Maize (Zea mays) Hi‐II Transformation via Agrobacterium‐Mediated T‐DNA Transfer

Abstract

Genetic transformation of maize via Agrobacterium tumefaciens is still more art than science, with different researchers achieving substantially different transformation results. This article describes our advanced Agrobacterium‐mediated transformation system in Hi‐II maize. The system utilizes simple binary vectors and immature embryos for the transformation, employing the bar gene as a plant selectable marker in combination with bialaphos for subsequent culture selection. The transformation process is efficient and highly reproducible. Certain inbred maize lines can also be transformed with some modification of the system. © 2016 by John Wiley & Sons, Inc.

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties

“…A basic protocol based on published data ( Ishida et al, 1996 ; Frame et al, 2002 ; Zhao et al, 2002 ; Huang and Wei, 2005 ; Frame et al, 2006 ; Ishida et al, 2007 ; Vega et al, 2008 ; Lee and Zhang, 2016 ) is currently being used for routine maize transformation using IZEs as explant in many laboratories worldwide. The main steps of this routine maize transformation ( Figure 1 ) involve plant growth under controlled conditions ( Figure 1A ), harvesting ears at 10–16days post-pollination, selecting 1.2–2-mm IZEs ( Figures 1B – C ), infecting with Agrobacterium harbouring the desired construct ( Figure 1D ), monitoring the infection rate using GUS-harbouring vectors ( Figure 1E ), incubating the infected embryos in the dark at 21°C for resting ( Figure 1F ), transferring the infected embryos to the first-round selective medium in the dark at 25°C ( Figure 1G ), transferring the infected embryos to the second-round selective medium in the dark at 25°C ( Figure 1H ), transferring the resistant embryogenic calli to the first-round regeneration medium in the dark at 25°C ( Figure 1I ), transferring the regenerating plants to second-round regeneration medium to rooting in penumbra light (16hs) at 25°C ( Figures 1J – K ), transferring the regenerated plants to the acclimation room at 26°C/22°C day/night, 16h light ( Figure 1L ) and finally transferring the acclimated plants to the greenhouse for plant growth and T 1 seed production ( Figures 1M – O ).…”

<b>Increased transient genetic transformation <i>in immature</i> embryos of Brazilian BR 451 maize co-cultivated with <i>Agrobacterium tumefaciens

“…The first transgenic maize plants obtained by agrobacteria were reported by Ishida et al (1996) followed by other groups (Zhao et al, 2001;Frame et al, 2002;Ombori, Muoma, & Machuka, 2014;Lee & Zhang, 2016;Souza et al, 2017). To succeed in maize transformation, it is necessary to use target tissues with high rates of cell division, such as immature zygotic embryos.…”

“…While several maize genotypes have been effectively transformed (Ji, Xu, & Wang, 2013, Ombori et al, 2014Souza et al, 2017), Hi-II hybrid maize has been widely used for genetic transformation (Zhao et al, 2001;Frame et al, 2002;Frame, Main, Schick, & Wang, 2011;Lee & Zhang, 2016;Nahampun, Lopez-Arredondo, Xu, HerreraEstrella, & Wang, 2016) and is considered the model for maize plant transformation. However, the poor agronomic traits of the Hi-II hybrid (Ishida, Hiei, & Komari, 2007;Que et al, 2014) motivate the search for other maize genotypes that respond well to transformation.…”

Towards understanding maize domestication: Genetic transformation and genomic editing of teosinte (Zea mays ssp. parviglumis)

“…It also requires the production of immature zygotic embryos that are costly and timeconsuming. Additionally, the media and methods of callus selection and regeneration are not uniform between these lines (Raji et al 2018;Lee and Zhang, 2016). Automation of tissue culture could improve throughput and make transformation scalable and affordable to independent laboratories as a service, as well as making large-scale knock-out screens feasible (Altpeter et al 2016.…”