3.3 Packing
and Cryopreservation
of Orthodox Seeds
1. Once equilibrated, pack and seal samples in a suitable container
(see Note 13). Screw caps onto vials or seal foil bags using a
heat sealer. This should be done quickly so moisture is maintained once samples are removed from the equilibration
chamber.
2. Place vials/bags into a suitable cryobox.
3. Wearing cryo-gloves and protective eyewear, place this box into
the appropriate rack within the cryo-storage dewar (see Note
14) and lower directly into the LN vapor phase (see Note 15).
4. Where possible, the storage of subsamples (for germination
assays or seed interchange) should be included to avoid
repeated freeze/thaw cycles on bulk collections.
3.4 Removing
Orthodox Seeds
from LN
1. Wearing cryo-gloves and protective eyewear, carefully lift the
appropriate stack, and remove the storage box, cane, cryovial,
or foil bag required, and replace the stack (see Note 14).
2. Place the cryovial into a temporary storage box containing
silica gel for around 20 min on a nearby bench to warm
under ambient conditions (~20
C) before moving to the dry
room or humidity box as appropriate (see Note 16).
3. Use samples after containers have been at ambient temperature
for about 24 h to ensure all seeds have been thawed.
3.5 Cryopreservation
of Fagus sylvatica
Whole Seeds
1. Following collection place seeds in a 47–50% RH atmosphere
at 18
C for 2–4 weeks (see Note 17). This drying step should
be kept as short as possible as seeds can deteriorate fast in this
atmosphere.
2. Monitor moisture during this period with a “Rotronic” sensor.
3. Once equilibrated, seal seeds in foil cryobags, and place into
the vapor phase of LN at ~À185
C to À190
C.
4. To thaw, remove foil bags from storage, and leave to warm in
ambient conditions for 24 h before opening.
3.6 Cryopreservation
of Whole Seed
of Coffea sp. in LN
This protocol is adapted from [14, 22].
1. Remove Coffea seed testa (endocarp).
2. Desiccate seeds between 75% and 81% RH for 3 weeks at 25
C
(see Note 18) in a humidity- and temperature-controlled
hydration cabinet or using salt solutions (see Note 2).
3. Seal seeds inside 10 mL cryovials or foil bags.
4. For seeds of C. pseudozanguebariae, C. racemosa, and
C. sessiliflora, immerse the cryovials in LN.
5. For seeds of C. arabica and C. eugenioides, precool the cryovials to À50
C at 1
C/min prior to immersion in LN (see
Note 19). For the precooling step, a controlled rate freezer or
Cryopreservation of Seeds and Seed Embryos
671
and Cryopreservation
of Orthodox Seeds
1. Once equilibrated, pack and seal samples in a suitable container
(see Note 13). Screw caps onto vials or seal foil bags using a
heat sealer. This should be done quickly so moisture is maintained once samples are removed from the equilibration
chamber.
2. Place vials/bags into a suitable cryobox.
3. Wearing cryo-gloves and protective eyewear, place this box into
the appropriate rack within the cryo-storage dewar (see Note
14) and lower directly into the LN vapor phase (see Note 15).
4. Where possible, the storage of subsamples (for germination
assays or seed interchange) should be included to avoid
repeated freeze/thaw cycles on bulk collections.
3.4 Removing
Orthodox Seeds
from LN
1. Wearing cryo-gloves and protective eyewear, carefully lift the
appropriate stack, and remove the storage box, cane, cryovial,
or foil bag required, and replace the stack (see Note 14).
2. Place the cryovial into a temporary storage box containing
silica gel for around 20 min on a nearby bench to warm
under ambient conditions (~20
C) before moving to the dry
room or humidity box as appropriate (see Note 16).
3. Use samples after containers have been at ambient temperature
for about 24 h to ensure all seeds have been thawed.
3.5 Cryopreservation
of Fagus sylvatica
Whole Seeds
1. Following collection place seeds in a 47–50% RH atmosphere
at 18
C for 2–4 weeks (see Note 17). This drying step should
be kept as short as possible as seeds can deteriorate fast in this
atmosphere.
2. Monitor moisture during this period with a “Rotronic” sensor.
3. Once equilibrated, seal seeds in foil cryobags, and place into
the vapor phase of LN at ~À185
C to À190
C.
4. To thaw, remove foil bags from storage, and leave to warm in
ambient conditions for 24 h before opening.
3.6 Cryopreservation
of Whole Seed
of Coffea sp. in LN
This protocol is adapted from [14, 22].
1. Remove Coffea seed testa (endocarp).
2. Desiccate seeds between 75% and 81% RH for 3 weeks at 25
C
(see Note 18) in a humidity- and temperature-controlled
hydration cabinet or using salt solutions (see Note 2).
3. Seal seeds inside 10 mL cryovials or foil bags.
4. For seeds of C. pseudozanguebariae, C. racemosa, and
C. sessiliflora, immerse the cryovials in LN.
5. For seeds of C. arabica and C. eugenioides, precool the cryovials to À50
C at 1
C/min prior to immersion in LN (see
Note 19). For the precooling step, a controlled rate freezer or
Cryopreservation of Seeds and Seed Embryos
671