the same levels as orthodox seeds, exhibit anomalous longevity
responses to temperatures between +10 and À30
C, or have
short lifespans no matter how they are dried or cooled (reviewed
in [3]).
Following international standards of gene banking [6, 7],
orthodox seeds are routinely stored dry (after drying in a controlled
environment of 10–25% relative humidity [RH] at 5–20
C) in
hermetically sealed containers at À18 Æ 3
C [6]. For example, in
the Millennium Seed Bank (MSB), Royal Botanic Gardens (RBG),
Kew, seeds are routinely stored at À20 Æ 3
C after drying at 15%
RH Æ 3% at 15
C Æ 3% [8]. This standard storage method is
potentially valid for seeds of most plant species as it has been
predicted that about 90% of them may produce orthodox seeds
[9]. However, some orthodox seeds, when stored at the standard
seed storage conditions, present a short lifespan and deteriorate
faster than expected, dying in just a few years [10–12]. In addition,
intermediate seeds store poorly or do not tolerate storage at
À18 Æ 3
C (even if they can be dried to levels at which water
does not freeze), and recalcitrant seeds do not tolerate the drying
needed for safe storage at sub-zero temperatures. For all these
species, cryopreservation is recommended for their long-term
preservation [4].
In the MSB, subsamples of the seed collections of orthodoxseeded species/accessions that are relatively short-lived at standard
seed storage conditions (e.g., Salix, Populus, Primula, Anemone,
and Corylus; [11, 12]) are stored dry at ~À185
C to À190
C [i.e.,
in the vapor phase of liquid nitrogen (LN)] [12]. The drying
conditions for LN storage of orthodox seeds are slightly different
to those used for standard storage at À20
C. This consideration
has been taken to avoid overdrying problems that may arise when
seeds are dried to low moisture contents at ambient temperature
and then stored at LN temperatures ([13], see Note 9). In addition,
the MSB also stores intermediate seeds of some species at LN
temperatures. The drying levels for LN storage of intermediate
seeds are higher than those used for standard storage at À20
C
to avoid drying damage (intermediate seeds tend to present tolerances to desiccation between orthodox and recalcitrant seeds
[14, 15]), but lower than the water contents at which water can
freeze [14, 15]. For recalcitrant seeds, the cryopreservation of the
whole seed is not usually possible due to their large size (>1 cm).
Large seeds tend to be dried, cryoprotected, and cooled slower
than desired to reach the maximum survival rates [16]. In this case
the embryonic axes of the seeds are excised and cryopreserved. The
drying, cryoprotection, and cooling procedures must be adjusted,
and are often species specific [4, 6].
664
Daniel Ballesteros et al.
responses to temperatures between +10 and À30
C, or have
short lifespans no matter how they are dried or cooled (reviewed
in [3]).
Following international standards of gene banking [6, 7],
orthodox seeds are routinely stored dry (after drying in a controlled
environment of 10–25% relative humidity [RH] at 5–20
C) in
hermetically sealed containers at À18 Æ 3
C [6]. For example, in
the Millennium Seed Bank (MSB), Royal Botanic Gardens (RBG),
Kew, seeds are routinely stored at À20 Æ 3
C after drying at 15%
RH Æ 3% at 15
C Æ 3% [8]. This standard storage method is
potentially valid for seeds of most plant species as it has been
predicted that about 90% of them may produce orthodox seeds
[9]. However, some orthodox seeds, when stored at the standard
seed storage conditions, present a short lifespan and deteriorate
faster than expected, dying in just a few years [10–12]. In addition,
intermediate seeds store poorly or do not tolerate storage at
À18 Æ 3
C (even if they can be dried to levels at which water
does not freeze), and recalcitrant seeds do not tolerate the drying
needed for safe storage at sub-zero temperatures. For all these
species, cryopreservation is recommended for their long-term
preservation [4].
In the MSB, subsamples of the seed collections of orthodoxseeded species/accessions that are relatively short-lived at standard
seed storage conditions (e.g., Salix, Populus, Primula, Anemone,
and Corylus; [11, 12]) are stored dry at ~À185
C to À190
C [i.e.,
in the vapor phase of liquid nitrogen (LN)] [12]. The drying
conditions for LN storage of orthodox seeds are slightly different
to those used for standard storage at À20
C. This consideration
has been taken to avoid overdrying problems that may arise when
seeds are dried to low moisture contents at ambient temperature
and then stored at LN temperatures ([13], see Note 9). In addition,
the MSB also stores intermediate seeds of some species at LN
temperatures. The drying levels for LN storage of intermediate
seeds are higher than those used for standard storage at À20
C
to avoid drying damage (intermediate seeds tend to present tolerances to desiccation between orthodox and recalcitrant seeds
[14, 15]), but lower than the water contents at which water can
freeze [14, 15]. For recalcitrant seeds, the cryopreservation of the
whole seed is not usually possible due to their large size (>1 cm).
Large seeds tend to be dried, cryoprotected, and cooled slower
than desired to reach the maximum survival rates [16]. In this case
the embryonic axes of the seeds are excised and cryopreserved. The
drying, cryoprotection, and cooling procedures must be adjusted,
and are often species specific [4, 6].
664
Daniel Ballesteros et al.