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Transformation of statice (Limonium sinuatum Mill.) by Agrobacterium tumefaciens-mediated gene trans

Transformation of statice (Limonium sinuatum Mill.) by Agrobacterium tumefaciens-mediated gene trans

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The Plant Journal (1998) 13 (5), 707–716 TECHNICAL ADVANCE Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension Jaideep Mathur 1 , La ´szlo ´ Szabados 2 , Sabine Schaefer 1 , Britta Grunenberg 1 , Andrea Lossow 1 , Esther Jonas-Straube 1 , Jeff Schell 1 , Csaba Koncz 1,2 and Zsuzsanna Koncz-Ka ´lma ´n 1, * 1 Max-Planck Institut fu ¨ r Zu ¨ chtungsforschung, D-50829 Ko ¨ ln, Carl-von-Linne ´-Weg 10, Germany, and 2 Institute of Plant Biology, Biological Research Center of Hungarian Academy of Sciences, H-6701 Szeged, PO Box 521, Temesva ´ri krt. 62, Hungary Summary A protocol for establishment and high-frequency Agrobac- terium -mediated transformation of morphogenic Arabi- dopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T- DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirec- tional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J- domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen- specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis. Introduction Following large-scale sequencing of expressed sequence tags (ESTs) and construction of physical maps, genome projects in Arabidopsis and other plants are advancing towards sequencing of whole chromosomes in conjunction with functional analysis of the sequenced genes (Cooke et al., 1996; Ho ¨ fte et al. , 1993; Huang and Miao, 1997; Newman et al. , 1994; Sasaki et al., 1994; Schmidt et al., 1995; Zachgo et al. , 1996). As gene replacement is not yet routine in higher plants, the T-DNA of Agrobacter- ium and transposons are used for saturating the genome Received 8 July 1997; revised 30 October 1997; accepted 7 November 1997. *For correspondence (fax 1 49 221 5062 213; e-mail [email protected]). © 1998 Blackwell Science Ltd 707 with insertional mutations in order to establish correlations between sequence data, mutant phenotypes and gene functions by reverse genetics (for reviews see Feldmann et al. , 1994; Long et al. , 1993; Miao and Lam, 1995; Osborn et al. , 1991; Souer et al. , 1995). Application of reporter gene fusion, enhancer trap, suicide gene and activator tagging technologies facilitate both targeted tagging of single genes and mutagenesis of particular gene classes charac- terized by well-defined regulatory functions and/or expres- sion patterns (Franzmann et al. , 1995; Hayashi et al. , 1992; Kertbundit et al. , 1991; Knapp et al., 1994; Koncz et al. , 1989, 1994; Sunderesan et al. , 1995; Topping et al. , 1991).
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