3.6 Cryopreservation
1. Loading phase: transfer explants into Petri dish containing
6–10 mL loading solution. Incubate at room temperature for
20 min. Shoot tips of garlic and shallots are incubated on a
shaker (80–100 rpm) for 20 min. Garlic inflorescences are
incubated on a shaker (80–100 rpm) for 1 h.
2. Cryoprotection: transfer explants into 2–8 mL PVS3 solution.
Shoot tips of potato and mint are incubated at room temperature for 2 h. Shoot tips of garlic and shallots are incubated on a
shaker (80–100 rpm) for 2 h. Garlic inflorescences are incubated on a shaker (80–100 rpm) for 2.5 h.
3. For potato, mint, and shallots, droplet vitrification is used.
Therefore, prepare aluminum stripes (5 Â 20 mm) and pipette
ten droplets of 2 μL PVS3 solution on the stripes. Transfer
shoot tips into the droplets, one shoot tip for each droplet.
Transfer the aluminum stripe into the cryovials.
4. For explants derived from garlic, a vitrification approach is
used. Therefore, add 0.5 mL PVS3 solution in one cryovials
and add ten explants.
5. Close the tubes and plunge them into a Dewar vessel containing liquid nitrogen. Afterward, keep the vials above liquid
nitrogen and avoid any temperature increase!
6. Arrange cryovials for storage in the liquid nitrogen tank.
3.7 Rewarming
and Regeneration
1. Rewarm cryovials (including aluminum stripes) quickly in a
water bath at 40
C for 3–5 s (potato, mint, shallot) or for
explants derived from garlic for 2–3 min (see Note 10).
2. Washing of potato, mint, and shallot explants: open cryovials,
transfer the aluminum stripe into 4–10 mL washing solution,
remove the aluminum stripe, and incubate at room temperature for 20 min.
3. Washing of garlic explants: open cryovials, remove PVS3 solution, and replace it by 1 mL washing solution and transfer into
a Petri dishes containing 10 mL washing solution. Shake
(80–100 rpm) at room temperature for 30 min.
4. Use washing solution for garlic inflorescences for explants
derived from inflorescences and shake (80–100 rpm) at room
temperature for 30 min.
5. Remove liquid medium, place the explants shortly on a filter
paper, transfer explants to solid regeneration medium, and seal
the Petri dishes with Parafilm.
6. Allow explants to grow on the specific regeneration medium in
the dark at 25/20
C (16/8 h) for 1–2 days (mint) or 7 days
(potato, shallot, garlic) (see Note 11).
7. Thereafter, cultivate the explants at low light (16 h) at
20–25
C.
Cryopreservation of Shoot Tips
657
1. Loading phase: transfer explants into Petri dish containing
6–10 mL loading solution. Incubate at room temperature for
20 min. Shoot tips of garlic and shallots are incubated on a
shaker (80–100 rpm) for 20 min. Garlic inflorescences are
incubated on a shaker (80–100 rpm) for 1 h.
2. Cryoprotection: transfer explants into 2–8 mL PVS3 solution.
Shoot tips of potato and mint are incubated at room temperature for 2 h. Shoot tips of garlic and shallots are incubated on a
shaker (80–100 rpm) for 2 h. Garlic inflorescences are incubated on a shaker (80–100 rpm) for 2.5 h.
3. For potato, mint, and shallots, droplet vitrification is used.
Therefore, prepare aluminum stripes (5 Â 20 mm) and pipette
ten droplets of 2 μL PVS3 solution on the stripes. Transfer
shoot tips into the droplets, one shoot tip for each droplet.
Transfer the aluminum stripe into the cryovials.
4. For explants derived from garlic, a vitrification approach is
used. Therefore, add 0.5 mL PVS3 solution in one cryovials
and add ten explants.
5. Close the tubes and plunge them into a Dewar vessel containing liquid nitrogen. Afterward, keep the vials above liquid
nitrogen and avoid any temperature increase!
6. Arrange cryovials for storage in the liquid nitrogen tank.
3.7 Rewarming
and Regeneration
1. Rewarm cryovials (including aluminum stripes) quickly in a
water bath at 40
C for 3–5 s (potato, mint, shallot) or for
explants derived from garlic for 2–3 min (see Note 10).
2. Washing of potato, mint, and shallot explants: open cryovials,
transfer the aluminum stripe into 4–10 mL washing solution,
remove the aluminum stripe, and incubate at room temperature for 20 min.
3. Washing of garlic explants: open cryovials, remove PVS3 solution, and replace it by 1 mL washing solution and transfer into
a Petri dishes containing 10 mL washing solution. Shake
(80–100 rpm) at room temperature for 30 min.
4. Use washing solution for garlic inflorescences for explants
derived from inflorescences and shake (80–100 rpm) at room
temperature for 30 min.
5. Remove liquid medium, place the explants shortly on a filter
paper, transfer explants to solid regeneration medium, and seal
the Petri dishes with Parafilm.
6. Allow explants to grow on the specific regeneration medium in
the dark at 25/20
C (16/8 h) for 1–2 days (mint) or 7 days
(potato, shallot, garlic) (see Note 11).
7. Thereafter, cultivate the explants at low light (16 h) at
20–25
C.
Cryopreservation of Shoot Tips
657