Glycine-rich proteins (GRPs) containing more than 60% glycine have been found in different tissues from many eukaryotic species. Despite the availability of literature on different groups of GRPs, there are few reports in which they are all considered and compared together. Some of these proteins are components of the cell walls of many higher plants. In most cases, it has been shown that they are accumulated in the vascular tissues and that their synthesis is part of the plant's defense mechanism. Other distinct types of GRPs are characterized by having structures and functions similar to animal cytokeratins or by a domain with typical RNA-binding motifs. The availability of cloned GRP genes facilitates the study of the function of this diverse class of proteins, which is expected to enhance the understanding of cell physiology.

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L-1 kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.

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The physical barrier imposed by the thick microspore wall constitutes an obstacle for an efficient Agrobacterium-mediated transformation of vacuolate microspores prior to androgenic induction and haploid embryogenic commitment. It is thus necessary to implement additional methods to overcome this drawback. In this study, we focused on the optimization of a protocol to allow for the exogenous DNA to enter the microspore in an efficient manner. We tested different options, based on microprojectile bombardment, to be applied prior to agroinfiltration. From them, the best results were obtained through co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by Agrobacterium tumefaciens infection. This method provides an efficient means to integrate extraneous DNA into rapeseed microspores prior to androgenesis induction.

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