An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos

TLDR

Maize genome editing requires efficient transformation of inbred lines, yet most are recalcitrant; the B104 inbred, closely related to the reference B73, has become a popular but traditionally slow and low‑efficiency target, taking 16–22 weeks and yielding only ~4 % transformation. This method paper presents an accelerated B104 transformation protocol that reduces production time to 7–10 weeks and increases transformation frequency to ~6.4 %. The protocol employs an Agrobacterium ternary vector system comprising a T‑DNA binary vector and a compatible helper plasmid, following a detailed, stepwise procedure that can be readily adopted by other researchers. Using this protocol, over 66 % of transgenic plants carried CRISPR/Cas9‑induced indel mutations, demonstrating its suitability for genome‑editing applications.

Abstract

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.