Cultivos vegetales

Plant Molecular Biology Reporter 22: 63-70, March 2004 9 2004 International Society for Plant Molecular Biology. Printed in Canada. Protocols

Improving Transformation Efficiency of Arabidopsis thaliana by Modifying the Floral Dip Method
MIGUEL MARTINEZ-TRUJILLO 1, VERONICA LIMONES-BRIONES 2, JOSI~ LUIS CABRERA-PONCE 2 and LUIS HERRERA-ESTRELLA 2'* lFacultad de Biologfa, UniversidadMichoacana de San Nicolds de Hidalgo, Ciudad Universitaria, Morelia, Michoacdn, Mdxico, 58066; eDepartamento de Ingenier& Gendtica de Plantas, Centro de Investigaci6n y de Estudios Avanzados del lnstituto Politdcnico Nacional, Unidad Irapuato, Km 9.6 carretera Irapuato-Le6n, Irapuato, Guanajuato, M~xico, 36500
Abstract. Arabidopsis thaliana transformation with the floral dip method was improved bymodifying the cell density and mode of application of the Agrobacterium inoculum. Drops of inoculum were applied 4 times to closed flower buds. The inoculum OD600 was increased from 0.8 to more than 2.0. These modifications improved reproducibility and increased transformation efficiencies to 2-3%.

Key words: Arabidopsis, efficiency, transformation
Abbreviations: GUS, []-glucuronidase; MS,Murashige and Skoog; RT-PCR, reverse

transcriptase-polymerase chain reaction.
Introduction

The feasibility of producing transgenic organisms has profoundly affected the understanding of molecular, cellular, and developmental processes in eukaryotes. Efficient and reproducible transformation protocols facilitate the study of any model system. In 1983, Herrera-Estrella et al. first introduced andexpressed heterologous genes into plants by using the natural system of Agrobacterium tumefaciens. This procedure, along with bombardment with microparticles (Klein et al., 1987), has been used successfully in many dicotyledonous and monocotyledonous plant species, generating diverse transgenic lines in the interest of agriculture, medicine, and industry (Martfnez-Trujillo et al., 2002).Arabidopsis thaliana, a member of the Cruciferae family, is used as a model for molecular and physiological studies because of its short life cycle, small size, small genome, and ease in mutagenesis analysis (Meinke et al., 1998). Transformation of plants usually requires sophisticated regeneration methods that imply technical abilities, which are complicated with the somaclonal *Author for correspondence,e-mail: lherrera@ira.cinvestav.mx; fax: (52-462) 6245849; ph: (52-462) 6239600.

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variation and epigenetic changes, such as DNA methylation (Bent, 2000). The first Arabidopsis transformation method required tissue culture and regeneration (Valvekens et al., 1988); however, in planta transformation protocols have been developed to eliminate these steps (Changet al., 1994; Katavic et al., 1994). Low in planta transformation efficiencies have been improved to 1% by using vacuum infiltration to inoculate Arabidopsis plants with Agrobacterium (Betchtold et al., 1993). This improved transformation efficiency was maintained by a simplified method that uses detergent instead of a vacuum (Clough and Bent, 1998). We have modified the floral dip method toimprove the transformation of A. thaliana ecotype Col-0.
Materials and Methods

Strains and plasmids
We used a GPV2260 strain of A. tumefaciens (McBride and Summerfelt, 1990) and binary vector p46UTR-BIN (Martfnez-Trujillo, 2002). This plasmid, derived from the commercial vector pCAMBIA2300, harbors in its T-DNA the gene that confers kanamycin resistance and the -46 truncated 35S promoter of thecauliflower mosaic virus fused to the leader region of the rice spsl gene and the reporter uidA gene.

Disinfection of seeds and growth of plantlets Arabidopsis ecotype Col-0 seeds were disinfected with 95% ethanol for 10 min and 6% chloride and 0.1% Tween 20 detergent for 15 min and rinsed twice with sterile distilled water. Seeds were refrigerated in water for 48 h and placed in Petri dishes...