The cooling rates reached with these procedures do not seem
to affect fern spore viability. Use appropriate protective gloves
and glasses.
7. For the warming process, retrieve the cryovials or foil bags
containing spores from the boxes/canisters that are in the LN
and allow them to warm at room temperature (25 Æ 2
C) for
20–40 min before use (see Note 13).
8. Storing spores in different subsamples is recommended to
avoid the repeated freeze/thawing of the core spore collection
as this may reduce spore viability [7]. Subsamples within a
cryovial can be retrieved without warming the cryovial and
the other subsamples therein if this is held in LN in a small
Dewar, while the subsample is removed with forceps.
3.4 Drying and
Cryopreservation
(Cooling and Warming)
of DesiccationTolerant Pollen
1. Collect pollen at its optimal maturity stage (see Note 14) and
analyze the initial viability of the pollen collected (see Note 15).
2. Dry the pollen before cryo-storage. To avoid over-desiccation
during LN storage, dry it in an open container in a RH chamber set up at 30% and 20
C (see Note 1).
3. Dry pollen for 24–72 h (see Note 16).
4. For small pollen samples (see Note 17), prepare small aluminum foil packets (see Fig. 1g). These must be small enough to
insert them into the cryovials.
5. To avoid moisture changes in the pollen samples, quickly place
a small amount of dried pollen (about 5 mg) in the aluminum
foil packet and fold appropriately.
6. For large pollen samples (see Note 17), place the dried pollen
directly into cryovials. Ideally 70% of the cryovial should be
filled with pollen to prevent a large void in the vial.
7. Label each cryovial with a unique code for identification.
8. The following steps should be performed using appropriate
protective gloves and glasses.
9. For cooling, plunge the cryovials (large samples) or the foil
packets (small samples) into LN with suitable forceps in a small
Dewar for 30 s (see Fig. 1h).
10. For small samples, plunge the cryovial in the LN and fill it with
LN. Place the foil packets in the cryovial, remove any excess LN
from the cryovials, and close the cryovial rapidly.
11. Place the cryovials into the appropriate boxes or canisters
(these should be precooled in LN to avoid temperature fluctuations in the sample) (see Note 13).
12. Insert boxes/canisters in the storage cryo-Dewar in the vapor
of LN (around À180
C).
13. For the warming process, retrieve the cryovials from the cryoDewar and leave them at room temperature (25 Æ 2
C) for
20–40 min before use.
Fern Spores and Pollen Cryopreservation
629
to affect fern spore viability. Use appropriate protective gloves
and glasses.
7. For the warming process, retrieve the cryovials or foil bags
containing spores from the boxes/canisters that are in the LN
and allow them to warm at room temperature (25 Æ 2
C) for
20–40 min before use (see Note 13).
8. Storing spores in different subsamples is recommended to
avoid the repeated freeze/thawing of the core spore collection
as this may reduce spore viability [7]. Subsamples within a
cryovial can be retrieved without warming the cryovial and
the other subsamples therein if this is held in LN in a small
Dewar, while the subsample is removed with forceps.
3.4 Drying and
Cryopreservation
(Cooling and Warming)
of DesiccationTolerant Pollen
1. Collect pollen at its optimal maturity stage (see Note 14) and
analyze the initial viability of the pollen collected (see Note 15).
2. Dry the pollen before cryo-storage. To avoid over-desiccation
during LN storage, dry it in an open container in a RH chamber set up at 30% and 20
C (see Note 1).
3. Dry pollen for 24–72 h (see Note 16).
4. For small pollen samples (see Note 17), prepare small aluminum foil packets (see Fig. 1g). These must be small enough to
insert them into the cryovials.
5. To avoid moisture changes in the pollen samples, quickly place
a small amount of dried pollen (about 5 mg) in the aluminum
foil packet and fold appropriately.
6. For large pollen samples (see Note 17), place the dried pollen
directly into cryovials. Ideally 70% of the cryovial should be
filled with pollen to prevent a large void in the vial.
7. Label each cryovial with a unique code for identification.
8. The following steps should be performed using appropriate
protective gloves and glasses.
9. For cooling, plunge the cryovials (large samples) or the foil
packets (small samples) into LN with suitable forceps in a small
Dewar for 30 s (see Fig. 1h).
10. For small samples, plunge the cryovial in the LN and fill it with
LN. Place the foil packets in the cryovial, remove any excess LN
from the cryovials, and close the cryovial rapidly.
11. Place the cryovials into the appropriate boxes or canisters
(these should be precooled in LN to avoid temperature fluctuations in the sample) (see Note 13).
12. Insert boxes/canisters in the storage cryo-Dewar in the vapor
of LN (around À180
C).
13. For the warming process, retrieve the cryovials from the cryoDewar and leave them at room temperature (25 Æ 2
C) for
20–40 min before use.
Fern Spores and Pollen Cryopreservation
629