Comparative analysis of maize leaf whorl-based transformation using ABA-induced Bbm/Wus2 removal in two Agrobacterium ternary vector systems

Abstract

Zea mays L. (maize) transformation is important for crop improvement and advancing plant genetics. One of the most commonly used methods is Agrobacterium-mediated transformation of immature maize embryos. However, this approach is often inefficient and requires costly greenhouse infrastructure for the production of high-quality ears. Moreover, immature embryo tissue culture response exhibits seasonal variation even under optimal greenhouse conditions. Recently, a novel approach using seedling-leaf whorl explants, combined with morphogenic genes BABY BOOM (Bbm) and WUSCHEL2 (Wus2) and an Agrobacterium ternary vector system, has emerged as a promising alternative. In this study, the leaf whorl-based transformation method was evaluated on two maize lines, PHR03 and B73, across two different institutions. Additionally, the performance of two helper plasmids, the proprietary PHP71539 and the publicly available pKL2299, was compared. Importantly, the study also utilized a maize drought-inducible Rab17 promoter to activate the Cre/loxP system, enabling the excision of the Bbm and Wus2 cassettes to mitigate negative effects on plant development in regenerated shoots. The comparative analysis revealed no significant differences in transformation efficiency or usable transgenic events between the two helper plasmids. These findings confirm the successful use of the publicly available pKL2299 plasmid for maize leaf whorl-based transformation. This approach offers a faster, more accessible, and cost-effective method that does not require high-quality greenhouse conditions typically required for explant preparation using immature embryos.