only in vitro plants are used for the
preparation of explants for the cryopreservation. For garlic
and shallots, plants can derive from in vitro plants among
other sources.
2. Use well-developed in vitro plantlets and prepare explants
under the dissection microscope.
3. For potato: Use apical shoot tips well-developed in vitro plants
and excise explants (see Fig. 3a; 2 mm thick and 3 mm long).
4. For mint: Use axillary shoot tips from nodal segments and
excise explants of 1–2 mm (see Fig. 3b).
5. For garlic: Excise shoot tips of about 1–2 mm size (see Fig. 3c).
6. For shallot: Excise shoot tips of about 0.8–1 mm size.
7. Preculture shoot tip explants in 2 mL preculture medium (solid
or liquid). Seal Petri dishes with Parafilm and keep at 25
C for
20–24 h.
3.5.2 Garlic Bulbils,
Cloves, and Shallot Cloves
1. For garlic cloves or bulbils, excise shoot tips of about 1–2 mm
size under the dissection microscope (see Fig. 2f, i).
2. For shallot cloves, excise shoot tips of about 0.8–1 mm size
under the dissection microscope (see Fig. 2m).
3. Preculture shoot tip explants in 2 mL preculture medium (solid
or liquid). Seal Petri dishes with Parafilm and keep at 25
C for
20–24 h.
3.5.3 Garlic
Inflorescences
1. Remove the flower buds, pedicel, and longer leaf primordia and
trim the inflorescences so that the inflorescence bases remain
(see Fig. 3d).
2. Cut the inflorescence in parts of 3 Â 3 mm.
3. Transfer the inflorescence explants on the solidified “preculture
medium for garlic inflorescences.” Seal Petri dishes with Parafilm and keep for a 12 h photoperiod (60 μmol/m
2 s) at 10
C
for 1–3 days.
Cryopreservation of Shoot Tips
655
2
s) with a day/night temperature regime of 25/20
C for
6–8 weeks.
5. Check the cultures regularly and discard contaminated (see
Note 6) and hyperhydric plantlets (see Note 9).
6. Duration of a subculture depends on the genotype, and some
types will need longer time. Continue propagation until the
required number of plants for cryopreservation is achieved (see
Note 7).
7. Prior cryopreservation, in vitro plants are exposed to cold
hardening for a 16 h photoperiod (60 μmol/m
2 s) at
25/À1
C for 4 weeks.
3.5 Shoot Tip
Preparation
and Pretreatment
for Cryopreservation
3.5.1 In Vitro Plantlets
1. For potato and mint, only in vitro plants are used for the
preparation of explants for the cryopreservation. For garlic
and shallots, plants can derive from in vitro plants among
other sources.
2. Use well-developed in vitro plantlets and prepare explants
under the dissection microscope.
3. For potato: Use apical shoot tips well-developed in vitro plants
and excise explants (see Fig. 3a; 2 mm thick and 3 mm long).
4. For mint: Use axillary shoot tips from nodal segments and
excise explants of 1–2 mm (see Fig. 3b).
5. For garlic: Excise shoot tips of about 1–2 mm size (see Fig. 3c).
6. For shallot: Excise shoot tips of about 0.8–1 mm size.
7. Preculture shoot tip explants in 2 mL preculture medium (solid
or liquid). Seal Petri dishes with Parafilm and keep at 25
C for
20–24 h.
3.5.2 Garlic Bulbils,
Cloves, and Shallot Cloves
1. For garlic cloves or bulbils, excise shoot tips of about 1–2 mm
size under the dissection microscope (see Fig. 2f, i).
2. For shallot cloves, excise shoot tips of about 0.8–1 mm size
under the dissection microscope (see Fig. 2m).
3. Preculture shoot tip explants in 2 mL preculture medium (solid
or liquid). Seal Petri dishes with Parafilm and keep at 25
C for
20–24 h.
3.5.3 Garlic
Inflorescences
1. Remove the flower buds, pedicel, and longer leaf primordia and
trim the inflorescences so that the inflorescence bases remain
(see Fig. 3d).
2. Cut the inflorescence in parts of 3 Â 3 mm.
3. Transfer the inflorescence explants on the solidified “preculture
medium for garlic inflorescences.” Seal Petri dishes with Parafilm and keep for a 12 h photoperiod (60 μmol/m
2 s) at 10
C
for 1–3 days.
Cryopreservation of Shoot Tips
655