Cathodic amelioration of the adverse effects of oxidative stress accompanying procedures necessary for cryopreservation of embryonic axes of recalcitrant-seeded species

Abstract

Several of the procedures necessary for cryopreservation of excised zygotic embryonic axes are known to be accompanied by emission of damaging levels of reactive oxygen species (ROS). These have been shown to be associated with shoot apical meristem necrosis, curtailing subsequent axis development to root production only, particularly for tropical/subtropical recalcitrant-seeded species. Here we report on the application of the principles of electrochemistry in the generation of strongly reducing, high pH cathodic water, by electrolysis of a solution containing calcium and magnesium chloride. The cathodic water in which Strychnos gerrardii axes were immersed for 30 min following dehydration, and importantly, after dehydration followed by cryopreservation, was shown to have strongly antioxidative properties in counteracting the damaging effects of ROS bursts and promoting shoot development. In a parallel experiment, axes of Boophane disticha exhibited enhanced total antioxidant activity when exposed to cathodic water both immediately following excision, and after flash-drying. For both species, the efficacious effects of cathodic treatment were manifest after the axes had been in culture for 4 h, suggesting that ROS were not quenched at source, but probably counteracted by enhancement of activity of endogenous antioxidants. Cathodic water therefore affords a non-toxic means of amelioration of oxidative, stress-related damage, which, coupled with the strongly fungicidal activity of the acidic, anionic water fraction, offers significant, and apparently non-injurious, advances towards successful cryopreservation of germplasm – and probably generally improved success of in-vitro-based procedures for plant tissues.

The use of plant stress biomarkers in assessing the effects of desiccation in zygotic embryos from recalcitrant seeds: challenges and considerations

“…The data also point to the value of characterising the type and quantity of oxidative stress associated with dehydration to particular WCs, as exogenous antioxidant application may be required to facilitate post‐cryo survival ( e.g ., Berjak et al . ).…”

“…Additionally, these data suggest that recalcitrant seeds of monocotyledonous/herbaceous and dicotyledonous/woody species may differ in terms of detecting and coping with ROS. The data also point to the value of characterising the type and quantity of oxidative stress associated with dehydration to particular WCs, as exogenous antioxidant application may be required to facilitate post-cryo survival (e.g., Berjak et al 2011).…”

Photo-oxidation modulates green fern spore longevity during dry storage

“…TAA activity was measured on 50 mg (per replicate, n = 3) of spores, which were ground in liquid nitrogen with 10 mg polyvinylpyrrolidone and 1 mL phosphate extraction buffer as described by Berjak et al (2011). The contents were transferred into chilled Eppendorf tubes, which were vortexed at 5 min intervals for 15 min and centrifuged at 14,000 rpm at 4 °C for 1 h. The supernatant was then decanted and placed on ice.…”

Implications of the lack of desiccation tolerance in recalcitrant seeds

“…This entails excision of the embryos/axes from the seeds which imposes the first of the oxidative stresses inflicted during processing for, and retrieval from, cryostorage. [However, as the subject of this review focuses on the implications of the lack of desiccation tolerance, excision-related oxidative injury as first suggested by Goveia et al (2004) and studied in detail by Roach et al (2008), Whitaker et al (2010), Berjak et al (2011), Naidoo et al (2011), and Varghese et al (2011), will not presently be considered further.…”

“…In this regard, Bai et al (2011) have reported that nitric oxide enhances survival of A. toxicaria axes and Pukacka et al (2011) have found that provision of selenium protects Acer saccharinum axes subjected to desiccation. The use of cathodic water (produced by electrolysis of a dilute solution of calcium- and magnesium chloride) that has strong reducing properties, is promising in terms of ROS quenching (Berjak et al, 2011). …”