Electroporation of maize embryogenic calli with the trehalose-6-phosphate synthase gene from Arabidopsis thaliana
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Trehalose is a non-reducing disaccharide of glucose that occurs in a large number of organisms, playing an important role in desiccation and heat stress protection. Trehalose accumulation has proven to be an effective way of increasing drought tolerance in both model plants such as tobacco and important crops such as potato or rice. In this work we aim to genetically engineer maize with the Arabidopsis thaliana trehalose phosphate synthase gene (AtTPS1), involved in trehalose biosynthesis via electroporation. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene Bar as a selective agent was inserted in the plasmid vector pGreen0229 and used to transform maize inbred line Pa91 via electroporation. Fifteen putative transgenic plants (T0 generation) were obtained. Transgene integration in T0 plants was analyzed by Southern-blot analysis. T0 plants had normal phenotypes, although smaller than wild type plants. Contrary to wild type plants, when sexual organs emerged, tassels appeared at least 15 days earlier than ears in the same plant, rendering impossible the self-pollination of the T0 plant. These plants were then crossed with wild type plants and in some cases T1 seeds were obtained. T1 seeds presented deformities, especially the lack of endosperm, but it was still possible to germinate some of these seeds. The so obtained plants were tested by Northern blot but no AtTPS1 gene expression was detected, a fact possibly due to the incomplete insertion of the AtTPS1 gene or an extremely low gene expression level.
KeywordsMaize Electroporation Trehalose
Authors acknowledge financial support from Fundação para Ciência e a Tecnologia (PRAXISXXI/BD/21270/99 A.M. Almeida), H. Schleppmann (Utrech University, Utrech, The Netherlands) and P. Mullineaux (John Innes, Norwich, England) for supplying, respectively, the AtTPS1 gene and the pGreen 0229 plasmid, as well as contributions from S. Duque, J. Paiva and A. Campos, the greenhouse work of P. Fontanet and A. Sanz and photography work of A. Sanchez.
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