Rapid and Highly Efficient Morphogenic Gene-Mediated Hexaploid Wheat Transformation

Abstract

The successful employment of morphogenic regulator genes, Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2), for Agrobacterium-mediated transformation of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) has been reported to improve transformation by inducing rapid somatic embryo formation. Here, we report two morphogenic gene-mediated wheat transformation methods, either with or without morphogenic and marker gene excision. These methods yield independent-transformation efficiency up to 58% and 75%, respectively. In both cases, the tissue culture duration for generating transgenic plants was significantly reduced from 80 to nearly 50 days. In addition, the transformation process was significantly simplified to make the procedure less labor-intensive, higher-throughput and more cost-effective, by eliminating the requirement for embryonic axis excision, bypassing the necessity for prolonged dual-selection steps for callus formation, and obviating the prerequisite of cytokinin for shoot regeneration. Furthermore, we have demonstrated the flexibility of the methods and generated high-quality transgenic events across multiple genotypes using herbicide (phosphinothricin, ethametsulfuron)- and antibiotic (G418)-based selections.

Diversity of Transgene Integration and Gene Editing Events in Wheat (T<em>riticum aestivum</em> L.) Transgenic Plants Generated Using Agrobacterium-Mediated Transformation

“…This is a significant increase from the biolistic-mediated transformation (0.1-1%) previously used in our study [17]. Similarly to the recently developed "QuickWheat" transformation method with overexpression of maize WUS and BBM genes [7], we observed a low 5% escape rate on hygromycin selection, as confirmed by PCR. The quality of transgenic events was evaluated by estimating the transgene copy number and integrity of the T-DNA borders.…”

“…Until recently, the stable wheat transformation was relatively challenging with high variability, genotype-dependent, resulting in low transformation efficiency (1-2%). Delivery of growth-regulating and regenerationrelated genes such as TaWox5 [6], maize Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2) [7] and GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) GRF-GIF [8] results in a significant boost in transformation efficiency ranging from 58% to 75%. Although higher transformation efficiency allows for reproducible generation of transgenic plants carrying the CRISPR/Cas9 constructs in different experiments, editing efficiency at different loci still varies significantly.…”

A framework for date palm (Phoenix dactylifera L.) tissue regeneration and stable transformation

“…Morphogenic regulators are instrumental in improving tissue culture. For instance, overexpressing these genes promotes somatic embryo formation without additional hormones (Gordon‐Kamm et al, 2019; Horstman et al, 2017; Lian et al, 2022; Liu et al, 2014; Lowe, et al, 2016; Yarra & Krysan, 2023) and significantly enhances the transformation efficiency in monocotyledonous plants, including maize, sorghum, sugarcane, and rice (Duan et al, 2022; Johnson et al, 2023; Kausch et al, 2021; Mookkan et al, 2017; Nalapalli et al, 2021; Nelson‐Vasilchik et al, 2022; Zhou et al, 2022; Lowe et al, 2016). Furthermore, the recent development of the non‐invasive reporter RUBY has facilitated screening in genetically transformed plants by producing a red coloring (He et al, 2020; Yu et al, 2023).…”

Using Gene Editing Strategies for Wheat Improvement

“…One such tool is wheat transformation, a prerequisite for many CRISPR-Cas gene editing applications, which until lately had been languishing with low transformation efficiency ~5% and suffered with genotype dependence. Developments in open access robust transformation protocols and the use of morphological genes to improve regeneration, transformation efficiency and reduce genotype dependence have recently made outstanding improvements [9][10][11].…”