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An oxidative burst of superoxide in embryonic axes of recalcitrant sweet chestnut seeds as induced by excision and desiccation

Authors

  • Thomas Roach,

    1. Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom
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  • Mariyana Ivanova,

    1. School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Pietermaritzburg, Scottsville 3209, Republic of South Africa
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  • Richard P. Beckett,

    1. School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Pietermaritzburg, Scottsville 3209, Republic of South Africa
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  • Farida V. Minibayeva,

    1. Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, PO Box 30, Kazan 420111, Russian Federation
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  • Ian Green,

    1. Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom
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  • Hugh W. Pritchard,

    1. Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom
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  • Ilse Kranner

    Corresponding author
    1. Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, Ardingly, West Sussex RH17 6TN, United Kingdom
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*e-mail: i.kranner@kew.org

Abstract

Recalcitrant seeds are intolerant of desiccation and cannot be stored in conventional seed banks. Cryopreservation allows storage of the germplasm of some recalcitrant seeded species, but application to a wide range of plant diversity is still limited. The present work aimed at understanding the stresses that accompany the first steps in cryopreservation protocols, wounding and desiccation, both of which are likely to lead to the formation of reactive oxygen species (ROS). Extracellular ROS production was studied in isolated embryonic axes of sweet chestnut (Castanea sativa). Axis excision was accompanied by a burst of superoxide (O2·−), demonstrated by a colorimetric assay using epinephrine, electron spin resonance and staining with nitroblue tetrazolium. Superoxide was immediately produced on the cut surface after isolation of the axis from the seed, with an initial ‘burst’ in the first 5 min. Isolated axes subjected to variable levels of desiccation stress showed a decrease in viability and vigour and increased electrolyte leakage, indicative of impaired membrane integrity. The pattern of O2·− production showed a typical Gaussian pattern in response to increasing desiccation stress. The results indicate a complex interaction between excision and subsequent drying and are discussed with a view of manipulating ROS production for optimisation of cryopreservation protocols.

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