Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

Abstract

Transformation in grass species has traditionally relied on immature embryos and has therefore been limited to a few major Poaceae crops. Other transformation explants, including leaf tissue, have been explored but with low success rates, which is one of the major factors hindering the broad application of genome editing for crop improvement. Recently, leaf transformation using morphogenic genes Wuschel2 (Wus2) and Babyboom (Bbm) has been successfully used for Cas9-mediated mutagenesis, but complex genome editing applications, requiring large numbers of regenerated plants to be screened, remain elusive. Here we demonstrate that enhanced Wus2/Bbm expression substantially improves leaf transformation in maize and sorghum, allowing the recovery of plants with Cas9-mediated gene dropouts and targeted gene insertion. Moreover, using a maize-optimized Wus2/Bbm construct, embryogenic callus and regenerated plantlets were successfully produced in eight species spanning four grass subfamilies, suggesting that this may lead to a universal family-wide method for transformation and genome editing across the Poaceae.

Sorghum: A Multipurpose Crop

“…Recent studies have identified the basal leaf as an alternative explant for young embryos, and the use of a new promoter combination to enhance the expression of the morphogenetic genes Babyboom ( Bbm ) and Wuschel 2 ( Wus2 ) was able to obtain high transformation efficiency. This method is sufficient to support Cas9-mediated genomic modification techniques with success rates similar to those obtained with immature embryos, and this method demonstrates the advantages of basal leaves as starting explants, opening up new ways of sorghum transformation . In addition, techniques that bypass tissue culture offer new opportunities for rapid genome editing and refinement.…”

“…This method is sufficient to support Cas9-mediated genomic modification techniques with success rates similar to those obtained with immature embryos, and this method demonstrates the advantages of basal leaves as starting explants, opening up new ways of sorghum transformation. 123 In addition, techniques that bypass tissue culture offer new opportunities for rapid genome editing and refinement. In this method, the junction of plant rhizomes was cut under nonsterile conditions, and the upper part was infected with Agrobacterium rhizomes, then cultured on vermiculite medium, and the positive new roots were taken, cut into segments and then cultured, to obtain stable transformation of regenerated transformation plants.…”

Exploring the frontier of rapid prototyping technologies for plant synthetic biology and what could lie beyond

“…This challenge can be overcome using a recently demonstrated approach that enabled de novo formation of transgenic shoot tissue via agrobacterium‐based delivery of plant developmental regulator genes to both explants and adult dicot plants (Maher et al ., 2020). Similarly, the delivery of developmental regulator genes has been recently demonstrated to enable de novo generation of large numbers of somatic embryos from leaf tissue in monocots (Wang et al ., 2023).…”

Carbon Dot Nanozyme for Enhancement of Yield and Quality in Maize through Multimechanism Joint Effects

“…(2) Pesticide spraying can effectively control maize diseases and pests, but some studies have demonstrated that even registered and widely used safety pesticides can cause environmental pollution when improperly applied . (3) Gene editing technology can enhance maize resistance to diseases, pests, and disasters . However, this technology is time-consuming and raises biosafety concerns.…”

“…4 (3) Gene editing technology can enhance maize resistance to diseases, pests, and disasters. 5 However, this technology is time-consuming and raises biosafety concerns. (4) Foliar fertilizers, 6 such as ZnO, 7 can promote nutrient absorption by enhancing metabolization; however, their efficiency needs improvement.…”