20. The use of a Mr. Frosty is convenient when a programmable
freezer is not available. Nalgene
® Mr. Frosty containers are
inexpensive, reusable, and convenient devices for freezing
biological samples at a controlled cooling rate of À1
C/min.
To use a Mr. Frosty:
(a) Remove the tube holder and foam insert from the
Mr. Frosty.
(b) Add 100% isopropyl alcohol to the fill line.
(c) Replace tube holder.
(d) Place cryovials containing the seed samples into holes in
the tube holder.
(e) Place the Mr. Frosty in the bottom of a À80
C mechanical freezer.
(f) Leave undisturbed for 3–4 h to reach the desired temperature (À50
C).
(g) Remove frozen cryovials from the Mr. Frosty and submerge in LN.
21. For some species (e.g., C. liberica and C. stenophylla), freezing
injury was restricted to the endosperm only, with embryos still
recoverable by extraction and tissue culture (when above a
critical water content of 0.27 and 0.25 g H 2 O/g dry
weight) [14].
22. The optimal hydration level for cryopreservation of nonorthodox oily seeds will vary by species but is generally found to
correspond to the upper limit of the seed’s unfrozen water
content. For species within Citrus, this can be obtained by
equilibrating seeds at 75–85% RH and 25
C.
23. Seeds of some species, including Cuphea sp., neem (Azadirachta indica), and papaya (Carica papaya), may require a short
period of high temperatures to melt lipids after storage. This is
required to prevent imbibition damage and promote germination. In some cases, the method of slow rehydration in a high
RH atmosphere (as used for Coffea sp.) may damage the
seed [42].
24. A drying curve must be done in advance for each species/
accession to determine the appropriate drying time to reach
the target water content. Target water contents found in the
literature are species specific (range between 0.25 and 0.40 g
H 2 O/g dry weight) (see Table 3).
25. Water content determination: The water content is determined
at each drying time to find the appropriate drying time to reach
the desired water content. The fresh weight is measured with a
microbalance before the partial desiccation in the flash drier.
After drying, the embryonic axes are then placed in the drying
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Daniel Ballesteros et al.
freezer is not available. Nalgene
® Mr. Frosty containers are
inexpensive, reusable, and convenient devices for freezing
biological samples at a controlled cooling rate of À1
C/min.
To use a Mr. Frosty:
(a) Remove the tube holder and foam insert from the
Mr. Frosty.
(b) Add 100% isopropyl alcohol to the fill line.
(c) Replace tube holder.
(d) Place cryovials containing the seed samples into holes in
the tube holder.
(e) Place the Mr. Frosty in the bottom of a À80
C mechanical freezer.
(f) Leave undisturbed for 3–4 h to reach the desired temperature (À50
C).
(g) Remove frozen cryovials from the Mr. Frosty and submerge in LN.
21. For some species (e.g., C. liberica and C. stenophylla), freezing
injury was restricted to the endosperm only, with embryos still
recoverable by extraction and tissue culture (when above a
critical water content of 0.27 and 0.25 g H 2 O/g dry
weight) [14].
22. The optimal hydration level for cryopreservation of nonorthodox oily seeds will vary by species but is generally found to
correspond to the upper limit of the seed’s unfrozen water
content. For species within Citrus, this can be obtained by
equilibrating seeds at 75–85% RH and 25
C.
23. Seeds of some species, including Cuphea sp., neem (Azadirachta indica), and papaya (Carica papaya), may require a short
period of high temperatures to melt lipids after storage. This is
required to prevent imbibition damage and promote germination. In some cases, the method of slow rehydration in a high
RH atmosphere (as used for Coffea sp.) may damage the
seed [42].
24. A drying curve must be done in advance for each species/
accession to determine the appropriate drying time to reach
the target water content. Target water contents found in the
literature are species specific (range between 0.25 and 0.40 g
H 2 O/g dry weight) (see Table 3).
25. Water content determination: The water content is determined
at each drying time to find the appropriate drying time to reach
the desired water content. The fresh weight is measured with a
microbalance before the partial desiccation in the flash drier.
After drying, the embryonic axes are then placed in the drying
678
Daniel Ballesteros et al.