Transformation of Teosinte (Zea mays ssp. parviglumis) via Biolistic Bombardment of Seedling-Derived Callus Tissues.

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the maize domestication process and facilitate breeding for future crop improvement. However, a protocol of genetic transformation, which is essential for gene functional analyses, is not available in teosinte. In this study, we report the establishment of a robust callus induction and regeneration protocol using whorl segments of seedlings germinated from mature seeds of Zea parviglumis. We also report, for the first time, the production of fertile, transgenic teosinte plants using the particle bombardment. Using herbicide resistance genes such as mutant acetolactate synthase (Als) or bialaphos resistance (bar) as selectable markers, we achieved an average transformation frequency of 4.17% (percentage of independent transgenic events in total bombarded explants that produced callus). Expression of visual marker genes of red fluorescent protein tdTomato and ß-glucuronidase (gus) could be detected in bombarded callus culture and in T1 and T2 progeny plants. The protocol established in this work provides a major enabling technology for research toward the understanding of this important plant in crop domestication.

Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes.

Switchgrass (Panicum virgatum) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, 'Summer' and 'Blackwell', by ectopic overexpression of morphogenic genes, Baby boom (Bbm) and Wuschel2 (Wus2). Two auxotrophic Agrobacterium strains, LBA4404Thy- and EHA105Thy-, each harboring the same construct containing ZmBbm, ZmWus2, and a green fluorescence protein (GFP) gene, ZsGreen1, were used to infect immature leaf segments derived from in vitro grown seedlings. The Agrobacterium strains also contain a transformation helper plasmid that carry additional copies of Agrobacterium virulence genes. GFP-expressing calli were identified and selected for regeneration. The highest transformation efficiency of 6% was obtained for the tetraploid cultivar Summer when LBA4404Thy- was used for infection, which is twice of that for the octoploid cultivar Blackwell. LBA4404Thy- consistently outperformed EHA105Thy- on transformation frequency across the two cultivars. Fifteen randomly selected putative transgenic plants of Summer and Blackwell, representing independent callus events, were confirmed as transgenic by the presence of the transgene, ZmAls, and the absence of AtuFtsZ, a chromosomal gene specific to the Agrobacterium strain LBA4404 using polymerase chain reaction. Transgene integration and expression was further confirmed by the detection of GFP in roots, and the resistance to herbicide injury to leaves of selected putative transgenic plants. The ZmBbm and ZmWus2 genes were successfully removed from 40 to 33.3% of the transgenic plants of Summer and Blackwell, respectively, via the Cre-Lox recombination system upon heat treatment of GFP-expressing embryogenic calli. Our successful transformation of recalcitrant upland switchgrass provides a method for gene function analysis and germplasm enhancement via biotechnology.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

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

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.