• Report on first successful alteration of seed protein gene 04/18. Report on first successful alteration of seed protein profile 08/18. This will be based on glycinin knockout and Kas1 overexpression.

For 2018/9
• Transgenic soybean events are now in the greenhouse that contain the methionine pathway from E. coli. These soybean events will be phenotyped in 2019 under both greenhouse and if promising small plots will be planted in the field.
• The genetic construct harboring the arabidopsis sucrose transporter AtSUC2, under control of an early embryo promoter has been introduced into soybean, with transgenic events expected to be established in the greenhouse early summer 2018. Progeny populations derived from these will be characterized for changes in protein/oil and sucrose reserves.
• Modulating programmed cell death in developing embryos. A rebuild of two vectors that harbor the arabidopsis gene call, AtBAG4, a known determinant of cell death in plants was placed under control of a seed specific promoter, either targeting the AtBAG4 protein to the cytosol or to the plastid. The objective is to alter the cell/organelle fate during embryogenesis to impact levels of seed reserves
(protein/oil/sucrose). Soybean transformations with these plasmids were initiated in winter 2018 with expected transgenic events to be established in the greenhouse by late summer 2018. The derived progeny from these events will be characterized for changes in protein/oil/sucrose levels, along with effects on cell fate during embryogenesis.
• High throughput means for editing in soybean. Two constitutive expression cassettes were assembled that carry the Cas9 and Cpf1 variant for genome editing. Soybean transformations have been initiated with these. Selected events will be carried down to homozygosity and seeds bulked. These bulked events will be used as a source to evaluate strategies for throughput introduction of guide RNAs, that bypass tissue culture, as a means to develop a throughput pipeline for genome editing in soybean.

Updated December 9, 2019:
Pretty much all crop seeds exhibit negative correlations between seed components, such that increasing one (e.g. oil) decreases another (e.g. protein). Yield and protein content also tend to be inversely correlated. The prevalence of these phenomena across crops suggests that they are due to the basic biochemistry underlying seed development, and this phenomenon is not well understood. Therefore, a multi-pronged approach to improve seed protein quantity and quality was undertaken. The premise was that since so little was known about increasing protein quantity in seeds, multiple strategies needed to be explored in order to determine the best strategy. These strategies included:
• Altering carbon flow into protein and oil through changes in sucrose transport
• Expressing a gene to impact storage of protein and oil
• RNAi and gene editing technologies to down-regulate a protein-limiting gene
• Gene editing to alter the protein balance through the simultaneous creation of knock-outs that target seed component combinations
• Gene editing to knockout proteins that have a low level of sulfur-containing amino acids. In their absence they could be compensated for by proteins with higher methionine or cysteine content.
• Increasing seed methionine levels by expressing a four-gene metabolic pathway
• Identifying molecular factors that regulate seed growth and development
Several engineered and edited soybean lines are now available for analysis. The results from this analysis should be very useful to select and help design future strategies aimed at improving soybean quality and quantity.

Because genome editing is a new technology, there is still room for improvement. Thus, supporting research aimed to improve knock-out technology for soybean, and develop knock-in technology, as well as to improve transformation efficiency in soybean. The vectors and other tools developed during this proposal are also available and ready for future use.