Solanum (Potato)

- In vitro plants are initiated from nodal segments and placed in either liquid or solid media. Plantlets are cultured at 25°C with a 8hr/16hr light (55 µm/m^-2/s^-1)/dark cycle. Plantlets are subcultured for further propagation at a height of ~ 6-10 cm (3-5 weeks).

- For cryopreservation testing, shoot tips (~ 50 meristems) consisting of the apical dome and 1 adjacent leaf (0.8-1.3 mm in length) are dissected with a hypodermic needle under a dissecting microscope and placed in 2% sucrose until all meristems are harvested. Shoot tips are then placed in 0.3M sucrose-MS medium for 13-20 hr at 25°C.

- Shoot tips are then placed in 0.4M sucrose + 2M glycerol MS media for 60 minutes at 25°C. Shoot tips are next placed in PVS2 for 30 minutes*. Just prior to the 30-minute PVS2 incubation*, transfer the shoot tips + approximately 2-3 µl PVS2 to heat sterilized aluminum foil strips. The foil strips are directly immersed in LN₂ and placed into LN₂-filled, 2 ml cryovials. Samples are held in LN₂ for at least 30 minutes.

Retrieval

- Frozen vials are directly immersed into 40°C water, uncapped and 1.2M sucrose (40°C) is placed into the vial to quickly thaw the meristems. After 20 minutes, shoot tips are placed on a bead of low-melting agarose with MSTo liquid medium added to the edge of the bead and placed under low light for ~ 24-48 hr.

- Transfer shoot tips to solid media into 150 ml tubes, place under regular lighting (as described previously) and evaluate after 6-8 weeks.

* 10 shoot tips serve as non-LN₂ exposure controls and 25 shoot tips are used for LN₂ viability testing.

** Generally, one technician can manage approximately 100-200 shoot tips a day.