Influence of anti- and prooxidants on rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured in vitro

The enrichment with antioxidants (glutathione or ascorbate) or prooxidants (alloxan, methylviologen, hydrogen peroxide) of root inducing medium significantly decreased rhizogenesis frequency (alloxan, hydrogen peroxide) or inhibited roots regeneration (ascorbate, methylviologen) during the in vitro culture of Mesembryanthemum crystallinum L. hypocotyls. The adventitious roots morphology, root hairs length and density, was also influenced. Changes in the rhizogenesis course were related to the differences in hydrogen peroxide concentration during following days of culture between explants exhibiting morphogenic potential and those without the ability to form adventitious roots. In explants with morphogenic potential, rhizogenesis induction was always accompanied by a high level of hydrogen peroxide followed by the decrease in H_{2}O_{2} content in following days. In contrast, in the explants without regeneration potential, the level of hydrogen peroxide was increasing during the culture period. Activity patterns of superoxide dismutase (SOD) and guaiacol peroxidase (POX) in the following days of culture were similar in the explants exhibiting regeneration potential cultured on different media. Total activity of SOD decreased during initial days of culture and then increased due to the activation of additional SOD isoform described as MnSODII. The activity of POX was low during the rhizogenesis induction, and then increased during following days of culture; the increase was correlated with the decrease in hydrogen peroxide content. In the explants without the ability to regenerate roots, the total activity of SOD was low throughout the whole culture period, whereas the POX activity was significantly higher than in hypocotyls with regeneration potential. It might be concluded that the increase in hydrogen peroxide during initial stages of rhizogenesis and the induction of MnSODII are prerequisites for adventitious roots formation from hypocotyls of M. crystallinum, independently in the presence of anti- or prooxidant in the culture medium

The localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L. hypocotyls discloses their differing roles in rhizogenesis

This work demonstrated how reactive oxygen species (ROS) are involved in the regulation of rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured on a medium containing 1-naphthaleneacetic acid (NAA). The increase of NADPH oxidase activity was correlated with an increase of hydrogen peroxide (H2O2) content and induction of mitotic activity in vascular cylinder cells, leading to root formation from cultured hypocotyls. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, inhibited H2O2 production and blocked rhizogenesis. Ultrastructural studies revealed differences in H2O2 localization between the vascular cylinder cells and cortex parenchyma cells of cultured explants. We suggest that NADPH oxidase is responsible for H2O2 level regulation in vascular cylinder cells, while peroxidase (POD) participates in H2O2 level regulation in cortex cells. Blue formazan (NBT) precipitates indicating superoxide radical (O2 •−) accumulation were localized within the vascular cylinder cells during the early stages of rhizogenesis and at the tip of root primordia, as well as in the distal and middle parts of newly formed organs. 3,3′-diaminobenzidine (DAB) staining of H2O2 was more intense in vascular bundle cells and in cortex cells. In newly formed roots, H2O2 was localized in vascular tissue. Adding DPI to the medium led to a decrease in the intensity of NBT and DAB staining in cultured explants. Accumulation of O2 •− was then limited to epidermis cells, while H2O2 was accumulated only in vascular tissue. These results indicate that O2 •− is engaged in processes of rhizogenesis induction involving division of competent cells, while H2O2 is engaged in developmental processes mainly involving cell growth

Steviol glycosides profile in Stevia rebaudiana Bertoni hairy roots cultured under oxidative stress-inducing conditions

The ability to synthesize particular steviol glycosides (SvGls) was studied in Stevia rebaudiana Bertoni hairy roots (HR) grown in the light or in the dark under the influence of different osmotic active compounds. Manipulation of culture conditions led to changes in the morphology and growth rate of HR, as well as to an increase in oxidative stress manifested as an enhancement in endogenous hydrogen peroxide concentration in the cultured samples. The highest level of H2O2 was noted in HR cultured under light or in the medium with the highest osmotic potential. This correlated with the highest increase in the expression level of ent-kaurenoic acid hydroxylase, responsible for the redirection of metabolic route to SvGls biosynthesis pathway. An analysis of transcriptional activity of some UDPglucosyltransferase (UGT85c2, UGT74g1, UGT76g1) revealed that all of them were upregulated due to the manipulation of culture conditions. However, the level of their upregulation depended on the type of stress factor used in our experiment. Analysis of SvGls content revealed that HR grown under all applied conditions were able to synthesize and accumulate several SvGls but their concentration differed between the samples across the different conditions. The level of rebaudioside A concentration exceeded the content of stevioside in HR in all tested conditions. Concomitantly, the presence of some minor SvGls, such as steviolbioside and rebaudioside F, was confirmed only in HR cultured in the lowest osmotic potential of the medium while rebaudioside D was also detected in the samples cultured in the media supplemented with NaCl or PEG.Key Points● Several steviol glycosides are synthesized in hairy roots of S. rebaudiana.● Light or osmotic factors cause enhancement in oxidative stress level in hairy roots.● It correlates with a significant increase in the level of KAH expression.● UGTs expression and steviol glycosides content depends on culture conditions