GENETIC TRANSFORMATION AND HYBRIDIZATION Expression of Escherichia coli heat-labile enterotoxin b subunit (LTB) in carrot (Daucus carota L.) Sergio Rosales-Mendoza Ruth Elena Soria-Guerra Marı´a Teresa de Jesu ´ s Olivera-Flores Rube ´n Lo ´ pez-Revilla Gerardo Rafael Argu ¨ ello-Astorga Juan Francisco Jime ´ nez-Bremont Ramo ´n Fernando Garcı´a-de la Cruz Juan Pablo Loyola-Rodrı´guez A ´ ngel Gabriel Alpuche-Solı´s Received: 12 September 2006 / Revised: 11 January 2007 / Accepted: 18 January 2007 / Published online: 20 February 2007 Ó Springer-Verlag 2007 Abstract We expressed the B subunit of enterotoxi- genic Escherichia coli heat-labile enterotoxin (LTB) encoded by a synthetic codon-optimized gene in carrot. An Agrobacterium-mediated transformation method was used. Thirty independent transgenic lines were regenerated via somatic embryogenesis after 6 months in culture and were transferred to a greenhouse. GM1- ELISA assay was used to assess LTB protein content in mature taproots. Some transgenic lines expressed LTB up to 0.3% of the total soluble protein, which is tenfold higher than the expression levels reported earlier using the native bacterial gene in plants. Immunological assay confirmed proper assembly of the pentameric complex and in vitro activity of the re- combinant LTB protein, suggesting that it can be functional in prevention of diarrhea. Keywords Diarrhea prevention Á Plant based-vaccine Á Synthetic gene Á Expression levels Á Somatic embryogenesis Abbreviations CaMV 35S Cauliflower mosaic virus 35S RNA promoter GM1 Galactosyl-N-acetylgalactosamyl-sialyl- galactosylglucosyl ceramide LTB B subunit of the heat-labile enterotoxin of Escherichia coli TSP Total soluble protein Introduction The use of molecular biology concepts and plant tissue culture techniques for expression of antigens in plants has advanced significantly over the last decade. This research aims to develop edible plant-based vaccines that elicit an immune response via the mucosal system. The oral immunogenicity of antigens expressed in di- verse transgenic plants has been reported by several groups (Curtis and Cardineau 1990; Haq et al. 1995; Mason et al. 1996, 2002). Viral proteins such as the respiratory syncytial virus G protein (Balenger et al. 2000) and hepatitis B surface antigen HBsAg (Kapusta et al. 1999) have been expressed in plants and mucosal immune response tested in mice has been achieved. Communicated by P. Lakshmanan. S. Rosales-Mendoza Á R. E. Soria-Guerra Á R. Lo ´ pez-Revilla Á G. R. Argu ¨ ello-Astorga Á J. F. Jime ´ nez-Bremont Á A ´ . G. Alpuche-Solı´s (&) Divisio ´n de Biologı´a Molecular, Instituto Potosino de Investigacio ´n Cientı´fica y Tecnolo ´ gica, Camino a la Presa San Jose ´ 2055, 78216 San Luis Potosı´, S.L.P., Mexico e-mail: alpuche@ipicyt.edu.mx M. T. de Jesu ´ s Olivera-Flores Facultad de Quı´mica, Universidad Nacional Auto ´ noma de Me ´ xico, Ciudad Universitaria, 04510 Me ´ xico, D.F., Mexico R. F. Garcı´a-de la Cruz Facultad de Ciencias Quı´micas, Universidad Auto ´noma de San Luis Potosı´, Av. Dr. Manuel Nava No. 6, 78210 San Luis Potosı´, S.L.P., Mexico J. P. Loyola-Rodrı´guez Maestrı´a en Ciencias Odontolo ´ gicas, Universidad Auto ´noma de San Luis Potosı´, Av. Dr. Manuel Nava No. 2, 78290 San Luis Potosı´, S.L.P., Mexico 123 Plant Cell Rep (2007) 26:969–976 DOI 10.1007/s00299-007-0310-2