The success of orthodox seed storage at sub-zero temperatures
(À20
C and LN) is mostly based in the natural seed tolerance to
low water contents (up to 3–7% moisture content) without viability
loss [3]. At these low water contents, the water molecules in the
seed tissues are strongly bonded to macromolecules [17] and do
not freeze (i.e., do not form ice crystals) when seeds are stored at
sub-zero temperatures [18]. This water fraction is known as
“unfreezable” or “unfrozen” water [19]. Knowing the window of
water contents at which water is “unfreezable” or “unfrozen” can
be important for the development of seed cryopreservation protocols for orthodox and intermediate seeds but also for different
tissues of recalcitrant-seeded species [14, 15, 20, 21]. Differential
scanning calorimetry is commonly used to determine the maximum
moisture level of the optimal hydration window (see Chapter 10 by
Sun for more details).
In this chapter, the methods for cryopreservation of orthodox
seeds and some intermediate seeds (e.g., Fagus sylvatica) followed
in the MSB are detailed. Methods for other intermediate seeds
(e.g., Citrus sp., Coffea sp.) have been extracted from the literature
[14, 15, 22]. In addition, a method used for research at RBG Kew
that is successful in the cryopreservation of the embryonic axes of
diverse Aesculus sp. and Quercus sp. is described.
2 Materials
Please note that the equipment mentioned in this chapter is used by
the MSB and has been chosen carefully using our many years’
experience. The list of suppliers is for guidance only and does not
represent an endorsement by the Royal Botanic Gardens, Kew. The
manufacturer’s instructions must be followed when using any of
the equipment referred to in this chapter.
2.1 Drying
of Orthodox Seeds
for Cryopreservation
1. Humidity- and temperature-controlled hydration cabinet set
up at 32% RH and 18
C (e.g., climate chamber Memmert
HPP110, Memmert GmBh) (see Note 1).
2. Water activity analyzer and probe for measuring seed moisture
(henceforth called as “Rotronic,” e.g., Hygropalm 3 portable
water activity analyzer with an AW-DIO station probe,
Rotronic UK).
2.2 Drying
of Intermediate Seeds
for Cryopreservation
1. Humidity- and temperature-controlled hydration cabinets set
up at a range of 50–75% RH and 20
C (e.g., climate chamber
Memmert HPP110, Memmert GmBh) (see Note 2).
2. “Rotronic” (e.g., Hygropalm 3 portable water activity analyzer
with an AW-DIO station probe, Rotronic UK).
Cryopreservation of Seeds and Seed Embryos
665
(À20
C and LN) is mostly based in the natural seed tolerance to
low water contents (up to 3–7% moisture content) without viability
loss [3]. At these low water contents, the water molecules in the
seed tissues are strongly bonded to macromolecules [17] and do
not freeze (i.e., do not form ice crystals) when seeds are stored at
sub-zero temperatures [18]. This water fraction is known as
“unfreezable” or “unfrozen” water [19]. Knowing the window of
water contents at which water is “unfreezable” or “unfrozen” can
be important for the development of seed cryopreservation protocols for orthodox and intermediate seeds but also for different
tissues of recalcitrant-seeded species [14, 15, 20, 21]. Differential
scanning calorimetry is commonly used to determine the maximum
moisture level of the optimal hydration window (see Chapter 10 by
Sun for more details).
In this chapter, the methods for cryopreservation of orthodox
seeds and some intermediate seeds (e.g., Fagus sylvatica) followed
in the MSB are detailed. Methods for other intermediate seeds
(e.g., Citrus sp., Coffea sp.) have been extracted from the literature
[14, 15, 22]. In addition, a method used for research at RBG Kew
that is successful in the cryopreservation of the embryonic axes of
diverse Aesculus sp. and Quercus sp. is described.
2 Materials
Please note that the equipment mentioned in this chapter is used by
the MSB and has been chosen carefully using our many years’
experience. The list of suppliers is for guidance only and does not
represent an endorsement by the Royal Botanic Gardens, Kew. The
manufacturer’s instructions must be followed when using any of
the equipment referred to in this chapter.
2.1 Drying
of Orthodox Seeds
for Cryopreservation
1. Humidity- and temperature-controlled hydration cabinet set
up at 32% RH and 18
C (e.g., climate chamber Memmert
HPP110, Memmert GmBh) (see Note 1).
2. Water activity analyzer and probe for measuring seed moisture
(henceforth called as “Rotronic,” e.g., Hygropalm 3 portable
water activity analyzer with an AW-DIO station probe,
Rotronic UK).
2.2 Drying
of Intermediate Seeds
for Cryopreservation
1. Humidity- and temperature-controlled hydration cabinets set
up at a range of 50–75% RH and 20
C (e.g., climate chamber
Memmert HPP110, Memmert GmBh) (see Note 2).
2. “Rotronic” (e.g., Hygropalm 3 portable water activity analyzer
with an AW-DIO station probe, Rotronic UK).
Cryopreservation of Seeds and Seed Embryos
665