removing the box. This needs to be done quickly and
usually takes less than 20 s. After the box is inserted or
removed, the rack should be placed back inside the cryodewar. It is recommended to insert just one box per rack
at a time to avoid warming up the tank or the adjacent
boxes.
(b) If a sample is added or removed from a box or a cane that
already holds samples, these containers should remain in
LN or in the vapor phase of LN. This can be accomplished
by adding a 3–4 cm of LN into a polystyrene foam box, in
which the box or cane is moved from the rack or canister.
When cryovials are added or removed, the other samples
will remain in contact with LN. The use of forceps is
recommended to avoid the heat of fingers warming up
adjacent cryovials.
15. No pre-freeze step at À20
C is necessary as this may be
harmful to some seeds, such as those with high oil contents
or freeze-sensitive properties.
16. If samples are in vials, ice may have formed on the outside. This
step will prevent excess water entering the vial during thawing.
17. Desiccation sensitivity, and therefore storage behavior, can vary
considerably across accessions for Fagus sylvatica. This may be
due to maturity at collection, provenance, post-harvest
handling, genetic, or other factors [41]. Some accessions can
maintain viability of 70–80% after desiccation to equilibrium to
as low as 15% RH at 15–18
C, though typically around 50%
viability is lost after desiccation below ~47% RH (MSB unpublished data). Therefore, drying conditions of at least 47% RH
are recommended to maintain maximum viability across multiple collections.
18. The optimal hydration level for cryopreservation of Coffea seed
will vary by species but is typically between 75% and 81%
RH. This is higher than that for conventional storage at
À20
C, which varies between 43% and 50% RH. Critical
water contents for cryo-storage will increase with decreasing
temperature and may not correspond to the critical water
content for desiccation sensitivity at room temperature. Some
useful tables can be found in [14, 22].
19. Coffea species vary considerably in response to cryopreservation.
Species
with
high
cryo-survival
include
C. pseudozanguebariae, C. racemosa, and C. sessiliflora; some
survival is reported for C. arabica and C. eugenioides and no
survival for C. brevipes, C. canephora, C. liberica, and
C. stenophylla [14]. Survival of C. arabica and C. eugenioides
seeds is enhanced by slow cooling (1
C/min to À50
C)
before immersion in LN.
Cryopreservation of Seeds and Seed Embryos
677
usually takes less than 20 s. After the box is inserted or
removed, the rack should be placed back inside the cryodewar. It is recommended to insert just one box per rack
at a time to avoid warming up the tank or the adjacent
boxes.
(b) If a sample is added or removed from a box or a cane that
already holds samples, these containers should remain in
LN or in the vapor phase of LN. This can be accomplished
by adding a 3–4 cm of LN into a polystyrene foam box, in
which the box or cane is moved from the rack or canister.
When cryovials are added or removed, the other samples
will remain in contact with LN. The use of forceps is
recommended to avoid the heat of fingers warming up
adjacent cryovials.
15. No pre-freeze step at À20
C is necessary as this may be
harmful to some seeds, such as those with high oil contents
or freeze-sensitive properties.
16. If samples are in vials, ice may have formed on the outside. This
step will prevent excess water entering the vial during thawing.
17. Desiccation sensitivity, and therefore storage behavior, can vary
considerably across accessions for Fagus sylvatica. This may be
due to maturity at collection, provenance, post-harvest
handling, genetic, or other factors [41]. Some accessions can
maintain viability of 70–80% after desiccation to equilibrium to
as low as 15% RH at 15–18
C, though typically around 50%
viability is lost after desiccation below ~47% RH (MSB unpublished data). Therefore, drying conditions of at least 47% RH
are recommended to maintain maximum viability across multiple collections.
18. The optimal hydration level for cryopreservation of Coffea seed
will vary by species but is typically between 75% and 81%
RH. This is higher than that for conventional storage at
À20
C, which varies between 43% and 50% RH. Critical
water contents for cryo-storage will increase with decreasing
temperature and may not correspond to the critical water
content for desiccation sensitivity at room temperature. Some
useful tables can be found in [14, 22].
19. Coffea species vary considerably in response to cryopreservation.
Species
with
high
cryo-survival
include
C. pseudozanguebariae, C. racemosa, and C. sessiliflora; some
survival is reported for C. arabica and C. eugenioides and no
survival for C. brevipes, C. canephora, C. liberica, and
C. stenophylla [14]. Survival of C. arabica and C. eugenioides
seeds is enhanced by slow cooling (1
C/min to À50
C)
before immersion in LN.
Cryopreservation of Seeds and Seed Embryos
677