2.8 Warming
and Rehydration
of Embryonic Axes
of Recalcitrant Seeds
1. Ca/Mg solution: 0.5 mM CaCl 2 and 0.5 mM MgCl 2 [24].
2. Water bath set up at 45
C.
3. Thermometer.
4. Beaker or polystyrene foam cup.
2.9 Sterilization
of Embryonic Axes
and In Vitro Culture
1. Sterilization solution: sodium hypochlorite at 1%, prepared by
adding 200 mL of NaClO and 1 mL Tween 20 to 800 mL of
H 2 O. It can be stored in the fridge for up to 5 days.
2. McCown Woody Plant Medium (WPM) [25]: 2.3 g/L WPM
salts, 30 g/L sucrose, and MS vitamins. Adjust pH to 5.6 Æ 0.1
before autoclave and add Phytagel (2.5 g/L). Some authors
also add 0.3% charcoal to the medium when working with
Quercus sp. [26].
3. Sterile tubes (e.g., 2 cm diameter  15 cm length) and lids for
plant in vitro culture or sterile petri dishes (5–6 cm diameter).
4. Long sterile forceps.
3 Methods
3.1 Drying Orthodox
Seeds
for Cryopreservation
1. Place the seeds in an appropriately sized “Rotronic” measurement pan or another suitable open container. Seeds should be
spread and filled no more than 50% of the dish or container to
allow efficient drying.
2. Label all samples adequately, such as by placing an empty
pre-labelled cryovial in the container.
3. Orchids and other small seeds which fill less than 1 mL of a
2 mL vial can be equilibrated in a storage cryovial or a folded
paper packet prior to storage in a foil bag.
4. Measure and record the initial eRH of the seeds using a
“Rotronic” water activity sensor (see Subheading 3.2) or
another suitable hygrometer [27]. Where sample volumes are
too small to measure, a surrogate sample of seeds may be used.
5. Place orthodox seeds at 32% RH and 18
C or 24% RH and
5
C (see Note 1). This drying will be equivalent to 5–9% MC
(see Note 9 and Tables 1 and 2).
6. Measure and record eRH of the seeds during drying using a
“Rotronic” sensor (see Subheading 3.2) or another suitable
hygrometer [27]. Alternatively, other low-cost monitors of
seed moisture status can be used [28]. Most samples should
be tested every 2 weeks and very short-lived samples (e.g., Salix
sp., Populus sp., orchids and ferns) every 2 days.
Cryopreservation of Seeds and Seed Embryos
669
and Rehydration
of Embryonic Axes
of Recalcitrant Seeds
1. Ca/Mg solution: 0.5 mM CaCl 2 and 0.5 mM MgCl 2 [24].
2. Water bath set up at 45
C.
3. Thermometer.
4. Beaker or polystyrene foam cup.
2.9 Sterilization
of Embryonic Axes
and In Vitro Culture
1. Sterilization solution: sodium hypochlorite at 1%, prepared by
adding 200 mL of NaClO and 1 mL Tween 20 to 800 mL of
H 2 O. It can be stored in the fridge for up to 5 days.
2. McCown Woody Plant Medium (WPM) [25]: 2.3 g/L WPM
salts, 30 g/L sucrose, and MS vitamins. Adjust pH to 5.6 Æ 0.1
before autoclave and add Phytagel (2.5 g/L). Some authors
also add 0.3% charcoal to the medium when working with
Quercus sp. [26].
3. Sterile tubes (e.g., 2 cm diameter  15 cm length) and lids for
plant in vitro culture or sterile petri dishes (5–6 cm diameter).
4. Long sterile forceps.
3 Methods
3.1 Drying Orthodox
Seeds
for Cryopreservation
1. Place the seeds in an appropriately sized “Rotronic” measurement pan or another suitable open container. Seeds should be
spread and filled no more than 50% of the dish or container to
allow efficient drying.
2. Label all samples adequately, such as by placing an empty
pre-labelled cryovial in the container.
3. Orchids and other small seeds which fill less than 1 mL of a
2 mL vial can be equilibrated in a storage cryovial or a folded
paper packet prior to storage in a foil bag.
4. Measure and record the initial eRH of the seeds using a
“Rotronic” water activity sensor (see Subheading 3.2) or
another suitable hygrometer [27]. Where sample volumes are
too small to measure, a surrogate sample of seeds may be used.
5. Place orthodox seeds at 32% RH and 18
C or 24% RH and
5
C (see Note 1). This drying will be equivalent to 5–9% MC
(see Note 9 and Tables 1 and 2).
6. Measure and record eRH of the seeds during drying using a
“Rotronic” sensor (see Subheading 3.2) or another suitable
hygrometer [27]. Alternatively, other low-cost monitors of
seed moisture status can be used [28]. Most samples should
be tested every 2 weeks and very short-lived samples (e.g., Salix
sp., Populus sp., orchids and ferns) every 2 days.
Cryopreservation of Seeds and Seed Embryos
669