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.
Highlights
Recent years have ushered in rapid advances in precise gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems (Jinek et al, 2012; Zetsche et al, 2015)
We demonstrate that these lines can be transformed using an Agrobacterium-mediated standard transformation method for efficient targeted mutagenesis by a CRISPR-Cas9 system
Efforts in attempting to transform Fast-Flowering Mini-Maize (FFMM)–A and –B lines were unsuccessful in our hands because they are unable to produce embryogenic callus using standard conventional tissue culture and transformation protocols
Summary
Recent years have ushered in rapid advances in precise gene editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems (Jinek et al, 2012; Zetsche et al, 2015). The most successful methods have traditionally relied on transformation of embryogenic callus derived from the scutellum of immature zygotic embryos (IZEs). This method has been widely used in maize transformation, few inbred maize lines are capable of readily producing embryogenic callus that can be transformed and regenerated into plants. Type-II callus in maize is typically induced by an N6-based medium and was originally derived from embryos of A188 or B73 × A188 hybrids (Armstrong and Green, 1985; Tomes and Smith, 1985). The regenerant seed (R1) plants were grown and tested for ∼100% Type-II callus formation in half ears and the remaining R2 seed from two plants of each embryo lineage were used to produce sib populations that comprise “Parent A” and “Parent B” (Armstrong et al, 1991)
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