a freezing container such as Mr. Frosty can be used. Follow the
instructions of the instrument or the supplier (see Note 20).
6. Thaw by plunging the cryotubes in a 40
C water bath for
2 min.
7. Place seeds in a high RH atmosphere for 24 h to allow slow
rehydration prior to placing in germination conditions (e.g.,
30
C dark) (see Note 21).
3.7 Cryopreservation
of Whole Seed of Citrus
sp. in LN
This protocol is adapted from [15].
1. Dry seeds at 75–85% RH and 25
C for a period of 4 weeks (see
Note 22) in a humidity- and temperature-controlled hydration
cabinet or using salt solutions (see Note 2).
2. Monitor moisture during this period with a “Rotronic” sensor.
3. Place seeds in 2 mL polypropylene cryotubes and close
screw cap.
4. Immerse directly in LN or in the LN vapor phase.
5. After removal from cryo-storage, place seeds immediately in a
37
C water bath for 5 min.
3.8 Melting Lipids
After Cryo-Storage
1. Expose dry seed to a heat pulse of 45
C for at least 1 h prior to
hydration (see Note 23).
2. Place directly into germination conditions.
3.9 Excision
of Embryonic Axes
(Quercus
sp. and Aesculus sp.)
1. Retrieve the seeds from cold storage and wash under the tap to
remove excess dirt.
2. Remove the pericarp and seed coat using a scalpel and forceps,
and carefully excise the embryonic axes from the cotyledon
tissue (see Fig. 2). Dissection of embryonic axes can be done
under a laminar flow hood on sterile paper for maximum sterile
conditions or, alternatively, on a lab bench previously cleaned
with ethanol at 70% or another surface disinfectant.
3. The excised embryonic axes are then placed in a Petri dish on
the sterile blot paper soaked with antioxidant solution. Embryonic axes should be used within 2 h after excision.
3.10 Rapid (Flash)
Drying of Embryonic
Axes
of Recalcitrant Seeds
1. Blot dry the embryonic axes carefully.
2. Add the activated silica gel to the flash drier.
3. Introduce the embryonic axes in the ring with a mesh attached
to both ends of the flash drier (see Fig. 1a).
4. Dry the embryonic axes in the flash drier for a certain amount
of time until water contents are in the 0.25–0.30 g H 2 O/g dry
weight range; the tested drying times in this lab range from 1 to
8 h, but in most species studied, the targeted water content is
reached between 2 and 4 h of rapid drying (see Notes 24–26).
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Daniel Ballesteros et al.
instructions of the instrument or the supplier (see Note 20).
6. Thaw by plunging the cryotubes in a 40
C water bath for
2 min.
7. Place seeds in a high RH atmosphere for 24 h to allow slow
rehydration prior to placing in germination conditions (e.g.,
30
C dark) (see Note 21).
3.7 Cryopreservation
of Whole Seed of Citrus
sp. in LN
This protocol is adapted from [15].
1. Dry seeds at 75–85% RH and 25
C for a period of 4 weeks (see
Note 22) in a humidity- and temperature-controlled hydration
cabinet or using salt solutions (see Note 2).
2. Monitor moisture during this period with a “Rotronic” sensor.
3. Place seeds in 2 mL polypropylene cryotubes and close
screw cap.
4. Immerse directly in LN or in the LN vapor phase.
5. After removal from cryo-storage, place seeds immediately in a
37
C water bath for 5 min.
3.8 Melting Lipids
After Cryo-Storage
1. Expose dry seed to a heat pulse of 45
C for at least 1 h prior to
hydration (see Note 23).
2. Place directly into germination conditions.
3.9 Excision
of Embryonic Axes
(Quercus
sp. and Aesculus sp.)
1. Retrieve the seeds from cold storage and wash under the tap to
remove excess dirt.
2. Remove the pericarp and seed coat using a scalpel and forceps,
and carefully excise the embryonic axes from the cotyledon
tissue (see Fig. 2). Dissection of embryonic axes can be done
under a laminar flow hood on sterile paper for maximum sterile
conditions or, alternatively, on a lab bench previously cleaned
with ethanol at 70% or another surface disinfectant.
3. The excised embryonic axes are then placed in a Petri dish on
the sterile blot paper soaked with antioxidant solution. Embryonic axes should be used within 2 h after excision.
3.10 Rapid (Flash)
Drying of Embryonic
Axes
of Recalcitrant Seeds
1. Blot dry the embryonic axes carefully.
2. Add the activated silica gel to the flash drier.
3. Introduce the embryonic axes in the ring with a mesh attached
to both ends of the flash drier (see Fig. 1a).
4. Dry the embryonic axes in the flash drier for a certain amount
of time until water contents are in the 0.25–0.30 g H 2 O/g dry
weight range; the tested drying times in this lab range from 1 to
8 h, but in most species studied, the targeted water content is
reached between 2 and 4 h of rapid drying (see Notes 24–26).
672
Daniel Ballesteros et al.