Cryopreservation of Fern Spores and Pollen

Nebot, Anna; Philpott, Victoria J.; Pajdo, Anna; Ballesteros, Daniel

doi:10.1007/978-1-0716-0783-1_33

Cited by 9 publications

(12 citation statements)

References 32 publications

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“…Fresh mature pollen was collected from 5 to 8 anthers of one spike, transferred to a mesh of pore size 30 µm and fixed with a second layer of mesh in a pollen flash-dryer according to Buitink, et al (1996) and Nebot, et al (2021). The pollen was exposed to a stream of dry air (10.7 ± 0.1 % RH) equilibrated above 250 g silica gel at room temperature which reached equilibrated RH 4 min after opening.…”

Section: Pollen Treatment and Water Contentmentioning

confidence: 99%

“…Dependent on genotype, some varieties retained high viability and seed set after drying to between 12 and 20 % WC corresponding to 0.14 and 0.25 mg H2O mg -1 DW (Barnabás and Rajki 1976;Barnabás and Rajki 1981). Inagaki and Mujeeb-Kazi (1994) achieved about 22.1 % of maize pollen germination after storage at -80 °C and 9.5 % WC for 4 weeks which was likely due to the removal of the freezable water fraction and the formation of a glassy state below -25°C at such WCs (Buitink, et al 1996).To cryopreserve desiccation-sensitive wheat pollen with high viability, existing procedures (Nebot, et al 2021) must be modified to favour the formation of a glassy state while overcoming the constraints of the low viability when WC is reduced below the freezable WC limit. This balance between vitrification without ice formation may be achieved by increasing cooling rates at the WCs at which wheat pollen still retain high viability.…”

Section: Wheat Pollen Can Partly Survive the Loss Of Freezable Watermentioning

confidence: 99%

“…Pollen grains that have WCs above 30 % (FW basis) at dispersal, termed desiccation-sensitive, partially hydrated or recalcitrant, are often bigger (15 to 150 µm) (Pacini and Franchi 2020) and hardly tolerate water loss below 30% (FW basis). To store dehydration-sensitive pollen, the levels of WC reached must be carefully balanced, and stay higher to those that generate desiccation damage and lower or close to the limit to those in which water can freeze (Nebot, et al 2021). For desiccation-sensitive pollen of some members of the Poaceae family, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…maize (Zea mays) (Barnabás and Rajki 1976;Nath and Anderson 1975), pearl millet (Pennisetum glaucum) (Hanna 1990), protocols for cryogenic storage were successfully developed. The additional application of rapid dehydration by a stream of dry air showed to increase the survival to low WCs and to improve the cryostorage success of embryonic axes of various desiccation sensitive seeds Pammenter, et al 1998;Pammenter, et al 1991;) and has been applied to pollen of maize (Buitink, et al 1996;Nebot, et al 2021).…”

Section: Introductionmentioning

confidence: 99%

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Fast Drying Combined With Rapid Cooling Can Enhance The Survival of The Desiccation Sensitive Wheat Pollen After Ultra-Low Temperature Storage

Impe

Ballesteros

Nagel

2021

Preprint

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Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially during hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-(flash)-drying and fast cooling (150°C min-1) compared to slow-(air)-drying and slow cooling (1°C min-1) would increase the rate of intracellular water content (WC) removal, decrease intracellular ice crystal formation, and increase viability after exposure to ultra-low temperatures. High correlations were found between pollen WC and viability analyzed by impedance flow cytometry (IFC viability: r=0.92, P<0.001) and pollen germination (r=0.94, P<0.001). After 10 min of air-drying, 66% WC was lost and pollen germination was at 12.2±12.3%. After 10 min of flash-drying, WC of pollen reduced by 74%. IFC viability decreased from 90.2±6.7 to 39.4±17.9%, and pollen germination dropped from 33.7±16.9 to 1.9±3.9%. After 12 min of flash-drying, WCs decreased to <0.34 mg H2O mg-1 DW, ice crystal formation was completely prevented (ΔH=0 J mg-1 DW), and pollen germination reached 1.2±1.0%. After slow and fast cooling, flash-dried pollen (WC 0.91±0.11 mg H2O mg-1 DW) showed less ice crystal formation during cryomicroscopic-video-recordings and had IFC viability of 4.5±7.0% (slow) and 6.1±8.8% (fast), respectively, compared to air-dried pollen which lost all viability. Generally, fast-(flash)-drying and increased cooling rates may enable the survival of wheat pollen likely due to (1) a fast rate of intracellular WC loss that reduces deleterious biochemical changes associated with the drying process and (2) a delay and reduction in intracellular ice crystal formation.

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Section: Pollen Treatment and Water Contentmentioning

confidence: 99%

Section: Wheat Pollen Can Partly Survive the Loss Of Freezable Watermentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Fast Drying Combined With Rapid Cooling Can Enhance The Survival of The Desiccation Sensitive Wheat Pollen After Ultra-Low Temperature Storage

Impe

Ballesteros

Nagel

2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Pollen grains that have WCs above 30% (FW basis) at dispersal, termed desiccation-sensitive, partially hydrated or recalcitrant, are often bigger (15 to 150 μm) (Pacini and Franchi 2020 ) and hardly tolerate water loss below 30% (FW basis). To store dehydration-sensitive pollen, the levels of WC reached must be carefully balanced, and stay higher to those that generate desiccation damage and lower or close to the limit to those in which water can freeze (Nebot et al 2021 ). For desiccation-sensitive pollen of some members of the Poaceae family, e.g.…”

Section: Introductionmentioning

confidence: 99%

Impact of drying and cooling rate on the survival of the desiccation-sensitive wheat pollen

2022

Self Cite

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Key message Fast-drying and cooling induce fast intracellular water loss and reduced ice-crystal formation, which may promote the formation of intracellular glasses that might improve the likelihood of wheat pollen survival. Abstract Long-term storage of pollen is important for the fertilization of spatially or temporally isolated female parents, especially in hybrid breeding. Wheat pollen is dehydration-sensitive and rapidly loses viability after shedding. To preserve wheat pollen, we hypothesized that fast-drying and cooling rates would increase the rate of intracellular water content (WC) removal, decrease intracellular ice-crystal formation, and increase viability after exposure to ultra-low temperatures. Therefore, we compared slow air-drying with fast-drying (dry air flow) and found significant correlations between pollen WC and viability (r = 0.92, P < 0.001); significant differences in WCs after specific drying times; and comparable viabilities after drying to specific WCs. Fast-drying to WCs at which ice melting events were not detected (ΔH = 0 J mg−1 DW, < 0.28 mg H2O mg−1 DW) reduced pollen viability to 1.2 ± 1.0%, but when drying to 0.39 mg H2O mg−1 DW, some viable pollen was detected (39.4 ± 17.9%). Fast cooling (150 °C min−1) of fast-dried pollen to 0.91 ± 0.11 mg H2O mg−1 DW induced less and a delay of ice-crystal formation during cryomicroscopic-video-recordings compared to slow cooling (1 °C min−1), but viability was low (4.5–6.1%) and comparable between cooling rates. Our data support that the combination of fast-drying and cooling rates may enable the survival of wheat pollen likely due to (1) a reduction of the time pollen would be exposed to drying-related deleterious biochemical changes and (2) an inhibition of intracellular ice-crystal formation, but additional research is needed to obtain higher pollen survival after cooling.

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Ex situ germplasm collections of exceptional species are a vital part of the conservation of Australia's national plant treasures

Martyn Yenson,

Sommerville,

Guja

et al. 2023

Plants People Planet

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Societal Impact StatementConservation seed banks maintain collections of many seed‐bearing plant species, providing germplasm and data to support management of wild populations. However, a proportion of plant species produce seeds that are difficult to collect, dry, store and utilise; these are known as ‘exceptional’ species. Here we tested a framework for identifying exceptional species, to document examples and provide case studies within the Australian flora. We present a workflow that may be used to identify additional exceptional species, and direct efforts to establish appropriate collection types (seeds and/or living collections, tissue culture or cryopreservation) for their ex situ conservation.SummarySeed banking is well established to contribute to the conservation of many seed‐bearing plant species ex situ for future use in restoration, translocation, agriculture and horticulture. In Australia, over 67% of currently listed threatened plants are represented in conservation seed banks. However, there are challenges to conserving the full extent of plant diversity in seed banks, with growing recognition that we need to think beyond conventional seed banking methods to conserve ‘exceptional’ plant species that are difficult to collect, store and germinate. We examine how the framework for identification of such species can be applied to the Australian flora, using examples from the recently published guidelines for ‘Plant Germplasm Conservation in Australia’ and case studies and data arising from the Australian Academy of Science Fenner Conference on the Environment ‘Exceptional Times, Exceptional Plants’. We present a workflow to assist conservation decision‐makers and practitioners in identifying exceptional species and overcoming barriers to storage of germplasm, enabling appropriate ex situ collection types to be established via seeds, living collections, tissue culture, cryopreservation or a combination of these. Australia's seed conservation sector continues to expand, with increasing expertise, facilities and networks established to conserve a diversity of plant species; however, resolving the challenges relating to each exceptionality factor requires significantly more time, labour and collaboration than current capacity allows. Understanding the barriers to conservation and production of healthy plants, via germination or other methods of propagation, is a critical component of conserving species long‐term and ultimately returning plants to the landscape.

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Cryopreservation of Fern Spores and Pollen

Cited by 9 publications

References 32 publications

Fast Drying Combined With Rapid Cooling Can Enhance The Survival of The Desiccation Sensitive Wheat Pollen After Ultra-Low Temperature Storage

Fast Drying Combined With Rapid Cooling Can Enhance The Survival of The Desiccation Sensitive Wheat Pollen After Ultra-Low Temperature Storage

Impact of drying and cooling rate on the survival of the desiccation-sensitive wheat pollen

Ex situ germplasm collections of exceptional species are a vital part of the conservation of Australia's national plant treasures

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