2. Begin surface sterilization by shaking the explants in 70% ethanol for 10–20 s.
3. Transfer to sodium hypochlorite solution and shake for
20–30 min. For garlic inflorescences, sterilization treatment
at maximum for 15 min.
4. For garlic bulbs, bulbils, and shallots: sterilization is carried out
under vacuum.
5. Wash explants with sterile water four to five times.
6. For garlic bulbs, bulbils, and shallots: use a dissection microscope and prepare final explants (2–3 mm diameter) by longitudinal cuts.
7. Transfer explants on the culture medium and cultivate them at
20–25
C and 16 h illumination of 60–80 μmol/m
2 /s (see
Note 6).
3.4 Culturing of In
Vitro Plants and Cold
Hardening
3.4.1 Potato In Vitro Plant
Propagation and Cold
Hardening
1. For micropropagation, apices and nodal segments of 2–3 cm
were used (see Fig. 2b).
2. Transfer to fresh culture medium (see Subheading 2.1.3) and
expose explants to a 16 h photoperiod (60 μmol/m
2
s) at 22
C
for 3–5 weeks (see Note 6).
3. Prior cryopreservation, in vitro plants are exposed to cold
hardening either at 5
C in the dark or at 22/8
C for
8/16 h day/night cycle for 1–3 weeks.
3.4.2 Mint In Vitro Plant
Propagation and Cold
Hardening
1. For micropropagation, apices and nodal segments of 2–3 cm
were used (see Fig. 2a).
2. Transfer to fresh culture medium (see Subheading 2.1.3) and
expose explants to a 16 h photoperiod (60 μmol/m
2 s) with a
day/night temperature regime of 25/20
C for 2–4 weeks (see
Note 7). Continue propagation until the required number of
plants for cryopreservation is achieved (see Note 6).
3. Prior cryopreservation, nodal segments are exposed to cold
hardening for a 16 h photoperiod (60 μmol/m
2 s) at
25/À1
C for 2 weeks.
3.4.3 Garlic and Shallot
In Vitro Plant Propagation
and Cold Hardening
1. Take bunches of multiple shoots or single bulbs from the
culture vessel and remove all dead parts (leaf sheaths) and
roots (see Fig. 2c).
2. Cut bunches longitudinally into smaller parts of 3–5 shoots.
Strong single bulbs of 2–3 mm diameter can be cut longitudinally (see Note 8).
3. Trim the shoots to a length of 1 cm and the roots.
4. Transfer the plantlets to fresh culture medium (see Subheading
2.1.3) and expose explants to a 16 h photoperiod (60 μmol/
654
Angelika Senula and Manuela Nagel
3. Transfer to sodium hypochlorite solution and shake for
20–30 min. For garlic inflorescences, sterilization treatment
at maximum for 15 min.
4. For garlic bulbs, bulbils, and shallots: sterilization is carried out
under vacuum.
5. Wash explants with sterile water four to five times.
6. For garlic bulbs, bulbils, and shallots: use a dissection microscope and prepare final explants (2–3 mm diameter) by longitudinal cuts.
7. Transfer explants on the culture medium and cultivate them at
20–25
C and 16 h illumination of 60–80 μmol/m
2 /s (see
Note 6).
3.4 Culturing of In
Vitro Plants and Cold
Hardening
3.4.1 Potato In Vitro Plant
Propagation and Cold
Hardening
1. For micropropagation, apices and nodal segments of 2–3 cm
were used (see Fig. 2b).
2. Transfer to fresh culture medium (see Subheading 2.1.3) and
expose explants to a 16 h photoperiod (60 μmol/m
2
s) at 22
C
for 3–5 weeks (see Note 6).
3. Prior cryopreservation, in vitro plants are exposed to cold
hardening either at 5
C in the dark or at 22/8
C for
8/16 h day/night cycle for 1–3 weeks.
3.4.2 Mint In Vitro Plant
Propagation and Cold
Hardening
1. For micropropagation, apices and nodal segments of 2–3 cm
were used (see Fig. 2a).
2. Transfer to fresh culture medium (see Subheading 2.1.3) and
expose explants to a 16 h photoperiod (60 μmol/m
2 s) with a
day/night temperature regime of 25/20
C for 2–4 weeks (see
Note 7). Continue propagation until the required number of
plants for cryopreservation is achieved (see Note 6).
3. Prior cryopreservation, nodal segments are exposed to cold
hardening for a 16 h photoperiod (60 μmol/m
2 s) at
25/À1
C for 2 weeks.
3.4.3 Garlic and Shallot
In Vitro Plant Propagation
and Cold Hardening
1. Take bunches of multiple shoots or single bulbs from the
culture vessel and remove all dead parts (leaf sheaths) and
roots (see Fig. 2c).
2. Cut bunches longitudinally into smaller parts of 3–5 shoots.
Strong single bulbs of 2–3 mm diameter can be cut longitudinally (see Note 8).
3. Trim the shoots to a length of 1 cm and the roots.
4. Transfer the plantlets to fresh culture medium (see Subheading
2.1.3) and expose explants to a 16 h photoperiod (60 μmol/
654
Angelika Senula and Manuela Nagel