Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation

Abstract

Three wild species exhibited a significant reduction in antioxidants throughout the cryopreservation protocol, whilst the half-cell reduction potential became more oxidised. Antioxidant content recuperated in recovering shoot tips. Cryopreservation is the most efficient and cost-effective long-term storage solution for the conservation of a wide range of plant species and material. Changes in the levels of antioxidants during the process of cryopreservation are known to reduce post-cryogenic survival due to oxidative stress. Low-molecular-weight thiols (cysteine, γ-glutamylcysteine, and glutathione) and ascorbic acid, which represent the two major water-soluble antioxidants in plants, were analysed at specific stages during cryopreservation of shoot tip material of three native Australian plant species [Anigozanthos viridis (Haemodoraceae), Lomandra sonderi (Asparagaceae), and Loxocarya cinerea (Restionaceae)] to quantify the oxidative stress experienced during cryopreservation. Post-cryogenic regeneration of shoot tips was greatest in A. viridis (78%) followed by L. sonderi (50%), whilst L. cinerea did not show any post-cryogenic regeneration. The application of a 3-week cold (5 °C) preconditioning regime, commonly used to increase post-cryogenic survival, resulted in significantly lower post-cryogenic regeneration for A. viridis (33%), but had little effect on the other two species. Total antioxidant concentration in shoot material decreased significantly with each step throughout the cryopreservation process, particularly in the cryoprotection and washing stages. Antioxidant levels in shoot tips then increased during the subsequent 7-day post-cryopreservation recovery period, with the greatest increase measured in A. viridis. Concentrations of thiols and their corresponding disulphides were used to calculate the corresponding half-cell reduction potentials, whereby the ability of these plant species to maintain a strong reducing environment in shoot tissues throughout the cryopreservation protocol was found to correlate with post-cryogenic survival.

Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species

“…Although antioxidants within the cells can balance the build-up of ROS (Mittler 2002), it has been shown that concentrations of antioxidants like glutathione (GSH) and ascorbic acid (AsA) decrease in plant tissues with each successive step of the cryopreservation process (Fig. 4) (Funnekotter et al 2017b). Although Chen et al (2015) also found a reduction in GSH and AsA antioxidants in thawing and recovery stages, levels of these antioxidants were found to increase in the desiccation period.…”

“…Cryopreservation is a multi-step process, involving (i) the initial excision of the germplasm such as seed embryos or shoot tips to obtain sufficiently small material; (ii) desiccation or preculture on osmotic media to reduce water content; (iii) cryoprotection through exposure to CPAs to promote vitrification; (iv) cryopreservation in LN; (v) re-warming; and finally (vi) the washing (unloading of CPA solutions) and recovery of the germplasm after cryopreservation ( Fig. 1; Funnekotter et al 2017b;Kaczmarczyk et al 2012). There is wide variation in cryo-capability among species, and also among different clonal lines of the same species, therefore each species requires its own cryopreservation protocol to achieve the maximum possible post-cryogenic survival (Reed 2008).…”

“…There is, however, a range of herbaceous and woody species from different ecosystems represented in the literature (Table 1). Cryopreservation of other Australian species has previously been reported by Bunn et al (2007), Kaczmarczyk et al (2011) and Funnekotter et al (2017b), while Merritt et al (2014a) has reviewed cryopreservation success with seeds of various orchid species. Due to the high diversity of endemic species in Australia, little is known about the physiology of each species, resulting in slow progress in the development of new cryopreservation protocols.…”

“…Plants have complex antioxidant systems, which are their natural defence against the accumulation of ROS and their byproducts, and which enable them to resist external stress (Chen et al 2015;Funnekotter et al 2017b). Although antioxidants within the cells can balance the build-up of ROS (Mittler 2002), it has been shown that concentrations of antioxidants like glutathione (GSH) and ascorbic acid (AsA) decrease in plant tissues with each successive step of the cryopreservation process (Fig.…”

“…Reduction in ascorbic acid (AsA) content in Lomandra sonderi during the five stages of the cryopreservation process. Three in vitro preconditioning temperatures (constant 25 C, 5 C and an alternating 25/5 C) for 3 weeks were tested before cryopreservation(Funnekotter et al 2017b).…”