Progress of cereal transformation technology mediated by Agrobacterium tumefaciens

Abstract

Monocotyledonous plants were believed to be not transformable by the soil bacterium Agrobacterium tumefaciens until two decades ago, although convenient protocols for infection of leaf disks and subsequent regeneration of transgenic plants had been well established in a number of dicotyledonous species by then. This belief was reinforced by the fact that monocotyledons are mostly outside the host range of crown gall disease caused by the bacterium and by the failures in trials in monocotyledons to mimic the transformation protocols for dicotyledons. However, a key reason for the failure could have been the lack of active cell divisions at the wound sites in monocotyledons. The complexity and narrow optimal windows of critical factors, such as genotypes of plants, conditions of the plants from which explants are prepared, tissue culture methods and culture media, pre-treatments of explants, strains of A. tumefaciens, inducers of virulence genes, transformation vectors, selection marker genes and selective agents, kept technical hurdles high. Eventually it was demonstrated that rice and maize could be transformed by co-cultivating cells of callus cultures or immature embryos, which are actively dividing or about to divide, with A. tumefaciens. Subsequently, these initial difficulties were resolved one by one by many research groups, and the major cereals are now transformed quite efficiently. As many as 15 independent transgenic events may be regenerated from a single piece of immature embryo of rice. Maize transformation protocols are well established, and almost all transgenic events deregulated for commercialization after 2003 were generated by Agrobacterium-mediated transformation. Wheat, barley, and sorghum are also among those plants that can be efficiently transformed by A. tumefaciens.

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

“…Likewise, the number of transformation sites (blue spots per embryo) was also lower for the BR 451 (11.7) compared to Hi-II (14.6) (Figure 1). Differences among maize genotypes in the capacity to be infected have been widely reported in the literature (Huang & Wei, 2005;Frame et al, 2006;Omer et al, 2013;Ombori et al, 2014;Souza et al, 2017), with genotype dependence being the main limitation in maize genetic transformation by this method (Hiei, Ishida, & Komari, 2014). Genotypic differences can be explained by compatibility variation between maize genotype and the bacterial strain employed in the transformation or the T-DNA transference machinery encoded by the bacteria and plant cell genomes (Ombori et al, 2014;Lowe et al, 2016).…”

“…According to Hiei et al (2014), high temperature during co-cultivation may also result in agrobacteria overgrowth in the co-cultivation medium. The present study showed that high temperature (28°C) was unfavorable for transient transformation but did not promoted bacterial overgrowth even during a prolonged co-culture period.…”

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

“…This result confirms the fact that the resistance of selected calli to chlorsulfuron is due to the expression of the ALS transgene. Despite the progress and the new interest in using Agrobacteriummediated methods for in vitro transformation of graminaceous (Jha et al, 2011;Sharma et al, 2011;Plaza-Wüthrich and Tadele, 2012;Hiei et al, 2014), the particles bombardment is still an effective technique for the introduction of transgenes in monocot plant cells with an average transformation frequency of more than 50% (Goldman et al, 2003). Thus, our results indicate that the biolistic method is suitable for in vitro transformation of P. glaucum varieties of Côte d'Ivoire because these varieties are recalcitrant to plants regeneration from protoplasts (Tiécoura et al, 2015).…”

“…As compared to dicotyledons plants, graminaceous are recalcitrant to transformation methods using the Ti plasmid as vector (Plaza-Wüthrich and Tadele, 2012;Hussain et al, 2013;Sah et al, 2014). The major obstacles to this gene delivery method were attributed to the limited host-range of Agrobacterium (Hwang et al, 2013;Qamar et al, 2015), and to the different responses of monocots (cell death) and dicots (cell division) to wounds (Potrykus, 1990;Hiei et al, 2014). A variety of techniques such as polyethylene glycol (PEG) method, electroporation method, mild ultrasonication and biolistic have been proposed for genetic transformation of cereals.…”

Advancing Crop Transformation in the Era of Genome Editing

“…Recalcitrance to tissue culture and transformation limits efforts to use transgenesis and genome editing for crop functional genomics research (Shrawat and Lörz, 2006;Hiei et al, 2014). Efficient Agrobacterium-mediated transformation is typically limited to a narrow range of genotypes within a species (Nam et al, 1997).…”