partially desiccated, the levels of water content reached must be
higher to those that generate desiccation damage and lower to
those in which water can freeze [17].
In this chapter we describe the protocol for fern spore collection and cryopreservation used in the Millennium Seed Bank of
Royal Botanic Gardens, Kew. In addition, two protocols for pollen
cryopreservation are described: one generic for desiccation-tolerant
pollen that can be used for multiple species (tested in our lab for
Aesculus sp., Betula sp., Camellia sinensis, Corylus sp., Hedera helix,
Ilex aquifolium, Magnolia quinquepeta, and Ravenea moorei) and
one specific for the desiccation-sensitive pollen of maize (Zea mays)
obtained from the literature [33] that could also be successful in
other desiccation-sensitive pollen from rice, rye, sugarcane, triticale, and wheat [17].
2 Materials
2.1 Fern Spore
Collection
and Cleaning
1. Plain paper (A4, A3).
2. Herbarium press.
3. Cleaning cabinet (e.g., dust hood with HEPA filter, XIT
Plus 804, Bigneat Ltd.).
4. Sieves of different pore size (106, 150, 212 μm).
5. Paint brush.
2.2 Drying of Fern
Spores and
Desiccation-Tolerant
Pollen
1. Tin foil, aluminum foil, or any other open container (ideally
with antistatic properties).
2. Humidity- and temperature-controlled hydration cabinet (e.g.,
climate chamber Memmert HPP110, Memmert GmBh) (see
Note 1).
3. 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) (see Note 2).
2.3 Cryopreservation
of Fern Spores and
Desiccation-Tolerant
Pollen
1. Aluminum foil or paper (e.g., paper from glassine bags) to
make small packets.
2. Cryovials (e.g., L-Brady) and boxes (e.g., Nalgene Cryobox
81 Â 5 mL Vials, Nalgene Cryobox 81 Â 2 mL Vials, Scientific
Laboratory Supplies Ltd, UK).
3. Foil bags (e.g., 85 Â 120 mm, Moore & Buckle Ltd, UK). Foil
bags can be placed inside a large vial box, with the vial insert
removed. One stack will fit 11 Â 2 mL vial boxes or 7 Â 5 mL
vial/foil bag boxes. 30–34 stacks will fit a large Dewar.
Fern Spores and Pollen Cryopreservation
625
higher to those that generate desiccation damage and lower to
those in which water can freeze [17].
In this chapter we describe the protocol for fern spore collection and cryopreservation used in the Millennium Seed Bank of
Royal Botanic Gardens, Kew. In addition, two protocols for pollen
cryopreservation are described: one generic for desiccation-tolerant
pollen that can be used for multiple species (tested in our lab for
Aesculus sp., Betula sp., Camellia sinensis, Corylus sp., Hedera helix,
Ilex aquifolium, Magnolia quinquepeta, and Ravenea moorei) and
one specific for the desiccation-sensitive pollen of maize (Zea mays)
obtained from the literature [33] that could also be successful in
other desiccation-sensitive pollen from rice, rye, sugarcane, triticale, and wheat [17].
2 Materials
2.1 Fern Spore
Collection
and Cleaning
1. Plain paper (A4, A3).
2. Herbarium press.
3. Cleaning cabinet (e.g., dust hood with HEPA filter, XIT
Plus 804, Bigneat Ltd.).
4. Sieves of different pore size (106, 150, 212 μm).
5. Paint brush.
2.2 Drying of Fern
Spores and
Desiccation-Tolerant
Pollen
1. Tin foil, aluminum foil, or any other open container (ideally
with antistatic properties).
2. Humidity- and temperature-controlled hydration cabinet (e.g.,
climate chamber Memmert HPP110, Memmert GmBh) (see
Note 1).
3. 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) (see Note 2).
2.3 Cryopreservation
of Fern Spores and
Desiccation-Tolerant
Pollen
1. Aluminum foil or paper (e.g., paper from glassine bags) to
make small packets.
2. Cryovials (e.g., L-Brady) and boxes (e.g., Nalgene Cryobox
81 Â 5 mL Vials, Nalgene Cryobox 81 Â 2 mL Vials, Scientific
Laboratory Supplies Ltd, UK).
3. Foil bags (e.g., 85 Â 120 mm, Moore & Buckle Ltd, UK). Foil
bags can be placed inside a large vial box, with the vial insert
removed. One stack will fit 11 Â 2 mL vial boxes or 7 Â 5 mL
vial/foil bag boxes. 30–34 stacks will fit a large Dewar.
Fern Spores and Pollen Cryopreservation
625