Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators

Abstract

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

The Promising Nanovectors for Gene Delivery in Plant Genome Engineering

“…DRs are activated during plant regeneration, with dramatic responses in different plant species. In recent years, transient or stable overexpression of DRs, such as BBM, WUS, GRF5, and GRF4-GIF1, have been proved to successfully overcome this bottleneck in many plants’ genetic transformation processes [ 6 , 7 , 8 , 9 , 10 ].…”

“…However, strong genotype dependence and highly technical requirements of these systems limit their broad application [ 4 , 5 ]. In recent years, the utilization of developmental regulators (DRs), which are also referred to as morphogenic regulators (MRs), such as the WUS (WUSCHEL), BBM (BABY BOOM) and GRFs (GROWTH-REGULATING FACTORs), have been proven to overcome genotype dependence in plant regeneration [ 6 , 7 , 8 , 9 , 10 , 11 ], while in many plant species, the delivery of exogenous DNA or RNA is genotype dependent and is the main bottleneck in genetic transformation. Therefore, there is an urgent need to develop new delivery vectors for genome engineering.…”

The combination of morphogenic regulators BABY BOOM and GRF-GIF improves maize transformation efficiency

“…In this study, we combined the morphogenic regulators ZmBBM and TaGRF4-GIF1 in a single binary vector carrying Cas9 to explore the potential of this combination to enhance transformation efficiency in two maize genetic backgrounds, Hi-II and B104, commonly used by transformation facilities. While Hi-II is a mixed genetic background, which can complicate phenotypic analysis of transgenic plants, B104 is an inbred line related to B73, the first maize genome to be sequenced and assembled (SCHNABLE et al 2009;RAJI et al 2018;AESAERT et al 2022;KANG et al 2022). In combination with a modified QuickCorn protocol (MASTERS et al 2020), we found that this system significantly enhanced the efficiency of transformation in both backgrounds, allowing the efficient generation of transgenic plants within a two-month time frame.…”

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

“…12,13 ), and two public maize lines, FFMM 14 and B104 (ref. 15 ). In addition, the expression of Wus2 alone has been shown to be effective for improving transformation in maize 16 and genome editing in sorghum 17 .…”

“…https://doi.org/10.1038/s41477-022-01338-0 was further extended to sorghum transformation 10,12 , sorghum genome editing 37 , maize public inbred transformation 14,15,38 and maize genome editing 15,35,36,39 . Recently, Wox5 has also been demonstrated to improve transformation in numerous wheat varieties, with potential application in other cereals such as Triticum monococcum, triticale, rye, barley and maize 19 .…”