Reduction of Total Steroidal Glycoalkaloids in Potato Tubers Using Antisense Constructs of a Gene Encoding a Solanidine Glucosyl Transferase

Accumulation of steroidal glycoalkaloid (SGA) toxicants in potatoes affects food quality and safety. High levels of SGAs hamper breeding efforts to develop new varieties of potatoes with improved agronomic and post harvest properties. To speed breeding efforts for reducing SGAs in breeding lines and correcting flawed selections we are taking a molecular genetic approach to reduce SGAs. A cDNA encoding a solanidine glucosyltransferase (SGT) was isolated and used to construct antisense transgenes transcribed from either the Cauliflower Mosaic Virus 35S or a tuberspecific granule bound starch synthase promoter. Transgenic lines of potato (Solanum tuberosum) cultivar ‘Lenape’ expressing these transgenes exhibit phenotypes with significantly lower SGAs. Field trails over several years in Idaho and Wisconsin resulted in reproducible and statistically significant reductions of up to 40% of tuber SGAs. Reduction in SGAs is accompanied by expression of Sgt antisense RNA transcripts and a reduction in SGT protein levels. Analysis of transgenic cultivar ‘Desiree’ lines expressing an antisense Sgt revealed essentially complete down-regulation of solanine biosynthesis in several transgenic lines. Chaconine accumulation in these lines was not significantly affected. Additional transgenes encoding SGT and other SGA related sequences are currently being evaluated. INTRODUCTION Steroidal glycoalkaloids (SGAs) are ubiquitous secondary products produced by potatoes and many other Solanaceae. SGAs are believed to play a role in pest resistance (Fragoyiannis et al., 2001; Rangarajan et al., 2000) and are an important component of potato flavor (Maga, 1980). When overproduced SGAs confer bitterness to the taste of potatoes and in very high concentrations present a food safety issue (Maga, 1980; McWilliams et al., 2000; Nitithamyong et al., 1999). Because of this new potato selections are routinely screened to ensure SGAs in tubers remain below established limits of 20 mg/g fresh weight (FW) or 1.0 mg/g dry weight (DW) (Nitithamyong et al., 1999). Excessive levels of SGAs are an impediment to breeding programs trying to Proc. XXVI IHC – Potatoes Healthy Food for Humanity Ed. R.Y. Yada Acta Hort. 619, ISHS 2003 Publication supported by Can. Int. Dev. Agency (CIDA) 78 introduce beneficial phenotypes from wild relatives. Some agronomically favorable cultivars such as ‘Lenape’ have been withdrawn from production due to a tendency to produce undesirable SGAs levels. The structure and occurrence of potato SGAs was confirmed by mass spectroscopy (Chen et al., 1994). The biosynthetic pathways and demonstration of the in vitro activities of the individual enzymes is still being investigated by several labs (Griffiths et al., 2000; Kozukue et al., 2001). The two major SGAs in potato are solanine and chaconine. Both are triglycosylated steroidal alkaloids derived from solanidine and are structurally related to similar steroidal alkaloids found in tomato. Steroidal alkaloids accumulate in leaves and in tubers naturally, and their deposition is increased in response to wounding, light and cold storage (Maga, 1980). Scientists and breeders have been seeking to lower SGAs and both conventional and non-conventional breeding can be used to produce lines with lower alkaloids (Esposito et al., 2002). We have chosen to use a direct genetic approach to lowering SGAs by manipulating the pathways responsible for their synthesis in a tuber specific manner. A gene encoding a solanidine glucosyltransferase (Sgt) was cloned from a cDNA library expressed in yeast challenged to grow on solanidine. The enzymatic activity of the Sgt cDNA was confirmed in vitro (Moehs et al., 1997). The Sgt gene was used in a series of antisense constructs. These constructs are being tested for their efficacy in reducing levels of SGAs in round white chipping cultivars ‘Lenape’ and ‘Desiree’. MATERIALS AND METHODS Plant Materials Round white potato (Solanum tuberosum) cultivars ‘Lenape’ and ‘Desiree’ were used in transgenesis experiments. Field evaluation of transgenic ‘Lenape’ lines was carried out in Aberdeen, Idaho in replicated plots (Coetzer et al., 2001). Additional materials for DNA, RNA and protein analysis grown in the glass house in Albany, California. Culture of transgenic ‘Desiree’ lines was carried out in a glass house in Invergowrie, Dundee, Scotland. Plant lines with reduced SGAs and growing true to type were selected from multiplication and replanting in subsequent years. Transgene Constructs Antisense transgenes with the Sgt gene were constructed using synthetic oligonucleotides that added BamHI sites the ends of the Sgt cDNA (Moehs et al., 1997) created by polymerase chain reaction and subsequent cloning into pCR2.1 (Stratagene). Transgenic ‘Lenape’ lines were initiated with antisense Sgt constructs under transcriptional control of either the 35S promoter or the potato granule bound starch synthase promoter (Gss) promoter (van der Steege et al., 1992)and the nopaline synthase (Nos) terminator (Beven et al., 1983), for (Lenape –35S-Sgt) LSS and (Lenape-Gss-Sgt) LGS plant lines, respectively, in the binary vector pCGN1547 (McBride and Summerfelt, 1990). A second generation of transgenic lines was produced with the Gss promoter, antisense Sgt, and the ubiquitin (Ubi3) terminator (Garbarino and Belknap, 1994) (Gssantisense Sgt-Ubi) GaSU lines in cultivars ‘Lenape’ and ‘Desiree’, in the binary vector pBINPLUS/ARS a modified expression cassette containing the Ubi3 promoter and terminator (Garbarino and Belknap, 1994) in pBINPLUS (Van Engelen et al., 1995). Empty vector transformed controls include pCGN1547 in ‘Lenape’ (Len 8.1) and pBINPLUS/ARS in Desiree (pBPA). Potato transformation was carried out as previously described (Snyder and Belknap, 1993). DNA and RNA Analysis Whole tubers were measured, weighed and the outer 1-2 mm peeled and frozen in liquid nitrogen. Aliquots of frozen peel were ground into a powder in an Omni Mixer Homogenizer (Omni International). Frozen peel or powder was stored at –80 ûC until