11. Using glass or metal funnels for transferring spores into cryovials is much easier as it prevents static problems.
12. Using small pieces of paper folded within a foil bag is easier as it
may prevent spores being lost.
13. Ideally samples stored in the cryo-Dewar should not be
exposed to room temperature to avoid temperature fluctuations. These fluctuations can increase the temperature of the
sample and could produce negative effects in the samples,
either by ice crystallization (in the case of samples stored at
high water contents such as embryonic axes) or by lipid melting
and recrystallization cycles (in intermediate seeds, orthodox
oily seeds, and fern spores). However, as moving racks, canisters, boxes, or canes from the cryo-Dewar may be unavoidable
when collections are inserted or removed from LN, some
recommendations must be followed to avoid damage to other
collections already stored in the cryo-Dewar. These recommendations follow those suggested in the Best Practices for
Repositories of the International Society for Biological and
Environmental Repositories—ISBER [44]:
(a) If a new box or cane is introduced in the cryo-Dewar, the
rack or the canister containing other boxes or canes
should be kept just above the cryo-Dewar only for the
brief time needed to insert the box in an empty space. In
samples going into large cryo-Dewars, two people may be
needed: one holding the rack and the other inserting or
removing the box. This needs to be done quickly and
usually takes less than 20 s. After the box is inserted or
removed, the rack should be placed back inside the cryoDewar. It is recommended to insert just one box per rack
at a time to avoid warming up the tank or the adjacent
boxes.
(b) If a sample is added or removed from a box or a cane that
already holds samples, these containers should remain in
LN or in the vapor phase of LN. This can be accomplished
by adding a 3–4 cm of LN into a polystyrene foam box, in
which the box or cane is moved from the rack or canister.
When cryovials are added or removed, the other samples
will remain in contact with LN. The use of forceps is
recommended to avoid the heat of fingers warming up
adjacent cryovials.
14. There are several ways to collect fresh pollen depending on the
species. For anemophilous species, pollen can be collected
directly from the flowers by shaking the inflorescences into a
vial as soon as the anthers open. Other collection strategies
include bagging catkins before pollen is dispersed (so pollen is
collected in the bags) or cutting the catkins when the anthers’
Fern Spores and Pollen Cryopreservation
633
12. Using small pieces of paper folded within a foil bag is easier as it
may prevent spores being lost.
13. Ideally samples stored in the cryo-Dewar should not be
exposed to room temperature to avoid temperature fluctuations. These fluctuations can increase the temperature of the
sample and could produce negative effects in the samples,
either by ice crystallization (in the case of samples stored at
high water contents such as embryonic axes) or by lipid melting
and recrystallization cycles (in intermediate seeds, orthodox
oily seeds, and fern spores). However, as moving racks, canisters, boxes, or canes from the cryo-Dewar may be unavoidable
when collections are inserted or removed from LN, some
recommendations must be followed to avoid damage to other
collections already stored in the cryo-Dewar. These recommendations follow those suggested in the Best Practices for
Repositories of the International Society for Biological and
Environmental Repositories—ISBER [44]:
(a) If a new box or cane is introduced in the cryo-Dewar, the
rack or the canister containing other boxes or canes
should be kept just above the cryo-Dewar only for the
brief time needed to insert the box in an empty space. In
samples going into large cryo-Dewars, two people may be
needed: one holding the rack and the other inserting or
removing the box. This needs to be done quickly and
usually takes less than 20 s. After the box is inserted or
removed, the rack should be placed back inside the cryoDewar. It is recommended to insert just one box per rack
at a time to avoid warming up the tank or the adjacent
boxes.
(b) If a sample is added or removed from a box or a cane that
already holds samples, these containers should remain in
LN or in the vapor phase of LN. This can be accomplished
by adding a 3–4 cm of LN into a polystyrene foam box, in
which the box or cane is moved from the rack or canister.
When cryovials are added or removed, the other samples
will remain in contact with LN. The use of forceps is
recommended to avoid the heat of fingers warming up
adjacent cryovials.
14. There are several ways to collect fresh pollen depending on the
species. For anemophilous species, pollen can be collected
directly from the flowers by shaking the inflorescences into a
vial as soon as the anthers open. Other collection strategies
include bagging catkins before pollen is dispersed (so pollen is
collected in the bags) or cutting the catkins when the anthers’
Fern Spores and Pollen Cryopreservation
633