Chapter 33
Cryopreservation of Fern Spores and Pollen
Anna Nebot, Victoria J. Philpott, Anna Pajdo, and Daniel Ballesteros
Abstract
Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate
full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs
(pollen). Due to their short life span in conventional storage (i.e., dry at À20
C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen
from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some
pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and
some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used
in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores
harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one
generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation
sensitive pollen (Zea mays).
Key words Aesculus sp., Corylus sp., Cryopreservation, Drying, Fern spores, Ilex aquifolium, Hedera
helix, Magnolia quinquepeta, Pollen, Quercus sp., Zea mays, Desiccation-tolerant pollen, Desiccationsensitive pollen, Drying, Fast cooling
1 Introduction
Dry seed storage at sub-zero temperatures in seed banks (commonly À20
C but also at liquid nitrogen [LN] temperatures) is a
widespread method to preserve ex situ large levels of plant genetic
diversity [1], and methods and international standards can be
found elsewhere in the literature [2–4] (see Chapter 36). However,
the storage of other germplasm forms may be essential in some
cases when plants do not produce seeds (e.g., ferns and lycophytes)
or when the preservation interests are focused in preserving specific
reproductive cells for breeding programs (e.g., pollen).
Spores are the dispersal units of ferns and lycophytes, and they
are an important source of plant germplasm for the ex situ preservation and propagation of these plants [5]. Fern and lycophytes’
spores are haploid, of microscopic nature, and can be used to
regenerate full plants (i.e., sporophytes) through germination and
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_33, © Springer Science+Business Media, LLC, part of Springer Nature 2021
623
Cryopreservation of Fern Spores and Pollen
Anna Nebot, Victoria J. Philpott, Anna Pajdo, and Daniel Ballesteros
Abstract
Fern spores and pollen are haploid plant germplasm of microscopic nature that can be used to regenerate
full plants through germination (fern spores) or to fertilize seed-bearing plants through breeding programs
(pollen). Due to their short life span in conventional storage (i.e., dry at À20
C), the use of cryopreservation has been indicated for long-term ex situ conservation. While fern spores of most species and pollen
from many seeded plants tolerate desiccation and can be stored dry at liquid nitrogen temperatures, some
pollen is desiccation sensitive, and cryopreservation protocols require controlled drying and cooling and
some level of cryoprotection. In this chapter we describe the cryopreservation process for fern spores used
in the Millennium Seed Bank of Royal Botanic Gardens, Kew, including some details of the fern spores
harvest and cleaning methods. In addition, two protocols for pollen cryopreservation are described, one
generic for desiccation-tolerant pollen that can be used for multiple species and one specific for a desiccation
sensitive pollen (Zea mays).
Key words Aesculus sp., Corylus sp., Cryopreservation, Drying, Fern spores, Ilex aquifolium, Hedera
helix, Magnolia quinquepeta, Pollen, Quercus sp., Zea mays, Desiccation-tolerant pollen, Desiccationsensitive pollen, Drying, Fast cooling
1 Introduction
Dry seed storage at sub-zero temperatures in seed banks (commonly À20
C but also at liquid nitrogen [LN] temperatures) is a
widespread method to preserve ex situ large levels of plant genetic
diversity [1], and methods and international standards can be
found elsewhere in the literature [2–4] (see Chapter 36). However,
the storage of other germplasm forms may be essential in some
cases when plants do not produce seeds (e.g., ferns and lycophytes)
or when the preservation interests are focused in preserving specific
reproductive cells for breeding programs (e.g., pollen).
Spores are the dispersal units of ferns and lycophytes, and they
are an important source of plant germplasm for the ex situ preservation and propagation of these plants [5]. Fern and lycophytes’
spores are haploid, of microscopic nature, and can be used to
regenerate full plants (i.e., sporophytes) through germination and
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_33, © Springer Science+Business Media, LLC, part of Springer Nature 2021
623