Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2.

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR-Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR-Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

Yield, Insect-Derived Ear Injury, and Aflatoxin Among Developmental and Commercial Maize Hybrids Adapted to the North American Subtropics.

The development of maize (Zea mays L.) hybrids that are adapted to subtropical areas of North America should consider yield potential under heat and moisture stress, and reduced susceptibility to insect herbivory and disease. To aid in this process, maize hybrids (43 developmental and seven non-Bt commercial hybrids) were evaluated for severity of ear injury to Helicoverpa zea (Boddie) and Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), susceptibility to Aspergillus flavus (Link) (Deuteromycetes: Moniliales), and yield. In subtropical Corpus Christi and College Station, TX, field experiments conducted over three years revealed significant differences among maize hybrids with the rank of the selected measurements differing across the two locations. When the location by maize hybrid interaction was not significant, variation across the main factors of maize hybrid genetics (in all cases) and location (in some cases) was detected. In 2014, a significant location by maize hybrid interaction in yield but not aflatoxin and ear injury were likely associated with differences in weather between locations. In Corpus Christi in 2015, a location by maize hybrid interaction was detected for ear injury only. Overall, experimental maize hybrids, containing the inbred line Tx777, displayed partial resistance to insect derived ear injury in both locations, and some hybrid testcrosses exhibited low rates of aflatoxin accumulation while maintaining relatively high yields. Tx777 was selected from populations originating in Bolivia and adapted to subtropical climates. The most promising hybrid testcrosses had lower ear injury and aflatoxin accumulation, and good yield under varying heat and moisture stress at the two subtropical maize growing areas in this study.