2.3 Cryopreservation
and Warming of
Orthodox and
Intermediate Seeds
1. Cryovials (2, 5, or 10 mL depending on the sample size, 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) or canes, depending on the cryodewar used.
2. Foil bags (e.g., 85 Â 120 mm, Moore and 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. Thirty to thirty-four stacks will fit
a large dewar.
3. Heat sealer for foil bags (e.g., heat sealer HM305CTD, Hulme
Martin).
4. Cryo-label printer (e.g., Brady BBP11, Brady, UK), Brady vial
labels (e.g., Admiral Labels & Print, Ltd, UK), 2500 per roll.
5. Cryo-dewar large/small (e.g., Archive Dewar, Biosystem
36 and Biorack 6000, Statebourne.com).
6. Cold-protective gloves (cryo-gloves) and protective glasses.
7. Controlled rate or programmable freezer (e.g., Minicool LC
40, Air Liquide) or freezing container (e.g., Nalgene
®
Mr. Frosty) (for intermediate seeds only).
8. Ultralow temperature mechanical freezer set at À80
C (e.g.,
U410, New Brunswick) (for Mr. Frosty).
9. Water bath set up at 37–40
C (for intermediate seeds only).
2.4 Excision
of Embryonic Axes
of Recalcitrant Seeds
1. Freshly harvested mature seeds (see Note 3).
2. Freshly prepared “antioxidant solution” (see Note 4); e.g.,
50 mg/L ascorbic acid prepared in water or liquid culture
medium (see Note 5).
3. Sterile blot paper.
4. Sterile tweezers.
5. Sterile scalpel.
6. 9-mm-diameter Petri dishes.
2.5 Partial and Rapid
(“Flash”) Drying
of Embryonic Axes
of Recalcitrant Seeds
1. “Flash” drier (see Figs. 1 and 2g) (see Note 6).
2. Activated silica gel cooled down to room temperature for
15 min (better if oven dried [activated] overnight before
being used in the morning) (see Note 7).
3. Forceps.
2.6 Moisture Content
Determination
1. Microbalance (e.g., XP6U/XP2U Ultra Micro Balance, Mettler Toledo).
2. Drying oven set up at 103
C.
3. Small (about 1 cm diameter) aluminum pans.
666
Daniel Ballesteros et al.
and Warming of
Orthodox and
Intermediate Seeds
1. Cryovials (2, 5, or 10 mL depending on the sample size, 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) or canes, depending on the cryodewar used.
2. Foil bags (e.g., 85 Â 120 mm, Moore and 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. Thirty to thirty-four stacks will fit
a large dewar.
3. Heat sealer for foil bags (e.g., heat sealer HM305CTD, Hulme
Martin).
4. Cryo-label printer (e.g., Brady BBP11, Brady, UK), Brady vial
labels (e.g., Admiral Labels & Print, Ltd, UK), 2500 per roll.
5. Cryo-dewar large/small (e.g., Archive Dewar, Biosystem
36 and Biorack 6000, Statebourne.com).
6. Cold-protective gloves (cryo-gloves) and protective glasses.
7. Controlled rate or programmable freezer (e.g., Minicool LC
40, Air Liquide) or freezing container (e.g., Nalgene
®
Mr. Frosty) (for intermediate seeds only).
8. Ultralow temperature mechanical freezer set at À80
C (e.g.,
U410, New Brunswick) (for Mr. Frosty).
9. Water bath set up at 37–40
C (for intermediate seeds only).
2.4 Excision
of Embryonic Axes
of Recalcitrant Seeds
1. Freshly harvested mature seeds (see Note 3).
2. Freshly prepared “antioxidant solution” (see Note 4); e.g.,
50 mg/L ascorbic acid prepared in water or liquid culture
medium (see Note 5).
3. Sterile blot paper.
4. Sterile tweezers.
5. Sterile scalpel.
6. 9-mm-diameter Petri dishes.
2.5 Partial and Rapid
(“Flash”) Drying
of Embryonic Axes
of Recalcitrant Seeds
1. “Flash” drier (see Figs. 1 and 2g) (see Note 6).
2. Activated silica gel cooled down to room temperature for
15 min (better if oven dried [activated] overnight before
being used in the morning) (see Note 7).
3. Forceps.
2.6 Moisture Content
Determination
1. Microbalance (e.g., XP6U/XP2U Ultra Micro Balance, Mettler Toledo).
2. Drying oven set up at 103
C.
3. Small (about 1 cm diameter) aluminum pans.
666
Daniel Ballesteros et al.