Improved soybean transformation for efficient and high throughput transgenic production.

Although genetic transformation of soybean dates back to over two decades, the process remains inefficient. Here, we report the development of an organogenesis-based transformation method of soybean that resulted in an average transformation frequency of 18.7%. This improved method resorts to Agrobacterium-mediated transformation of the split-seed explant with an attached partial embryonic axis obtained from an imbibed seed. In addition to the split-seed explant, Agrobacterium strain and preparation were shown to be important for improved transformation. Transformation with Agrobacterium tumefaciens EHA105 generated higher transformation frequencies and number of low copy events compared to the strain EHA101. In this system, phosphinothricin acetyl transferase conferring tolerance to glufosinate was successfully employed for efficiently producing transgenic events. Around 48% of the T1 progeny was demonstrated to be heritable based on molecular analysis and screening with the herbicide Liberty®. This method was shown to be applicable to different genotypes and a few elite lines showed high transformation frequencies. This split-seed system with an attached partial embryonic axis serves not only as an efficient means for high throughput transgenic production for basic research studies but also for the commercial development of transgenic soybean products.

Zinc finger nuclease-mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non-homologous end joining.

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence-specific endonucleases to generate DNA double strand breaks (DSBs) at user-specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non-homologous end joining (NHEJ)-mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology-directed repair (HDR)-mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2-1a (FAD2-1a) locus of embryogenic cells in tissue culture. We then describe ZFN- and NHEJ-mediated, targeted integration of two different multigene donors to the FAD2-1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T1 progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ-integrated donor were perfect or near-perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ-mediated targeted insertions of multigene donors at an endogenous genomic locus.

Analysis of xyloglucan fucosylation in Arabidopsis.

Xyloglucan (XyG) is a load-bearing primary wall component in dicotyledonous and non-graminaceous monocotyledonous plants. XyG fucosyltransferase (FUTase), encoded by the Arabidopsis gene AtFUT1, directs addition of fucose (Fuc) residues to terminal galactose residues on XyG side chains. Reverse transcription-polymerase chain reaction and analysis of promoter-beta-glucuronidase transgenic plants indicated highest expression of AtFUT1 in the upper portion of elongating inflorescence stems of Arabidopsis. XyG FUTase activity was highest in Golgi vesicles prepared from growing Arabidopsis tissues and low in those isolated from mature tissues. There was no discernible correlation between the Fuc contents of XyG oligosaccharides derived from different Arabidopsis organs and the level of AtFUT1 expression in the organs. Thus, organ-specific variations in AtFUT1 expression and enzyme activity probably reflect differential rates of cell wall biosynthesis, rather than differences in levels of XyG fucosylation. The effects of manipulating AtFUT1 expression were examined using an Arabidopsis mutant (atfut1) containing a T-DNA insertion in the AtFUT1 locus and transgenic plants with strong constitutive expression of AtFUT1. No Fuc was detected in XyG derived from leaves or roots of atfut1. Plants overexpressing AtFUT1 had higher XyG FUTase activity than wild-type plants, but the XyG oligosaccharides derived from the transgenic and wild-type plants contained comparable amounts of Fuc, indicating that suitable acceptor substrates are limiting. Galactosyl residues had slightly higher levels of O-acetylation in XyG from plants that overexpressed AtFUT1 than in XyG from wild-type plants. O-Acetylation of galactose residues was considerably reduced in Fuc-deficient mutants (atfut1, mur1, and mur2) that synthesize XyG containing little or no Fuc. These results suggest that fucosylated XyG is a suitable substrate for at least one O-acetyltransferase in Arabidopsis.