Chapter 36
Cryopreservation of Seeds and Seed Embryos in Orthodox-,
Intermediate-, and Recalcitrant-Seeded Species
Daniel Ballesteros, Natalia Fanega-Sleziak, and Rachael M. Davies
Abstract
Seeds are one of the preferable and most used sources of germplasm for the ex situ preservation of plant
genetic resources. They are generally stored dry at À20
C in seed banks following international standards.
However, some seeds do not tolerate drying and/or storage at À20
C, or present short lifespans at these
conditions. For them cryopreservation is indicated for long-term preservation. When seeds tolerate
desiccation (i.e., orthodox seeds), they can be dried at about 32 Æ 3% relative humidity at 18
C and
stored in the vapor phase of liquid nitrogen. This is the method followed in the Millennium Seed Bank of
the Royal Botanic Gardens, Kew, for wild species with short lifespans in the standard conditions of seed
banks. When seeds do not tolerate desiccation (i.e., recalcitrant seeds) or their tolerance to desiccation
and/or À20
C storage is limited (i.e., intermediate seeds), drying and cooling procedures must be
adjusted, and often, cryoprotection is also required. Some methods are detailed for diverse species of
temperate and tropical origin.
Key words Seeds, Embryonic axes, Drying, Flash drying, Aesculus sp., Quercus sp., Fagus sylvatica,
Citrus sp., Coffea sp., Differential scanning calorimetry (DSC)
1 Introduction
Seeds are one of the most used sources of germplasm for the ex situ
preservation of genetic resources for agriculture, horticulture, forestry, and biodiversity conservation [1]. For their storage behavior
in ex situ preservation facilities, seeds are classified as orthodox,
intermediate, and recalcitrant based on their tolerance to drying
and exposure to low temperatures. Orthodox seeds tolerate drying
to low moisture contents (e.g., less than about 0.07 g H 2 O/g dry
weight) and also tolerate storage at sub-zero temperatures
[2]. Recalcitrant seeds do not tolerate drying to water contents
less than about 0.20 g H 2 O/g dry weight and are killed by exposure to sub-zero temperatures due to massive ice crystal formation
[3–5]. In the intermediate seeds category, we find a heterogeneous
group that enclose seeds that may tolerate some drying but not to
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_36, © Springer Science+Business Media, LLC, part of Springer Nature 2021
663
Cryopreservation of Seeds and Seed Embryos in Orthodox-,
Intermediate-, and Recalcitrant-Seeded Species
Daniel Ballesteros, Natalia Fanega-Sleziak, and Rachael M. Davies
Abstract
Seeds are one of the preferable and most used sources of germplasm for the ex situ preservation of plant
genetic resources. They are generally stored dry at À20
C in seed banks following international standards.
However, some seeds do not tolerate drying and/or storage at À20
C, or present short lifespans at these
conditions. For them cryopreservation is indicated for long-term preservation. When seeds tolerate
desiccation (i.e., orthodox seeds), they can be dried at about 32 Æ 3% relative humidity at 18
C and
stored in the vapor phase of liquid nitrogen. This is the method followed in the Millennium Seed Bank of
the Royal Botanic Gardens, Kew, for wild species with short lifespans in the standard conditions of seed
banks. When seeds do not tolerate desiccation (i.e., recalcitrant seeds) or their tolerance to desiccation
and/or À20
C storage is limited (i.e., intermediate seeds), drying and cooling procedures must be
adjusted, and often, cryoprotection is also required. Some methods are detailed for diverse species of
temperate and tropical origin.
Key words Seeds, Embryonic axes, Drying, Flash drying, Aesculus sp., Quercus sp., Fagus sylvatica,
Citrus sp., Coffea sp., Differential scanning calorimetry (DSC)
1 Introduction
Seeds are one of the most used sources of germplasm for the ex situ
preservation of genetic resources for agriculture, horticulture, forestry, and biodiversity conservation [1]. For their storage behavior
in ex situ preservation facilities, seeds are classified as orthodox,
intermediate, and recalcitrant based on their tolerance to drying
and exposure to low temperatures. Orthodox seeds tolerate drying
to low moisture contents (e.g., less than about 0.07 g H 2 O/g dry
weight) and also tolerate storage at sub-zero temperatures
[2]. Recalcitrant seeds do not tolerate drying to water contents
less than about 0.20 g H 2 O/g dry weight and are killed by exposure to sub-zero temperatures due to massive ice crystal formation
[3–5]. In the intermediate seeds category, we find a heterogeneous
group that enclose seeds that may tolerate some drying but not to
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_36, © Springer Science+Business Media, LLC, part of Springer Nature 2021
663