Regular paper Phosphomannose isomerase gene for selection in lettuce (Lactuca sativa L.) transformation Jindřich Bříza 1,2* , Nina Růžičková 2 , Hana Niedermeierová 1 , Jana Dusbábková 1 and Josef Vlasák 1 1 Biology Centre of the Academy of Sciences of the Czech Republic, v.v.i., Institute of Plant Molecular Biology, České Budějovice, Czech Republic; 2 University of South Bohemia, Faculty of Science, České Budějovice, Czech Republic A positive selection system using phosphomannose iso- merase was employed for Agrobacterium tumefaciens mediated transformation of lettuce (Lactuca sativa L. var. ‘Achát’). It was shown that the mannose-based selec- tion system works very well with the lettuce genotype used, reaching up to 25 % transformation efciency on the medium with 20 g/L mannose and 20 g/L sucrose. The best transformation efcacy with the commonly- used kanamycin at 100 mg/L as a selection agent was 21 %. Southern blot analyses of thirteen chosen man- nose-resistant regenerants revealed that some of them have clonal origin, about one-half harbour a single T- DNA copy and one plant contains an incomplete T-DNA segment with only the left part of T-DNA with the pmi gene present in the genomic DNA. The following North- ern analysis showed transcriptional activity of the in- troduced pmi gene in all plants analysed with very high diferences in the level of pmi specifc mRNA. The results demonstrate that both mannose and kanamycin provide comparable transformation efciencies in our lettuce genotype. An alternative selection method with man- nose as a selection agent is now available for lettuce transgenosis. Keywords: phosphomannose isomerase, pmi, npt II, Lactuca sativa, kanamycin, transformation efciency Received: 30 January, 2010; revised: 25 February, 2010; accepted: 11 March, 2010; available on-line: 16 March, 2010 INTRODUCTION When Fraley and coworkers published in 1983 the frst successful transformation of a plant genome with foreign genes, a new key tool for basic plant research as well as applied research was created. It was shown that genes for antibiotic resistance or herbicide tolerance could be used as reliable markers for transgenic tissue selection. Such traditional and frequently used plant se- lectable marker genes are, for example, the nptII gene granting resistance to aminoglycoside antibiotics like kanamycin, neomycin, paromomycin and G-418, the hph gene conferring hygromycin B resistance, or the bialap- hos resistance genes bar and pat providing resistance to herbicides with phosphinothricin active compound. Un- fortunately, antibiotic resistance markers are not appro- priate for monocots (Wilmink & Dons, 1993) and other plant species, and they are not accepted by the public. The same is true for the markers based on herbicide re- sistance. In addition, the traditional selective agents of- ten adversely affect the transformed plant cells, bringing about a decrease in the regeneration of transformed cells by the accumulation of toxic compounds from killed, non-transformed cells (Hansen & Wright, 1999). Also, for the introduction of several genes into a single trans- genic plant, the development of further types of selecta- ble markers is desirable. Up to now, a number of marker genes have been employed for the development of alternative selection systems that avoid the use of either antibiotics or her- bicides (Sundar & Sakthivel, 2008). Such promising al- ternative systems were developed by Joersbo and Okkels (1996) with the selectable agent benzyladenine N-3-glu- curonide, Haldrup et al. (1998) with d-xylose, Kunze et al. (2001) with 2-deoxyglucose, Erikson et al. (2004) with d-amino acids, Erikson et al. (2005) with d-serine, Ya- mada et al. (2005) and Hsiao et al. (2007) with 5-methyl- trytophan. Also, You et al. (2003) used a ferredoxin-like protein gene and Ebmeier et al. (2004) the Escherichia coli threonine deaminase gene as selectable markers. The mannose-based selection system with phosphomannose isomerase (pmi) gene as a selectable marker was frst re- ported by Joersbo et al. (1998) for the transformation of sugar beet. In the following years, PMI was shown to be a useful marker in the transformation of a number of plant species such as cassava (Zhang et al., 2000), maize (Negrotto et al., 2000; Reed et al., 2001; Wright et al., 2001), Arabidopsis (Todd & Tague, 2001), wheat (Reed et al., 2001; Wright et al., 2001), barley and water- melon (Reed et al., 2001), durum wheat (Gadaleta et al., 2006), rice (Lucca et al., 2001), sweet orange (Boscariol et al., 2003), hemp (Feeney & Punja, 2003), pearl millet (O’Kennedy et al., 2004), tomato (Sigareva et al., 2004), bentgrass (Fu et al., 2005), papaya (Zhu et al., 2005), sor- ghum (Gao et al., 2005), almond (Ramesh et al., 2006), onion (Aswath et al., 2006), cucumber (He et al., 2006), Chinese cabbage (Ku et al., 2006; Min et al., 2007), Tore- nia hybrids (Seitz et al., 2007), fax (Lamblin et al., 2007), sugarcane (Jain et al., 2007), apple (Degenhardt et al., 2007), plum (Mikhailov et al., 2007), potato (Bříza et al., 2008), and citrus (Ballester et al., 2008). Cells of the majority of plant species take up mannose and convert it by endogenous hexokinase into mannose- 6-phosphate (M6P). This inhibits glycolysis, depletes the cells of inorganic phosphate and induces endonu- cleases to degrade DNA (Stein & Hansen, 1999). The non-transformed plant cells starve and cease growing. In PMI-harbouring cells, however, the enzyme catalyses * e-mail: briza@umbr.cas.cz Abbreviations: BAP, 6-benzylaminopurine; MS, Murashige and Skoog medium; NAA, α-naphthalene acetic acid; NPT II, neomycin phosphotransferase II; PMI, phosphomannose isomerase. Vol. 57, No 1/2010 63–68 on-line at: www.actabp.pl