Amylose content is not affected by overexpression of the Wx-B1 gene in durum wheat F RANCESCO S ESTILI , E RMELINDA B OTTICELLA , G LORIA P ROIETTI , M ICHELA J ANNI , R ENATO D Õ O VIDIO and D OMENICO L AFIANDRA Department of Agriculture, Forest, Nature and Energy, University of Tuscia, via S. Camillo de Lellis, SNC 01100 Viterbo, Italy Corresponding author, E-mail: lafiandr@unitus.it With 6 figures and 1 table Received September 28, 2011/Accepted June 1, 2012 Communicated by L. Hartl Abstract The granule-bound starch synthase (GBSS), known as waxy protein, is the sole enzyme responsible for amylose synthesis, whereas amylo- pectin is produced by concerted action of several isoforms, including starch synthases, branching and debranching enzymes. Durum wheat contains two waxy genes, which encode the Wx-A1 and Wx-B1 proteins, respectively. In order to investigate the effect of the overexpression of the waxy genes on amylose content and starch properties, durum wheat transgenic lines overexpressing the Wx-B1 gene were produced. Amylose content of the transgenic lines did not show any increase compared to the untransformed cultivar. One of the transgenic lines was crossed with a mutant waxy genotype of durum wheat lacking all the waxy protein isoforms. The screening of F 2 segregant plants permitted the identification of lines possessing various combinations of waxy genes, including the line containing only the construct used in the transformation. In this line, the normal vitreous seed phenotype, because of the presence of amylose, was restored confirming a correct functioning of the transgene. Key words: starch — waxy gene — amylose — Triticum durum Technological and nutritional properties of flours and semo- lina depend on various factors, such as storage protein, starch and lipid compositions. Reserve starch, the major component of the kernel, is a glucan carbohydrate that is formed by two different polymers known as amylose and amylopectin. Amy- lose is mainly composed of linear chains and constitutes about 25–30% of total starch; amylopectin is the main starch polymer (70–75%) and possesses a high number of branches owing to the presence of a-1,6 glucan linkages. Starch biosynthesis takes place in the amyloplasts of endosperm cells, and several classes of enzymes are involved. Amylose and amylopectin are synthesized from a common substrate, ADP-glucose, by two distinct pathways. Amylopec- tin is produced by the concerted action of different enzymes (starch synthases, branching and debranching enzymes), whereas a granule-bound starch synthase (GBSSI or waxy protein) catalyses the production of amylose (James et al. 2003, Stone and Morell 2009). In common wheat (genome AABBDD), three different GBSSI proteins are present which are encoded by three genes located on the short arm of the chromosome 7A (locus Wx-A1), 7D (locus Wx-D1) and on the long arm of chromosome 4A (locus Wx-B1), consequence of a translocation from the chromosome 7B (Chao et al. 1989, Miura et al. 1994, Yamamori et al. 1994). In further studies, the detection of bread and durum wheat lines characterized by the absence of one or two waxy proteins (partial waxy) has also been reported and their crossing has resulted in the isolation of complete waxy material in both species (Miura et al. 1994, Nakamura et al. 1995, Urbano et al. 2002, Slade et al. 2005, Lafiandra et al. 2010). The comparison of starch properties of waxy and wild-type lines has shown several differences in amylose content and viscosity characteristics as determined by Rapid Visco Analyzer (RVA) parameters both in durum and in bread wheat. Complete waxy lines have a low amount of amylose (0–3%), a higher viscosity peak and a lower peak time, setback and final viscosity compared to normal genotypes (Grant et al. 2001, Lafiandra et al. 2010). The partial waxy lines showed intermediate characteristics between complete waxy and wild-type cultivars (Yamamori and Quynh 2000, Lafiandra et al. 2010). The advances of the technologies for genetic transformation of wheat have opened up wide perspectives for starch manipulation. In particular, silencing and overexpression of starch biosynthetic genes represent effective and powerful tools to modify starch structure and composition. In this work, we have produced transgenic durum wheat plants overexpressing an endogenous waxy gene with the aim of evaluating the effects on starch properties. Materials and Methods Isolation of Wx-B1 gene: cDNA was produced by reverse transcrip- tase polymerase chain reaction (RT-PCR) on total RNA extracted from immature seeds (18 day postanthesis) of durum wheat cv. ÔSvevoÕ as reported in the study by Sestili et al. (2010). PCR amplification was carried out on 1 ll of cDNA in a volume of 50 ll, containing 2.5 units of Taq DNA Polymerase (Qiagen, GmbH, Hilden, Germany), 1X Qiagen PCR buffer, 200 lM of each dNTP and 0.5 lM of each primer (Wx3F 5¢cttgctgcaggtagccaca and Wx1R 5¢ctccccatacgagaaccct). Amplification conditions included an initial denaturation step at 94°C for 5 min, followed by 35 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 2 min, followed by a final incubation at 72°C for 5 min. PCR amplicon was purified and cloned in pGem-T Easy plasmid (Promega, Corporation, Madison, WI, USA). DNA sequence analyses were conducted by a commercial sequencing service (Eurofins MWG Operon, Ebersberg, Germany). The comparative analyses of nucleotide and amino acid sequences were performed by DNAMAN program. The comparison with known sequences in databank was made using an online blast program (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). Chromosomal localization of the isolated gene was carried out by PCR analysis on genomic DNA extracted from ditelosomic and Plant Breeding doi:10.1111/j.1439-0523.2012.02004.x Ó 2012 Blackwell Verlag GmbH wileyonlinelibrary.com