Mentha

(Mint)

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Vitrification

References

Media Recipes

Mentha species are cryopreserved through a variety of methodologies. The Vegetative Propagation Group uses two protocols for preservation: vitrification or cold hardening combined with vitrification.

Vitrification
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- In vitro plants are initiated from nodal segments, placed on Murashige & Skoog (1962), (Mint Medium) for 10-12 weeks at 25°C with 16hr light (55µm/m^-2/s^-1)/8hr dark cycle for development of shoot tips. Shoot tips should have dark green leaf primordia tightly wrapped around the apical dome vs. an olive green, smaller leaf primordia with exposed apical dome typically described in basic botany courses. Shoot tips should be 0.5 to1 mm in length.

- Nodal segments (one shoot segments) are cut and placed on Mint Medium for an additional 2-3 days prior to excision.

- Shoot tips are excised from axillary side shoots, consisting of an apical dome and 2-3 leaf primordia, using a hypodermic needle under a dissecting microscope. Approximately 25 shoot tips are placed in 2.5 ml of 2% sucrose (25°C) for 24 hr. Shoot tips are next placed in 2.5 ml of 3M sucrose (25°C) for 24hr. Petri dish size is 35x60mm.

- Shoot tips are placed in 2.5 ml of 2M glycerol + .4 M sucrose for 2-3 hr at 22°C . Shoot tips are next placed into PVS2 for 20-30 minutes at 22°C . Within the last 5 minutes of the PVS2 incubation period, shoot tips are transferred with 2-3 µl of PVS2 onto heat sterilized aluminum foil strips. In the last minute, PVS2 is removed from the shoot tips, foil strips are folded over the shoot tips, placed in cryovials and capped. Cryovials are plunged in LN₂, being careful to not let them float.

Retrieval (do one cryovial at a time)

- A cryovial is quickly removed from LN₂ and immersed into a 40° C water bath for 3-5 seconds. Immediately uncap cryovial and place 1.2 M sucrose (22°C) inside to warm. The folded foil strip is quickly removed and placed in 3ml (12x75 mm tube) of 1.2 M sucrose (22°C) for 20 minutes.

- Shoot tips are transferred to Mint Recovery Medium, placed in the dark for 48hr followed by in dim light (27µm/m^-2/s^-1) for 72hr then to full light (55µm/m^-2/s^-1).

- Shoot tips are grown for 4 to 6 weeks for viability evaluation. Viability is defined as full development of a plantlet.

Vitrification + Cold Hardening
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- After plants have received the prescribed cold hardening period*, shoot tips are excised from the axillary side shoots which consist of an apical dome and 2-3 leaf primordia and placed on Mint Medium containing 5% DMSO and returned to a cold hardening chamber for an additional 48hr.

- Shoot tips are placed in 2.5 ml 2M glycerol + 0.4 M sucrose (0°C) for 20 minutes. Keep on ice. Petri dish size is 35x60 mm.

- Remove 2M glycerol and place 2.5 ml PVS2 (0°C) over the shoot tips and transfer into precooled cryovials** containing 1ml of PVS2 (0°C) for 20, 25, or 30 minutes (cultivar dependent). In the last minute, 0.75ml PVS2 is removed and cryovials are submerged into LN₂ (do not let them float).

*Cold Hardening: Accessions are cold hardened for 1-6 weeks under the following alternating temperature and light conditions to simulate normal environmental conditions: 8hr light (55µm/m^-2/s^-1)@ 20°C/16hr dark @ -1.0°C.

**Note: Cryorack is frozen in a block of ice prior to treatments.

Retrieval

- Cryovials are quickly removed from LN₂ and placed into a 45°C water bath for 1 minute and then transferred to a 25°C water bath for an additional 1-2 minutes to warm. Cryovials are quickly uncapped and 1.2 M sucrose (22°C) is added to the cryovial. Shoot tips are washed twice with additional 1.2 M sucrose. After 20 minutes, shoot tips and 1.2 M sucrose is poured off onto filter paper to drain and shoot tips are transferred to Mint Recovery Medium.

- Shoot tips are placed in the dark for 48hr followed by dim light (2100 lux) for 72hr then to full light (4400 lux).

- Shoot tips are grown for 4 to 6 weeks for viability evaluation. Viability is defined as full development of a plantlet.

Additional information: 10 shoot tips serve as a control after exposure to PVS2 and 20 shoot tips are used for LN₂ viability testing. The remaining shoot tips remain in long-term LN₂ storage if viability is acceptable. Generally, one technician can manage approximately 100-200 shoot tips a day.

References for Vitrification
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Towill, L.E. 1988. Survival of shoot tips from mint species after short-term exposure to cryogenic conditions. HortScience 23:839-841.
Towill, L.E. 1990. Cryopreservation of isolated mint shoot tips by vitrification. Plant Cell Reports 9:178-180.
Subculture mint accessions every 12 weeks.