Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin

With the aim of increasing the rumen-protected level of the sulphur amino a cids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacteriu m tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four diff erent T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carb oxylase (Rubisco) from Arabidopsis thaliana (A(ssu)), the promoter of the g ene encoding the small subunit of Rubisco of Medicago sativa (L-ssu), or th e Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. r epens cv. Haifa. Transgenic T-0-plants and inter-transgenic hybrids were an alysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractab le leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with A(ssu) > CaMV35S > L-ssu. Results from t he inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally ap pears not to be additive with extra transgenic loci. During this work, we a ttempted to improve the efficiency of A. tumefaciens-mediated transformatio n of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-med iated Transformation) on cotyledons of T. repens. T-DNA transfer was in gen eral not enhanced by sonication compared to traditional A. tumefaciens-medi ated transformation. Furthermore, Southern blot analyses of plants regenera ted from the same cotyledon after A. tumefaciens treatment and under select ion, indicated that multiple shoots were usually derived from the same tran sformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic c lones.