hermetically sealed jar or container (e.g., electrical enclosure
box) when controlled environment chambers are not available.
Saturated salt solutions can also be used to generate RH >75%.
Tables for the relationship of RH and temperature of saturated
salt solutions can be found in [30, 32].
3. Seeds must be collected when fully mature. For example, for
Aesculus sp. and Quercus sp. seeds are collected from the
ground, right after falling from the tree. Seeds that remain a
long time after shedding on the ground can dry and increase
the chances of predation or infestation. Cryopreservation of
embryonic axes should be done on freshly harvested seeds, but
if needed, seeds of Aesculus sp. and Quercus sp. can be stored
for a few months in the dark in cold storage at 5
C in sealed
bags to avoid drying. For example, in such conditions, embryonic axes of Aesculus hippocastanum can be successfully cryopreserved after 6 months of seed storage, but the optimal
response to cryopreservation in A. hippocastanum embryonic
axes is only maintained for up to 3 months [33]. Indications for
short-term storage of Quercus sp. acorns can be found in
[34]. Sprouting seeds should not be used for cryopreservation
of the embryonic axes.
4. Several antioxidant solutions can be found in the literature,
e.g.:
(a) Filter-sterilized ascorbic acid solution (50 mg/L) [35].
(b) Citric and ascorbic acid (0.2 g/L) solution [26].
(c) A strongly reducing, high pH cathodic water produced by
electrolysis of a solution containing calcium and magnesium chloride (0.5 mM CaCl 2 ·2H 2 O and 0.5 mM
MgCl 2 ·6H 2 O) used with particular success in Strychnos
gerrardii axes [36].
5. For Aesculus sp. and Quercus sp., liquid medium containing
1/2 the macronutrients and micronutrients of MS [37] or the
macronutrients and micronutrients of the Woody Plant
Medium has been used [25].
6. Rapid (“flash”) drying using a “flash drier” has proved to be far
more effective for cryopreservation of embryonic axes of recalcitrant seeds than slow drying over silica gel [23]. Mainly, two
types of “flash” driers can be found in the literature, one that is
built in a jar with a computer fan (see Fig. 1a) [38] and one that
uses a stream of air or nitrogen gas through fish tank diffusers
(see Fig. 1b) [23, 26]. Drying of embryonic axes is also usually
done under the air flow or a laminar flow chamber [39].
7. 450 mL of activated silica gel is used in a 2 L container where
the flash drier is built (see Fig. 1a). This ratio creates a RH in the
container that is <15% during the drying time needed.
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Daniel Ballesteros et al.
box) when controlled environment chambers are not available.
Saturated salt solutions can also be used to generate RH >75%.
Tables for the relationship of RH and temperature of saturated
salt solutions can be found in [30, 32].
3. Seeds must be collected when fully mature. For example, for
Aesculus sp. and Quercus sp. seeds are collected from the
ground, right after falling from the tree. Seeds that remain a
long time after shedding on the ground can dry and increase
the chances of predation or infestation. Cryopreservation of
embryonic axes should be done on freshly harvested seeds, but
if needed, seeds of Aesculus sp. and Quercus sp. can be stored
for a few months in the dark in cold storage at 5
C in sealed
bags to avoid drying. For example, in such conditions, embryonic axes of Aesculus hippocastanum can be successfully cryopreserved after 6 months of seed storage, but the optimal
response to cryopreservation in A. hippocastanum embryonic
axes is only maintained for up to 3 months [33]. Indications for
short-term storage of Quercus sp. acorns can be found in
[34]. Sprouting seeds should not be used for cryopreservation
of the embryonic axes.
4. Several antioxidant solutions can be found in the literature,
e.g.:
(a) Filter-sterilized ascorbic acid solution (50 mg/L) [35].
(b) Citric and ascorbic acid (0.2 g/L) solution [26].
(c) A strongly reducing, high pH cathodic water produced by
electrolysis of a solution containing calcium and magnesium chloride (0.5 mM CaCl 2 ·2H 2 O and 0.5 mM
MgCl 2 ·6H 2 O) used with particular success in Strychnos
gerrardii axes [36].
5. For Aesculus sp. and Quercus sp., liquid medium containing
1/2 the macronutrients and micronutrients of MS [37] or the
macronutrients and micronutrients of the Woody Plant
Medium has been used [25].
6. Rapid (“flash”) drying using a “flash drier” has proved to be far
more effective for cryopreservation of embryonic axes of recalcitrant seeds than slow drying over silica gel [23]. Mainly, two
types of “flash” driers can be found in the literature, one that is
built in a jar with a computer fan (see Fig. 1a) [38] and one that
uses a stream of air or nitrogen gas through fish tank diffusers
(see Fig. 1b) [23, 26]. Drying of embryonic axes is also usually
done under the air flow or a laminar flow chamber [39].
7. 450 mL of activated silica gel is used in a 2 L container where
the flash drier is built (see Fig. 1a). This ratio creates a RH in the
container that is <15% during the drying time needed.
674
Daniel Ballesteros et al.