Biharean Biologist (2009) Vol. 3, No.1, Pp.: 51-58 P-ISSN: 1843-5637, E-ISSN: 2065-1155 Article No.: 031109 Investigation into the Arabidopsis transformant selection time and escapes frequency reduction Ali DEHESTANI 1,2,3 , Gholamreza AHMADIAN 2 , Ali Hatef SALMANIAN 3 , Nadali Babaeian JELODAR 1 , Mehdi SEYEDI 3 and Kamal KAZEMITABAR 1 * 1. Agronomy and plant breeding department, Sari Agriculture and Natural Resources University, Sari, Iran 2. Molecular genetics department, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran 3. Plant biotechnology department, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran * Corresponding Author: P.O. Box: 578, Sari, Iran; Fax: (+98)1513822715; Tel: (+98)9111116203; Email: kazemi_ka@yahoo.com Abstract. Selection of putative transformants after Arabidopsis in planta transformation and the main factors involved in are comprehensively discussed. Different antibiotic concentrations, basal medium composition, incubation condition of selection plates and various selection criteria were investigated. Results showed that although kanamycin concentrations from 35 to 75 mgl -1 could discriminate transformants from non-transformed seedlings, optimum selection was carried out on plates with 75 mgl -1 kanamycin. This concentration of kanamycin along with three days dark treatment before light incubation of selection plates resulted in etiolated seedlings which were distinguished quickly by their small dark green secondary leaves. Secondary leaves development was demonstrated to be the most reliable criteria for rapid and accurate selection of transformants with a selection efficiency of 97.5%. In contrast, variations in root length and seedling colour complicated the selection process resulting in more wrong selections. Addition of sucrose to selection medium deferred the selection procedure by repressing antibiotic toxicity; while sucrose omission accelerated discrimination of transformants form bleached non-transformed seedlings. We strongly recommend use of 75 mgl -1 kanamycin supplemented medium, 3 day dark treatment before light incubation of plates, and selection of transformants using secondary leaves development criteria for rapid and accurate selection of transformed seedling. These modifications would eliminate false positive transformants while reducing selection period to 6 days. Key words: In planta transformation, Secondary leaves, seedling morphology; selection efficiency, Arabidopsis, Antibiotic concentration. Introduction Heterologous expression of different genes and study of their effects on plant biology has provided plant biologists with an excellent tool for their research experiments. Various strategies have been used for plant transformation resulting in permanent or transient expression of the transgene. Agrobacterium tumefaciens- mediated gene transfer is undoubtedly one of the most frequently used techniques for stable or transient transformation of many plant species (Weising et al. 1988, Hooykaas & Shilperoort 1992). ©Biharean Biologist, Oradea, Romania, 2009 Biharean Biol. 3, 2009 To date, most of the fundamental molecular biology studies have been focused on two major plant models: Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) due to their intrinsic properties which made them more suitable for molecular biology experiments (Newell 2000). The latter, Arabidopsis, have gained more attention recently, after its complete genome was sequenced (The Arabidopsis Genome Initiative 2000) and subsequently, a wide array of information such as complete contig maps, YAC clones, cDNA libraries and EST sequences have been available for this species. Furthermore, T-tagging of the Arabidopsis genome (Feldmann 1991) has facilitated reverse-genetic study of Arabidopsis genes function and accelerated isolation and cloning of various genes (McKinney et al. 1995). Various Agrobacterium- mediated transformation techniques for Arabidopsis have been established, among them, so called in planta methods are most frequently used due to their apparent advantages over other methods including: omission of complicated tissue culture procedures, genetically uniform progenies, high rate of transformants obtained within a quite short time, and minimal labour and reagents required for transformation (Bent 2000). After the first report of Arabidopsis transformation through in planta method (Feldmann and Marks 1987), several in planta procedures for Arabidopsis transformation were designed, including: clip and squirt method (Chang et al. 1994, Katavic et al. 1994) vacuum infiltration (Bechtold et al. 1993), floral dip (Clough & Bent 1998), floral spray transformation (Chung et al. 2000). http://biologie-oradea.xhost.ro/BihBiol/index.html Oradea, Romania