4. Heat sealer for foil bags (e.g., Heat Sealer HM305CTD,
Hulme Martin).
5. Large and small forceps.
6. Cryo-gloves (e.g., manufactured by Scillabub, supplied by
BOC) and face visor/safety glasses (e.g., face shield, supplied
by BOC).
7. Cryo-Dewar large/small depending on space necessities (e.g.,
Archive Dewar, Biosystem 36 and Biorack 6000, Statebourne.
com).
2.4 Cryopreservation
of
Desiccation-Sensitive
Pollen (e.g., Zea mays)
1. Pollen “flash drier” (see Note 3 and Fig. 2).
2. Aluminum foil to make small packets.
3. Cryovials (e.g., L-Brady) and boxes (e.g., Nalgene Cryobox
81 Â 5 mL Vials, Nalgene Cryobox 81 Â 2 mL Vials, Scientific
Laboratory Supplies Ltd, UK).
4. Large and small forceps.
5. Cryo-gloves (e.g., manufactured by Scillabub, supplied by
BOC) and face visor/safety glasses (e.g., face shield, supplied
by BOC).
6. Cryo-Dewar large/small depending on space necessities (e.g.,
Archive Dewar, Biosystem 36 and Biorack 6000, Statebourne.
com).
3 Methods
3.1 Fern Spore
Collection
and Cleaning
1. Collect fern fronds (leaves) at the optimal developmental stage
to obtain mature spores of the highest quality (see Note 4 and
Fig. 1aÀc).
2. Place fern fronds on a piece of white A3/4 plain paper with
sporangia facing downward and then cover with another piece
of paper on top (like a paper sheet). This can then be layered
carefully with blotting paper on the lab bench or in a herbarium
press but with a light weight (not fully pressed). Place in a dry
room for sporangia dehiscence (ideally at RH between 30%
and 60%).
3. Keep the paper with the fern fronds flat, checking daily for
spores dehiscing up to a maximum of 7 days (3 days for
chlorophyllous spores) (see Note 5).
4. When sporangia open, spores are released onto the paper, and
spores can be seen as dust on the page when lifting the frond
(see Fig. 1d).
5. Carefully move the paper sheet with the spores to a cleaning
hood (see Note 6) or other suitable space (the fern frond can be
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Anna Nebot et al.
Hulme Martin).
5. Large and small forceps.
6. Cryo-gloves (e.g., manufactured by Scillabub, supplied by
BOC) and face visor/safety glasses (e.g., face shield, supplied
by BOC).
7. Cryo-Dewar large/small depending on space necessities (e.g.,
Archive Dewar, Biosystem 36 and Biorack 6000, Statebourne.
com).
2.4 Cryopreservation
of
Desiccation-Sensitive
Pollen (e.g., Zea mays)
1. Pollen “flash drier” (see Note 3 and Fig. 2).
2. Aluminum foil to make small packets.
3. Cryovials (e.g., L-Brady) and boxes (e.g., Nalgene Cryobox
81 Â 5 mL Vials, Nalgene Cryobox 81 Â 2 mL Vials, Scientific
Laboratory Supplies Ltd, UK).
4. Large and small forceps.
5. Cryo-gloves (e.g., manufactured by Scillabub, supplied by
BOC) and face visor/safety glasses (e.g., face shield, supplied
by BOC).
6. Cryo-Dewar large/small depending on space necessities (e.g.,
Archive Dewar, Biosystem 36 and Biorack 6000, Statebourne.
com).
3 Methods
3.1 Fern Spore
Collection
and Cleaning
1. Collect fern fronds (leaves) at the optimal developmental stage
to obtain mature spores of the highest quality (see Note 4 and
Fig. 1aÀc).
2. Place fern fronds on a piece of white A3/4 plain paper with
sporangia facing downward and then cover with another piece
of paper on top (like a paper sheet). This can then be layered
carefully with blotting paper on the lab bench or in a herbarium
press but with a light weight (not fully pressed). Place in a dry
room for sporangia dehiscence (ideally at RH between 30%
and 60%).
3. Keep the paper with the fern fronds flat, checking daily for
spores dehiscing up to a maximum of 7 days (3 days for
chlorophyllous spores) (see Note 5).
4. When sporangia open, spores are released onto the paper, and
spores can be seen as dust on the page when lifting the frond
(see Fig. 1d).
5. Carefully move the paper sheet with the spores to a cleaning
hood (see Note 6) or other suitable space (the fern frond can be
626
Anna Nebot et al.