8. Rapid cooling of the samples can be done following several
methods, each method providing different cooling rates
[26]. For embryonic axes of recalcitrant seeds, a common
method is to enclose 5–10 embryonic axes in a cryovial and
submerge the cryovial in LN. This method allows cooling
Quercus sp. embryonic axes to about 3–8
C/s [26]. Another
method used to provide a faster cooling rate is to enclose 2–3
axes in lightweight aluminum foil packets (e.g., 2 cm  3 cm)
that are submerged with forceps in N 2 slush (formed by exposing to vacuum LN, about À210
C). This method allows cooling Quercus sp. embryonic axes to about 30–80
C/s [26].
9. As storage temperature decreases, the critical or optimal moisture content for seed storage increases, so moisture requirements for cryo-storage are typically above those recommended
for conventional storage [13]. Prior to cryo-storage, seeds
must be dried at above zero temperatures to a suitable moisture. To reach 5–12% moisture content (fresh weight basis) or
0.05–0.14 g H 2 O/g dry weight (suggested moisture content
for cryo-stored seeds [7]), orthodox seeds can be dried at 32%
RH and 18
C (as performed in the MSB, RBG Kew, UK), at
24% RH and 5
C (as performed in the National Laboratory for
Genetic Resources Preservation [NLGRP], Fort Collins,
USA), or at 32% RH and 5
C [as suggested by the Center
for Plant Conservation (CPC)]. Moisture content at 30–32%
RH and 18
C will generally range from 5% to 9% depending
on oil content (see Table 1). Drying at 24% RH and 5
C or 35%
RH and 25
C will give a similar moisture range. The NLGRP
and the CPC use these drying conditions for both conventional
and cryo-storage. These moisture conditions are compared for
Ranunculus sceleratus using the SID (http://data.kew.org/
sid/viability/mc1.jsp) equations (see Table 2). However, it
could be possible that the optimal moisture contents for seed
cryo-storage are higher than the ones indicated in this chapter.
For example, [13] suggested that the optimal water contents
for cryo-storage (0.17 g H 2 O/g dry weight) would be
obtained after equilibration at 75% RH at room temperature.
10. “Rotronic” water activity sensors provide a nondestructive,
quick method for measuring sample moisture status, making
it ideal for small collections (e.g., those of rare and threatened
species). The method measures equilibrium RH (eRH), that is,
the RH of air at equilibrium with seeds held in a sealed chamber. RH is the ratio of actual water vapor pressure to the
saturation water vapor pressure at the same temperature.
11. Always note the exact instrument used and use this again for
the same sample. The temperature of the seed sample and
“Rotronic” chamber must be equal for an accurate measurement and should always be recorded with the eRH.
Cryopreservation of Seeds and Seed Embryos
675
methods, each method providing different cooling rates
[26]. For embryonic axes of recalcitrant seeds, a common
method is to enclose 5–10 embryonic axes in a cryovial and
submerge the cryovial in LN. This method allows cooling
Quercus sp. embryonic axes to about 3–8
C/s [26]. Another
method used to provide a faster cooling rate is to enclose 2–3
axes in lightweight aluminum foil packets (e.g., 2 cm  3 cm)
that are submerged with forceps in N 2 slush (formed by exposing to vacuum LN, about À210
C). This method allows cooling Quercus sp. embryonic axes to about 30–80
C/s [26].
9. As storage temperature decreases, the critical or optimal moisture content for seed storage increases, so moisture requirements for cryo-storage are typically above those recommended
for conventional storage [13]. Prior to cryo-storage, seeds
must be dried at above zero temperatures to a suitable moisture. To reach 5–12% moisture content (fresh weight basis) or
0.05–0.14 g H 2 O/g dry weight (suggested moisture content
for cryo-stored seeds [7]), orthodox seeds can be dried at 32%
RH and 18
C (as performed in the MSB, RBG Kew, UK), at
24% RH and 5
C (as performed in the National Laboratory for
Genetic Resources Preservation [NLGRP], Fort Collins,
USA), or at 32% RH and 5
C [as suggested by the Center
for Plant Conservation (CPC)]. Moisture content at 30–32%
RH and 18
C will generally range from 5% to 9% depending
on oil content (see Table 1). Drying at 24% RH and 5
C or 35%
RH and 25
C will give a similar moisture range. The NLGRP
and the CPC use these drying conditions for both conventional
and cryo-storage. These moisture conditions are compared for
Ranunculus sceleratus using the SID (http://data.kew.org/
sid/viability/mc1.jsp) equations (see Table 2). However, it
could be possible that the optimal moisture contents for seed
cryo-storage are higher than the ones indicated in this chapter.
For example, [13] suggested that the optimal water contents
for cryo-storage (0.17 g H 2 O/g dry weight) would be
obtained after equilibration at 75% RH at room temperature.
10. “Rotronic” water activity sensors provide a nondestructive,
quick method for measuring sample moisture status, making
it ideal for small collections (e.g., those of rare and threatened
species). The method measures equilibrium RH (eRH), that is,
the RH of air at equilibrium with seeds held in a sealed chamber. RH is the ratio of actual water vapor pressure to the
saturation water vapor pressure at the same temperature.
11. Always note the exact instrument used and use this again for
the same sample. The temperature of the seed sample and
“Rotronic” chamber must be equal for an accurate measurement and should always be recorded with the eRH.
Cryopreservation of Seeds and Seed Embryos
675