Control of Wild Carrot Somatic Embryo Development by Antioxidants

Abstract

ABSTRACrAs we previously reported for glutathione (GSH), both ascorbic acid (AA) and vitamin E were observed to suppress wild carrot (Daucus carota L.) somatic embryogenesis with little concomitant effect on biomass.Endogenous concentrations of AA were lower during embryo development than during cell proliferation, exhibiting a temporal pattern nearly identical to that of GSH. GSSG (oxidized GSH) MATERIALS AND METHODS Cell Culture. Wild carrot (Daucus carota L.) cell suspensions were grown proliferatively in the presence of 2,4-D and embryogenically in its absence as described previously (5).Determination of AA and DHA. Following extraction of cells with metaphosphoric/acetic acid and centrifugation as described for GSH/GSSG analyses (5), the resulting extracts were analyzed for both AA and DHA by the fluorimetric procedure of Deutsch and Weeks (15). In this assay AA is oxidized to DHA by shaking with activated charcoal before derivatization with o-phenylenediamine. Excitation is at 350 nm and emission at 430 nm. In addition to the specificity of this procedure which is provided by the wavelength selections and the formation of boric acid complexes, the activated charcoal used to promote the oxidation of AA to DHA is also a good adsorbent for potentially interfering substances such as phenolics found in plant cell extracts. Consequently, the resulting extracts were clear and colorless, and known amounts of AA added to wild carrot extracts as internal standards were recovered completely.Both -2,4-D and -2,4-D cultures were monitored for fresh weight, soluble protein (Bradford procedure), and cell numbers (Coulter counter) in early experiments. Since the +2,4-D and -2,4-D plots paralleled each other for each of these parameters over time, no interpretation differences were forthcoming due to the basis selected to express the results.

Variation in free-radical damage in rice cell suspensions with different embryogenic potentials

“…However, the interaction between antioxidant status and in-vitro morphogenesis may be quite complex, especially when manipulated by exogenous hormones. Earnshaw and Johnson (1987) report that somatic embryogenesis in wild carrot can be suppressed by the addition of glutathione, ascorbate and vitamin E, and that the concentration of the former two compounds can be affected by the presence of 2,4-D. Whilst this study provides evidence that a lowering of certain antioxidants is necessary for embryo development it is important to note that development is a dynamic process. The redox requirements necessary for the maintenance of embryogenic potential may be quite different from those required for embryo development per se.…”

Improved development of microspore‐derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

“…Besides hormonal supplementations in the culture medium, another important factor regulating embryogenesis in both animal and plant systems is the cellular redox state (Earnshaw and Johnson 1987, Gardiner et al 1998, Shi et al 2000, Stasolla et al 2004, Belmonte et al 2005). Alterations of the endogenous glutathione redox status [defined as the ratio of the concentration of the reduced form (GSH) to the concentration of the oxidized plus reduced forms (GSSG + GSH)] delineate specific stages of embryo development both in vivo and in vitro.…”

Oxidative events during in vitro regeneration of sunflower

“…Studies of Cui et al (1999) and Gupta andDatta (2003/2004) showed that an addition of H 2 O 2 to culture medium resulted in an increase of frequency of somatic embryogenesis in L. barbarum and G. hybridus. Antioxidants have been shown to suppress somatic embryo formation in culture of wild Daucus carota (Earnshaw and Johnson 1987). Taking it into consideration, it may not be ruled out that the enhancement of H 2 O 2 production in the sunflower embryos maintained for 1 day on EIM may be a part of signalling cascade leading to somatic embryo induction.…”