3.11 Cryopreservation of Embryonic
Axes of Recalcitrant
Seeds
1. Place five (Aesculus sp.) to ten (Quercus sp.) embryonic axes (see
Fig. 2h, i) in each 2 mL cryovial (cryovials should be properly
labelled).
2. Plunge cryovial into LN using a canister for 5 min.
3. Store the cryovial in a box for cryovials in the vapor phase of
LN. The box should be precooled in LN to avoid temperature
fluctuations (see Note 14).
3.12 Thawing
the Cryopreserved
Embryonic Axes
1. Remove cryovial from the LN. The box containing cryovials
should be kept on LN in a polystyrene foam box to avoid
heating of the other samples in the box (see Note 14).
2. Open cap of the cryovial, and place the cryopreserved embryonic axes in a beaker or polystyrene foam cup filled with
Ca/Mg solution preheated to 42 Æ 2
C in a bath.
3. Remove the embryonic axes from the warm bath, and gently
place into Ca/Mg solution for 2 min. Check with a thermometer that the temperature of the solution is around 40
C.
4. Transfer the thawed embryonic axes to a Petri dish with the
Ca/Mg solution for rehydration for 30 min in the dark.
3.13 In Vitro Culture
and Regeneration
of Embryonic Axes
1. Working under sterile conditions in the laminar flow, wash the
embryonic axes for 2 or 5 min (for Quercus sp. and Aesculus sp.,
respectively) with the sterilization medium, and rinse three
times with sterile water (60 s each rinse).
2. Plant the embryonic axes in test tubes or Petri dishes containing the proper culture medium, making sure the plumule or
shoot tip is a few millimeters above the culture medium and the
root tip is inserted in the medium.
3. The in vitro cultures are stored in the dark for 2 weeks in a light
incubator at 25
C.
4. Move the cultures into the light after 2 weeks in the dark and
checked for contamination and growth measurement.
4 Notes
1. The use of saturated salt solutions (e.g., MgCl 2 for 33% RH at
15
C [30]) or dissolutions of LiCl [31] can be an alternative to
generate an environment of 30% RH in a sealed jar when
controlled environment chambers are not available. Tables for
the relationship of RH and temperature of saturated salt solutions can be found in [30, 32].
2. The use of saturated salt solutions (e.g., Ca(NO 3 ) 2 for 51% and
56% RH at 15
C and 25
C, respectively, and NaCl for 75%
RH at 5–45
C [30]) or dissolutions of LiCl [31] can be an
alternative to generate an environment of 50–75% RH in a
Cryopreservation of Seeds and Seed Embryos
673
Axes of Recalcitrant
Seeds
1. Place five (Aesculus sp.) to ten (Quercus sp.) embryonic axes (see
Fig. 2h, i) in each 2 mL cryovial (cryovials should be properly
labelled).
2. Plunge cryovial into LN using a canister for 5 min.
3. Store the cryovial in a box for cryovials in the vapor phase of
LN. The box should be precooled in LN to avoid temperature
fluctuations (see Note 14).
3.12 Thawing
the Cryopreserved
Embryonic Axes
1. Remove cryovial from the LN. The box containing cryovials
should be kept on LN in a polystyrene foam box to avoid
heating of the other samples in the box (see Note 14).
2. Open cap of the cryovial, and place the cryopreserved embryonic axes in a beaker or polystyrene foam cup filled with
Ca/Mg solution preheated to 42 Æ 2
C in a bath.
3. Remove the embryonic axes from the warm bath, and gently
place into Ca/Mg solution for 2 min. Check with a thermometer that the temperature of the solution is around 40
C.
4. Transfer the thawed embryonic axes to a Petri dish with the
Ca/Mg solution for rehydration for 30 min in the dark.
3.13 In Vitro Culture
and Regeneration
of Embryonic Axes
1. Working under sterile conditions in the laminar flow, wash the
embryonic axes for 2 or 5 min (for Quercus sp. and Aesculus sp.,
respectively) with the sterilization medium, and rinse three
times with sterile water (60 s each rinse).
2. Plant the embryonic axes in test tubes or Petri dishes containing the proper culture medium, making sure the plumule or
shoot tip is a few millimeters above the culture medium and the
root tip is inserted in the medium.
3. The in vitro cultures are stored in the dark for 2 weeks in a light
incubator at 25
C.
4. Move the cultures into the light after 2 weeks in the dark and
checked for contamination and growth measurement.
4 Notes
1. The use of saturated salt solutions (e.g., MgCl 2 for 33% RH at
15
C [30]) or dissolutions of LiCl [31] can be an alternative to
generate an environment of 30% RH in a sealed jar when
controlled environment chambers are not available. Tables for
the relationship of RH and temperature of saturated salt solutions can be found in [30, 32].
2. The use of saturated salt solutions (e.g., Ca(NO 3 ) 2 for 51% and
56% RH at 15
C and 25
C, respectively, and NaCl for 75%
RH at 5–45
C [30]) or dissolutions of LiCl [31] can be an
alternative to generate an environment of 50–75% RH in a
Cryopreservation of Seeds and Seed Embryos
673