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

Abstract

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore-derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH þ GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO-treated embryos had well-organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL, a SAM molecular marker gene which was miss-expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO-treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.

Buthionine sulfoximine (BSO)-mediated improvement in cultured embryo quality in vitro entails changes in ascorbate metabolism, meristem development and embryo maturation

“…If applied in the medium after 7 days of culture, BSO induces an oxidized glutathione environment by inhibiting GSH synthesis (GriYth and Meister 1979), thereby lowering the cellular GSH/ GSH + GSSG ratio. This metabolic switch improves embryo quality, as measured by the enhanced conversion frequency, and induces major structural changes in the developing embryos (Belmonte et al 2006). Histological analyses revealed that compared to their control counterparts, BSO-treated embryos form well organized meristems and accumulate storage products in a similar pattern to that observed in seed embryos.…”

“…Induction of embryo development from B. napus cv Topas DH4079 microspores and applications of BSO were carried out exactly as reported by Belmonte et al (2006). For this experiment days in culture were counted after the imposition of the heat shock treatment.…”

“…Attempts to enhance the regeneration frequency of the embryos have mainly focused on optimizing the culture environment during the early phases of embryo development, with the assumption that once initiated the embryogenic pathway is completed successfully. Although often true, this notion cannot be generalized as cellular and tissue deterioration leading to embryo abortion have been reported in both angiosperms (Yeung et al 1996;Belmonte et al 2006) and gymnosperms (Kong and Yeung 1992). Such abnormalities mainly develop during mid-to late-embryogenesis and speciWcally aVect the integrity of the shoot apical meristem.…”

“…Alterations in the cellular redox status have been shown to control embryo development in several systems (Earnshaw and Johnson 1987;Gardiner et al 1998;Shi et al 2000;Stasolla et al 2004a;Belmonte et al 2006). In recent studies on B. napus, Belmonte et al (2006) show that the endogenous glutathione redox status [deWned as the ratio of the concentration of the reduced form (GSH) to the concentration of the oxidized plus reduced forms (GSSG + GSH)] can be easily manipulated in culture through applications of buthionine sulfoximine (BSO).…”

“…In recent studies on B. napus, Belmonte et al (2006) show that the endogenous glutathione redox status [deWned as the ratio of the concentration of the reduced form (GSH) to the concentration of the oxidized plus reduced forms (GSSG + GSH)] can be easily manipulated in culture through applications of buthionine sulfoximine (BSO). If applied in the medium after 7 days of culture, BSO induces an oxidized glutathione environment by inhibiting GSH synthesis (GriYth and Meister 1979), thereby lowering the cellular GSH/ GSH + GSSG ratio.…”