Preparation of Transgenic Barley Producing Essential Polyunsaturated Fatty Acids GLA and SDA
Marcela Gubisova1,3, Daniel Mihalik1, Katarína Ondreickova1, Lenka Klcova1,3,Tatiana Klempova2, Milan Certik2, Martina Hudcovicova1, Jozef Gubis1, Jan Kraic1,3 
1Plant Production Research Centre, Bratislavska 122, 921 68 Piestany, Slovak Republic; 2Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic; 3Faculty of Natural Sciences, Constantine the Philosopher University in Nitra, Nabrezie mladeze 91, 949 74 Nitra, Slovak Republic
 
Barley (Hordeum vulgare L.) is one of the most widely grown cereals in Europe used in the malting and brewing industry, but utilized also for animal feed as well as human food. Polyunsaturated fatty acids in barley grain are represented mainly by linoleic acid (LA; C18:2, n-6) and alpha-linolenic acid (ALA: C18:3, n-6). PUFAs with longer chain are not presented since their biosynthesis is dependent on desaturases and elongases not common for cereals. Changes in the composition of fatty acids in cereal grains are not feasible by classical breeding methods, but genetic transformation may be an alternative approach to reach this goal. In this work we used biolistics to transform immature barleys embryos of cv. Golden Promise with synthetic gene encoding delta-6-desaturase (D6D), an enzyme catalyzing conversion of LA to GLA (gamma-linolenic acid; C 18:3, n-6) and also conversion of ALA to SDA (stearidonic acid; C18:4, n-3). The synthetic gene was prepared according to gene sequence of filamentous fungus Thamnidium elegans. The codon usage was optimized for cereals and signal sequence from the gene encoding Dx5 high-molecular-weight glutenin was added. The gene was cloned into the pLRPT vector with endosperm-specific promoter Dx5. The goal of the work was to induce production of GLA in barley seeds as a result of the reaction catalysed by the product of transgene expression. Successful transformation was confirmed in T0 plants at the genomic level and in T1 seeds at the transcriptomic and metabolomic levels. Transformed plants produced up to 0.141% gamma-linolenic acid (GLA) and 0.294% stearidonic acid (SDA) of the total amount of fatty acids in their grains. 
The work was supported by the Slovak Research and Development Agency under the grants No. APVV-0294-11 and APVV-0662-11.