7. Gently tap the paper sheet over the sieve to remove the spores.
It should be possible to see the spores collecting below on the
clean piece of paper (see Fig. 1f). It is usually not necessary to
use a brush as the spores should fall out easily (see Note 7).
8. Once the spores appear to have been removed (you can check
under the microscope), carefully take the paper from underneath the sieves and fold in half. Then fold the sides in to create
a packet (see Note 8).
9. If a fern spore collection is received which contains spores
mixed with frond and sporangia debris, clean them by sieving
as indicated above (see Note 9).
3.2 Fern Spore
Drying
1. Place spores in a foil lined open container for drying purposes
(any open container, ideally with antistatic properties, will
also work).
2. Drying spores at 15% RH and 15
C (e.g., the MSB drying
room) is acceptable for storage at 25
C or 4
C but may be
detrimental for sub-zero storage, particularly LN storage (see
Note 10). Therefore, spores should be dried at a higher RH,
for example, at 30% RH and 20
C in a controlled environment
chamber in an open container (see Note 1).
3. Drying and moisture content manipulation can be monitored
regularly with an RH probe or “Rotronic” (see Chapter 36).
However, based on published data [36], it is likely that spores
are sufficiently dried in 5–7 days (see Note 2).
3.3 Fern Spore
Cryopreservation
(Cooling and Warming)
1. Once the spores have been dried, they can be transferred into
containers for storage. The spores can be stored either in
cryovials (see Note 11) or foil bags (see Note 12). It is important that cryovials are at least 70% filled to prevent a large void
in the vial.
2. For small fern spore samples (e.g., those occupying less than
half 2 mL cryovial), prepare small aluminum foil or paper
packets (see Fig. 1g). These must be small enough for insertion
into the cryovials. The use of aluminum foil or paper packets
will also help to retrieve the spores from the cryovial.
3. To avoid moisture changes in the pollen samples, quickly place
a small amount of dried spores (about 5–10 mg) in an aluminum foil or paper packet (see Fig. 1g).
4. Label each cryovial or foil bags with a unique code for identification purposes.
5. Place the cryovials or foil bags in the appropriate boxes or
canisters depending on the cryo-Dewar model used.
6. For the cooling process, store these boxes/canisters directly in
LN or in the vapor phase of LN (in the storage cryo-Dewar).
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Anna Nebot et al.
It should be possible to see the spores collecting below on the
clean piece of paper (see Fig. 1f). It is usually not necessary to
use a brush as the spores should fall out easily (see Note 7).
8. Once the spores appear to have been removed (you can check
under the microscope), carefully take the paper from underneath the sieves and fold in half. Then fold the sides in to create
a packet (see Note 8).
9. If a fern spore collection is received which contains spores
mixed with frond and sporangia debris, clean them by sieving
as indicated above (see Note 9).
3.2 Fern Spore
Drying
1. Place spores in a foil lined open container for drying purposes
(any open container, ideally with antistatic properties, will
also work).
2. Drying spores at 15% RH and 15
C (e.g., the MSB drying
room) is acceptable for storage at 25
C or 4
C but may be
detrimental for sub-zero storage, particularly LN storage (see
Note 10). Therefore, spores should be dried at a higher RH,
for example, at 30% RH and 20
C in a controlled environment
chamber in an open container (see Note 1).
3. Drying and moisture content manipulation can be monitored
regularly with an RH probe or “Rotronic” (see Chapter 36).
However, based on published data [36], it is likely that spores
are sufficiently dried in 5–7 days (see Note 2).
3.3 Fern Spore
Cryopreservation
(Cooling and Warming)
1. Once the spores have been dried, they can be transferred into
containers for storage. The spores can be stored either in
cryovials (see Note 11) or foil bags (see Note 12). It is important that cryovials are at least 70% filled to prevent a large void
in the vial.
2. For small fern spore samples (e.g., those occupying less than
half 2 mL cryovial), prepare small aluminum foil or paper
packets (see Fig. 1g). These must be small enough for insertion
into the cryovials. The use of aluminum foil or paper packets
will also help to retrieve the spores from the cryovial.
3. To avoid moisture changes in the pollen samples, quickly place
a small amount of dried spores (about 5–10 mg) in an aluminum foil or paper packet (see Fig. 1g).
4. Label each cryovial or foil bags with a unique code for identification purposes.
5. Place the cryovials or foil bags in the appropriate boxes or
canisters depending on the cryo-Dewar model used.
6. For the cooling process, store these boxes/canisters directly in
LN or in the vapor phase of LN (in the storage cryo-Dewar).
628
Anna Nebot et al.