ABSTRACT
To investigate the mechanism of auxin action during pattern formation in dicot embryos, we tested the effects of the natural auxin indole-3-acetic acid (IAA), the auxin transport inhibitor N-(1-naphthyl)thalamic acid (NPA) and the antiauxin p-chlorophenoxyisobutyric acid (PCIB). In vitro treatments of isolated zygotic Brassica juncea embryos with these substances led to a wide range of morphogenetic alterations. Treatment of globular embryos with exogenous auxin (10-40 microM) either completely inhibited morphogenesis, resulting in ball-shaped embryos, or caused the development of egg- and cucumber-shaped embryos, which only consisted of a shortened hypocotyl without any apical structures. Axis duplication was observed sometimes after inhibition of auxin transport in globular embryos, and led to the development of twin embryos. During the transition from globular to heart stage, changes in auxin distribution or activity frequently caused the development of either split-collar or collar-cotyledons. Antiauxin inhibited cotyledon growth, leading to embryos with single or no cotyledons, or inhibited the development of the hypocotyl and the radicle. Inhibition of auxin transport in transition embryos sometimes led to axis broadening, which resulted in the development of two radicles. The described changes in embryo shapes represent arrests in different auxin-regulated developmental steps and phenocopy some Arabidopsis morphogenetic mutants.
Subject(s)
Brassica/drug effects , Brassica/embryology , Indoleacetic Acids/pharmacology , Biological Transport, Active/drug effects , Brassica/metabolism , Cell Differentiation/drug effects , Clofibric Acid/pharmacology , Cotyledon/drug effects , Cotyledon/embryology , Hypocotyl/drug effects , Hypocotyl/embryology , Indoleacetic Acids/antagonists & inhibitors , Indoleacetic Acids/metabolism , Microscopy, Electron, ScanningABSTRACT
A procedure for the regeneration of fertile transgenic white mustard (Sinapis alba L.) is presented. The protocol is based on infection of stem explants of 7-9 day old plants with an Agrobacterium tumefaciens strain harboring a disarmed binary vector with chimeric genes encoding neomycin phosphotransferase and ß-glucuronidase. Shoots are regenerated from callus-forming explants within 3-4 weeks. Under selection, 10% of the explants with transgenic embryonic callus develop into fertile transgenic plants. Rooting shoots transferred to soil yield seeds within 14-16 weeks following transformation. Integration and expression of the T-DNA encoded marker genes was confirmed by histochemical ß glucuronidase assays and Southern-DNA hybridization using primary transformants and S1-progeny. The analysis showed stable integration and Mendelian inheritance of trans-genes in transformed Sinapis lines.
