Glutathione modulation of in vitro organized development in canola microspore derived and spruce somatic embryos

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Belmonte, Mark Findlay
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The production of good quality microspore derived embryos (MDEs) of Brassica napus cv. Topaz (canola) and somatic embryos of spruce including Picea glauca and P. abies (white spruce and Norway spruce, respectively) are of great importance for developmental and applied studies. One of the obstacles encountered during the culture process is the lack of organized developmet, especially within the shoot apical meristem (SAM). In the last few years, glutathione and its association in the ascorbate-glutathione cycle has been viewed as rational modulator of embryo development in vivo and in vitro. It is apparent that glutathione plays a fundamental role during embryogeny of both canola and spruce and that the ratio between the reduced (BSH) and oxidized (GSSG) forms of this redox compound have profound effects on development. Applications of buthionine sulfoximine (BSO), a specific glutathione biosynthesis inhibitor, to MDEs of B. napus shifts the glutathione redox status, i.e. GSH/(GSH+GSSG) in favour of an oxidized sate and significantly enhances the quality of embryos through alterations in hormone metabolism and gene expression. Improved development in accompanied by major changes in the ascorbate-glutathione redox system. Like canola, spruce somatic embryos cultured in the presence of BSO are depleted of GSH which creates a forced shift in the glutathione redox ratio towards a more oxidized status. This shift is necessary to maintain structural integrity of the shoot apex and to prevent deterioration of the subapical cells. To examine whether altered embryonic development affects the glutathione redox status, the function of a class I homeobox of knox gene, HBK3, was characterized from the conifer P. abies (L.) Karst. HBK3 is necessary for improved somatic embryo development in the promotion of a more stable SAM through alterations in PgAGO gene expression, a specific meristem marker. Cells down-regulating HBK3 failed to develop into somatic embryos. Futhermore, glutathione metabolism in embryos down regulating HBK3 failed to shift the GSH/GSH+GSSG ratio necessary for transdifferentiation of somatic embryos. Overall, the findings emerging from this thesis represent a solid contribution to the improvement of canola and spruce embryos produced in culture.