Establishing an Agrobacterium-mediated maize transformation protocol

Abstract

Maize (Zea mays) is one of the top five agricultural crops grown in the world. It is important to keep developing new and improved varieties to counter insecticide-resistant pests, increase yield, and develop varieties that can be grown in areas significantly impacted by climate change. Traditional breeding methods, which tend to be slow, require a large area, and large amounts of fertilizer and pesticide inputs. Novel methods using a vector in vitro to transform maize are also possible. Currently, possible ways to transform and increase transformability are done primarily with the use of commercially available genes, Wuschel (WUS2), Baby boom (BBM) and Growth Regulating Factor- GRF-Interacting Factor (GRF-GIF), which can be cost prohibitive. The aim of this project is to establish a transformation system that can be used efficiently and be made freely available as an alternative to currently available systems. To do this, several experiments were conducted. First, which type of maize explant to use was established. To do this, the most common type of explant, immature embryos, was compared to other potential explants, mature seed embryos, and leaf tissue. This was done with three different varieties, A188, B104 and KSU62. The best explant was determined to be the immature maize embryos. To determine the best way of selecting transformed embryos from untransformed, a selection pressure test comparing common herbicides and selection markers was done. This resulted in G418 Sulfide being the best selection pressure over the more common Bialaphos. Once the explant was determined, transformation using a classical protocol was attempted, and it was determined that several medias needed to be reevaluated, as no transgenic lines or events had occurred. Later, a modified Iowa State University’s ‘QuickCorn’ protocol was used as a modern control protocol, and several transgenic lines have been generated from our lab using this protocol. However, this protocol is not solely our labs, so media was formulated, tested, and compared to the QuickCorn method. The resulting best medias of the preliminary testing have been compiled as a recommended media for a protocol alternative moving forward. However, a modified QuickCorn protocol is a reliable, widely tested way to acquire transformed explants. To test the protocols, transformation needed to occur. Using Agrobacterium strains LBA4404 PYLCRISPR/Cas9_WAT1_Pubi-B, LBA4404 PYLCRISPR/Cas9_WAT1and Gl3_Pubi-B,. The first agrobacterium strains tested was plain LBA4404 with Bialaphos resistant single gene and double gene knock out constructs. Later, to update the materials, a ternary vector strain of LBA4404 harboring PKL2299 and plasmid PYLCRISPR/Cas9_WAT1_Pubi-B, and LBA4404 harboring PKL2299 and plasmid PYLCRISPR/Cas9_WAT1and Gl3_Pubi-B was used. Also, a strain just encoding the visual marker gene RUBY, LBA4404 harboring PKL2299 and plasmid PYLCRISPR/Cas9_PCL101_RUBY, was used to test inoculation media efficacy. From the background experiments it was determined that immature embryos remain the ideal transformation explant. Also, out of the lines tested, A188 and KSU62 had the best regeneration from immature, but B104 is more robust. Finally, it was determined that a reliable method for maize transformation is a modified QuickCorn method, but preliminary data suggests a protocol specifically tailored for our lab that can produce results of equal efficiency and quickness, using G418 as a selection pressure, and adding cysteine, DTT, and acetosyringone to media during the inoculation stage. However, this protocol requires more rigorous testing on different maize varieties using different expressed or excised genes to determine if it is truly a replacement for the known good protocol. Using the modified QuickCorn protocol, transformed maize had been generated, but only for the single gene deletion of WAT1, and only inoculation data for the new protocol has been tested, not generation of mature transgenic plants.