Evaluation of putative seed-specific promoters for Linum usitatissimum

The GUS reporter gene was used to test four different putative seed-specific promoters in developing and mature seeds, leaves and roots from linseed flax (Linum usitatissimum). The promoters tested included the regulatory regions of the beta-ketoacyl-CoA synthase gene (KCS) and the napin protein gene from Brassica napus, the promoter regions of the 'unknown seed protein' (USP), and a legumin protein gene (LeB4) from Vicia faba and the CaMV 35S promoter (positive control). The promoter-GUS constructs were inserted into L. usitatissimum via Agrobacterium mediated transformation, and GUS activity evaluated using histochemical and fluorimetrical assays. All the promoters showed some activity, but only CaMV 35S, LeB4 and USP exhibited an expression level high enough to be useful in linseed flax. Plants with USP-GUS showed the earliest GUS activity at 5 to 6 days after flowering (daf) and persisting until 40 daf. Expression of GUS under the control of the LeB4 promoter was measurable 11 daf and was still detectable at 40 daf. The KCS-GUS construct showed a low level of GUS activity between 14 daf and 40 daf. Plants transformed with USP-GUS or LeB4-GUS exhibited a low level of GUS activity in leaves and roots of some of the transformants, indicating the need for generating large numbers of primary transformants, followed by careful evaluation and selection for ones with not only the desired level of expression, but also the desired spatial and temporal expression.