Advancing Agrobacterium-Based Crop Transformation and Genome Modification Technology for Agricultural Biotechnology

Abstract

The last decade has seen significant strides in Agrobacterium-mediated plant transformation technology. This has not only expanded the number of crop species that can be transformed by Agrobacterium, but has also made it possible to routinely transform several recalcitrant crop species including cereals (e.g., maize, sorghum, and wheat). However, the technology is limited by the random nature of DNA insertions, genotype dependency, low frequency of quality events, and variation in gene expression arising from genomic insertion sites. A majority of these deficiencies have now been addressed by improving the frequency of quality events, developing genotype-independent transformation capability in maize, developing an Agrobacterium-based site-specific integration technology for precise gene targeting, and adopting Agrobacterium-delivered CRISPR-Cas genes for gene editing. These improved transformation technologies are discussed in detail in this chapter.

Site‐specific recombinase genome engineering toolkit in maize

“…Multiple transgenes in different loci become increasingly difficult to maintain in subsequent generations due to independent segregation in meiosis, requiring a labor‐intensive and time‐consuming process of introgression into agronomically significant cultivars. Additionally, recovered events from plant transformations exhibit differences in expression level, copy number, and can possibly interfere with endogenous gene function; therefore, it is necessary to screen each event for optimal insertion locations, eliminating upwards of 90% of T 0 plants under commercial parameters (Anand & Jones, 2018). Recent developments of precise genome editing techniques, such as zinc‐finger nucleases (ZFN) (Bibikova, Beumer, Trautman, & Carroll, 2003; Carroll, 2011), transcription activator‐like effector nucleases (TALENs) (Christian et al, 2010; Bogdanove and Voytas 2011), and CRISPR‐Cas9 (Jinek et al, 2012), create promising avenues for targeted modifications; however, they have a low frequency of transgene targeting.…”

“…Multiple transgenes in different loci become increasingly difficult to maintain in subsequent generations due to independent segregation in meiosis, requiring a labor-intensive and time-consuming process of introgression into agronomically significant cultivars. Additionally, recovered events from plant transformations exhibit differences in expression level, copy number, and can possibly interfere with endogenous gene function; therefore, it is necessary to screen each event for optimal insertion locations, eliminating upwards of 90% of T 0 plants under commercial parameters (Anand & Jones, 2018).…”

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus

“…However, integration of tandem repeats or truncated copies of T-DNA, and disruption of critical genomic regions cannot be ruled out. These features of random transformation methods pose major challenges for multigene transformation, rendering a high number of the recovered events unsuitable for product development (Anand and Jones, 2018;Halpin, 2005).…”

High efficiency Agrobacterium‐mediated site‐specific gene integration in maize utilizing the FLP‐FRT recombination system

“…Therefore, it is a common practice to discard events with transgenes in or near endogenous genes. Since 60%-75% of otherwise quality events fail to meet the above criteria, cumulatively >90% of the transgenic T0 events are discarded (Anand and Jones, 2018). One possible approach to reduce this attrition is to accurately insert transgenes into well characterized insertion sites through site-directed integration (Akbudak et al, 2010;Cardi and Neal Stewart, 2016;Rinaldo and Ayliffe, 2015).…”