Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing

Abstract

Maize (Zea mays ssp. mays) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium-mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos

“…Transformation with the construct pKL2013 carrying a Cas9/sgRNA cassette targeting the Gl2 gene in maize ( McCaw et al, 2021 ) resulted in the generation of 24 transgenic plants. These plants were characterized by Sanger sequencing to determine the targeted mutagenesis outcomes.…”

“…This phenotype can be observed in both T0 regenerated young plants and T1 seedlings of mutant progenies after misting their leaf surfaces. However, it is much easier to ascertain the phenotype in T1 seedlings than in the T0 plants ( McCaw et al, 2021 ). In this work, although we screened the gl2 mutant phenotype in the T0 plants after the regenerants were acclimated for 10 days ( Figure 7 ), we consider the water spraying assay as preliminary.…”

“…Binary vector pKL2013 ( McCaw et al, 2021 ; Figure 1A ): The T-DNA carries (1) 2X CaMV 35S promoter (P35S) driving the bialaphos resistant ( bar ) gene as a selectable marker, (2) a single P35S driving mCherry gene as a visual marker; (3) maize ubiquitin promoter driving a maize-codon-optimized Cas9 , and (4) rice U3 promoter driving a guide RNA (gRNA) targeting maize glossy2 ( Gl2 ) gene ( McCaw et al, 2021 ).…”

“… Schematic illustration of the ternary vector system used for maize B104 transformation. (A) Binary construct pKL2013 ( McCaw et al, 2021 ), 17,777 bp. PZmUbi, maize polyubiquitin gene promoter; zCas9, maize codon-optimized Cas9 from Streptococcus pyogenes ; TrbcS-E9, transcriptional terminator from Pisum sativum rbcS -E9 gene; POsU3, a promoter from Oryza sativa U3 small nucleolar RNA (snoRNA) gene; ZmGl2-sgRNA1, single-guide RNA targeting maize glossy2 gene ( Lee et al, 2019 ); TOsU6-2, terminator from O. sativa OsU6-2 snoRNA gene; Tvsp, terminator from soybean vegetative storage protein gene; mCherry, red fluorescent protein; P35S, CaMV 35S promoter; bar, bialaphos resistance gene; RB, the T-DNA right border repeat; LB, the T-DNA left border repeat; KanR, kanamycin resistance gene cassette; pBR322 ori, high copy number origin of replication for E. coli ; pVS1 ori, the origin of replication from the plasmid pVS1.…”

Plant genome engineering from lab to field—a Keystone Symposia report

“…They also were amenable to CRISPR/Cas‐mediated mutagenesis. Using this mini‐maize variety can reduce total transformation time from approximately 44 weeks for traditional maize to 22 weeks 82 …”