Establishment of a Central Crop Transformation Facility of the Interdisciplinary Center for Crop Plant Research (IZN)

A major concern of the IZN is the identification of genes and processes that are involved in the response of cereal crops to abiotic and biotic stress conditions. In addition to the crops currently studied, barley and maize, wheat will most likely be in the focus of upcoming research consortia. Direct evidence of the roles that candidate genes play in stress responses can only be obtained through either downregulating or upregulating their expression in the crop species of interest. Such a functional characterization of genes can be achieved by the transformation with overexpression or RNAi-knockdown contructs, respectively. Inducible and tissue-specific promoters are available for this purpose. Unfortunately, the genetic transformation of the aforementioned crop species is not straightforward. In the past, feasible yet laborious methods have been developed for certain cultivars of certain species. For barley, for example, Agrobacterium-mediated transformation of immature embryos has first been described in 1997 (Tingay et al., 1997) and has become the method of choice (Harwood, 2012). With certain barley genotypes, transformation efficiencies of more than 10% can be achieved (Bartlett et al., 2008). Similar to barley, reliable protocols for Agrobacterium-mediated transformation of immature maize embryos have been published (Ishida et al., 2007). Within the past funding period of the IZN, we have successfully established a barley transformation protocol in our laboratory, which uses the scutellum of immature embryos as target tissue. This groundwork was strongly promoted by the support of J. Imani (IPAZ, Justus-Liebig-Universität Giessen). The efficiency of our protocol compares very well with figures described in the literature. In addition to GFP control lines, barley lines that overexpress or are knocked down of cation transporter and polyamine metablism genes have been produced and are currently being genotypically and phenotypically analysed. In addition, transformants are being or will be generated to test candidate genes that have emerged in other IZN projects (K. Humbeck, S. Rosahl). Albeit very reproducible, the immature-embryo technique has some important drawbacks.
Based on the experience with barley transformation, we propose to maintain and extend this facility as an IZN service (“Z”) project. First, the established protocol will enable a continued functional characterization of genes in the barley cultivar “Golden Promise”. Second, the exploration and establishment of alternative, ideally non-tissue-culture-based methods, will propel the functional research on other cultivars and other crops.