Journal of Biotechnology 163 (2013) 61–68 Contents lists available at SciVerse ScienceDirect Journal of Biotechnology jou rn al hom epage: www.elsevier.com/locate/jbiotec Establishment of an efficient genetic transformation system in Scenedesmus obliquus Suo-Lian Guo a , Xin-Qing Zhao a,∗ , Ying Tang a , Chun Wan a , Md. Asraful Alam a , Shih-Hsin Ho b , Feng-Wu Bai a,∗ , Jo-Shu Chang b,c,d a School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China b Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan c University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan d Research Center for Energy Technology and Strategy Center, National Cheng Kung University, Tainan 701, Taiwan a r t i c l e i n f o Article history: Received 14 May 2012 Received in revised form 30 October 2012 Accepted 31 October 2012 Available online 9 November 2012 Keywords: Scenedesmus obliquus Green microalgae Genetic transformation system Electroporation GFP gene a b s t r a c t Scenedesmus obliquus belongs to green microalgae, which is attracting attention as a feedstock for biofuels production and biorefinery as well as in bioremediation of environmental pollutants, making its genetic modifications for more efficient growth and accumulation of aimed metabolites significant. However, the genetic transformation system of S. obliquus is still not well established. In the current work, S. obliquus was transformed via electroporation using a plasmid containing chloramphenicol resistance gene (CAT) as a selectable marker and the green fluorescent protein gene (gfp) as a reporter. Using the optimized transformation conditions, the transformation efficiency was 494 ± 48 positive transgenic clones per 10 6 recipient cells, which is more efficient comparing with those reported in other microalgal transformation studies. Green fluorescence was observed after six months of cultivation, and CAT-specific products were also detected in the transformants by PCR, Southern blot and RT-PCR analysis. This is the first report on establishing such an efficient and stable transformation system for S. obliquus, a prerequisite for both functional genomic studies and strain improvement for other biotechnology applications of this important microalgal species. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Microalgae have been traditionally used for aquatic feed, and in the recent years, they also have received renewed attention on account of their capacity to offer numerous value-added products (Harun et al., 2010), as well as acting as cell factories for the pro- duction of biofuel and recombinant proteins (Amaro et al., 2011; de Morais and Costa, 2007; Huang et al., 2010; Mata et al., 2010; Potvin and Zhang, 2010). Scenedesmus obliquus belongs to green microal- gae and contains high concentration of protein and has thus been found applications as animal feed additives (Hintz et al., 1966) and for pigment (including astaxanthin) production (Qin et al., 2008; Wiltshire et al., 2000). S. obliquus also possesses excellent ability of wastewater treatment, CO 2 sequestration and biodiesel produc- tion (Cheng et al., 2010; Ho et al., 2010, 2012; Kumar et al., 2010; Mandal and Mallick, 2009, 2011). The maximum CO 2 consumption ∗ Corresponding author at: School of Life Science and Biotechnology, Dalian University of Technology, Linggong Road 2, Dalian 116024, China. Tel.: +86 411 84706319; fax: +86 411 84706329. E-mail addresses: xqzhao@dlut.edu.cn (X.-Q. Zhao), fwbai@dlut.edu.cn (F.-W. Bai). rate of S. obliquus can reach 1782 mg L -1 d -1 and their lipid content is up to 55% of the dry cell weight (DCW) (Cheng et al., 2010; Ho et al., 2010, 2012), which indicates the great potential of coupling CO 2 fixation with biodiesel production. Moreover, S. obliquus was used to remove heavy metal for bioremediation (Chen et al., 2012; Fayed et al., 1983). Despite the great economic and environmental importance of S. obliquus, genetic studies of S. obliquus are very limited, which impedes strain development and improvement of production effi- ciency using advanced molecular tools. Therefore, there is an urgent need for fundamental and applied research based on genetic manipulation in S. obliquus. In microalgae, gene transfer proceeds at a slower rate as compared with bacteria, while genetic transfor- mation systems have been established only in some microalgae species (Hallmann, 2007; Potvin and Zhang, 2010; Kilian et al., 2011; Radakovits et al., 2010, 2012). Absence of the available genetic transformation method is a major bottleneck for genetic engineering of microalgae (Amaro et al., 2011). So far, several methods have been developed for microalgal transformations, including particle bombardment method (Hirata et al., 2011), sil- icon carbide whiskers method (Dunahay, 1993), Agrobacterium tumifaciens-mediated method (Anila et al., 2011; Rajam and Kumar, 2006), glass beads method (Feng et al., 2009; Wang et al., 2010) and 0168-1656/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2012.10.020