New vector systems for maize genetic transformation by CBC members
(August 28, 2023)
Maize transformation is a crucial technique for introducing desired genetic traits; however, this is a challenging process for many laboratores. To improve thisprocess, members of the Iowa State University Crop Bioengineering center built new vector systems for the effective selection of transgenic plants. The journal article, featured as a cover story in the August issue of Plant Physiology is titled "New T-DNA binary vectors with Nptll selection and RUBY reporter for efficient maize transformation and targeted mutagenesis." This work is a collaborative effort of multiple members of the Crop Bioengineering Center, including Keunsub Lee, Minjeong Kang, and Kan Wang of the Agronomy Department as well as Qing (Jessica) Ji and Sehiza Grosic of the Crop Bioengineering Laboratory.
One of the important components in plant genetic transformation and gene editing is a reliable visible marker. This marker facilitates the monitoring of successful advancement in transformation procedures and the subsequent generation's segregation of transgenes. A novel visible marker known as RUBY introduces a captivating purple betalain pigment, offering a distinct advantage over other markers like GUS or Green/Red Fluorescent Proteins (GFP/RFP). The former requires sacrificing source material (GUS), while the latter demands specialized equipment like a fluorescent microscope for marker gene visualization. Enhanced by the upgraded ternary vector system for Agrobacterium-mediated transformation, RUBY stands poised as an exceptional asset for generating transgenic and gene-edited plants. The cover image of Plant Physiology, contributed by the ISU CBC authors, showcases maize reproductive tissues expressing RUBY, each exhibiting unique patterns of betalain accumulation.
The new vector system generated by the CBC members is now available publicly via Addgene, a non-profit plasmid repository.
High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice
(December 26, 2019)
CBC co-director, Kan Wang, CBC members Dipali Sashital, Carolyn Lawrence-Dill and David Wright jointly published a paper in Scientific Reports describing observations made when comparing different methods in delivering CRISPR reagents into rice. This work highlights the importance of molecular screening and the strategy for screening both on- and off-target sites. When the focus and biased assumption is on intended edits, genome rearrangements and unintended insertions can go undetected. Successful identification of unintended insertion and genome rearrangements are important information for further improvement of the CRISPR-mediated genome editing for plants.
Transient gene editing in monocots using a viral delivery system
(November 22, 2019)
CBC co-director, Steve Whitham, has published a paper in Plant Direct describing the use of foxtail mosaic virus (FoMV) to deliver functional guide RNAs to plants expressing the Cas9 protein. FoMV carrying guide RNAs could induce site-specific gene edits in Nicotiana benthamiana, Seteria viridis (green foxtail), and maize plants expressing Cas9 protein. The gene edits were enhanced in the presence of viral silencing suppressors. This work demonstrates the feasibility of using viral systems to delivery gene editing reagents in crop plants, such as maize.
Broad-spectrum resistance to bacterial blight in rice using genome editing
(October 28, 2019)
CBC council member, Bing Yang, has published a paper in Nature Biotechnology describing the use of gene editing to develop rice lines with broad spectrum resistance to bacterial blight disease. Bacterial blight of rice caused by Xanthomonas oryzae pv. oryze (Xoo) is an important disease affecting rice production in Asia and Africa. Xoo produces several transcription activator-like effectors (TALEs) that bind elements within the promoters of rice sucrose transporter genes known as SWEET genes to enhance their expression and promote bacterial growth and disease susceptibility. The team used gene editing to modify the promoters of a set of critical SWEET genes, so that they cannot be activated by Xoo. In a companion paper, the team also developed the SWEETR kit 1.0, which is a diagnostic kit for analyzing bacterial blight occurring in the field and deploying the most effective resistant rice lines. Please see the Nature Biotechnology News & Views article by Varshney et al. for a discussion and summary of this work.