Advancing plant transformation and CRISPR/Cas editing: Strategies for recalcitrant maize

Plant genetic transformation is a pivotal tool for plant biology and crop improvement. While stable genetic transformation methods have been established, their applicability remains constrained to specific plant species and genotypes. The process is further characterized by labor-intensive procedures, particularly maize genetic transformation, which takes 165 days. Given the escalating requirements for genetic transformation and gene editing, there is a growing imperative for the development of a more efficient genetic transformation process. In addition, the selection of an efficient CRISPR/Cas based gene editing system and target sequence becomes imperative prior to stable genetic transformation, particularly when dealing with recalcitrant plant species. Comprehending the factors that impact genetic transformation and subsequently optimizing these factors are crucial steps in enhancing the overall success rate and precision of attaining desired traits. To improve the conventional B104 maize genetic transformation method, we incorporated the Agrobacterium ternary vector system aimed at enhancing T-DNA delivery. Medium with phytohormone profiles was tested to reduce the regeneration process. The new method drastically reduced the total transformation process to 51 days while maintaining a comparable transformation frequency. Our study demonstrated the efficacy of CRISPR/Cas-mediated targeted mutagenesis using this method. The result showed the successful induction of indel mutations at the target site, validating the robustness of the approach in achieving precise gene editing. Although the rapid transformation drastically reduced the tissue culture time with comparable transformation efficiency, the inefficacy of bialaphos selection in suppressing non-transgenic regenerants was observed due to shorten regeneration step. In response, we developed a new T-DNA binary vectors with neomycin phosphotransferase II (NptII) selection and RUBY reporter for efficient selection fits rapid transformation process. CRISPR/Cas constructs with engineered sgRNA scaffold was incorporated for efficient targeted mutagenesis as well. With the efficient targeted mutagenesis frequency and improved selection/visual marker system, the new T-DNA binary vectors can provide highly effective system for both the genetic transformation and targeted mutagenesis in maize. A continuous maize transformation improvement and genome editing work was developed for recalcitrant genotypes as well. Recalcitrant maize transformation and potential DNA-free genome editing using non-integrating Wuschel2 (NIW) were demonstrated in maize B104 and B73. The simple addition of an Agrobacterium carrying the NIW vector led to a doubling of the transformation frequency in B104. The NIW-mediated transformation of the recalcitrant B73 genotype was explored under selection-free conditions, aiming to achieve edited plants without T-DNA integration. Indeed, this approach introduces an option for DNA-free editing in maize transformation, representing a significant advancement in CRISPR-mediated genome editing. In addressing the challenges in plant transformation and gene editing systems for recalcitrant crops, we devised a strategy involving robust protoplast isolation and the effective application of protoplasts for CRISPR/Cas editing. We offered a detailed, sequential protocol for isolating mesophyll protoplasts from in vitro cultured pennycress and soil-grown camelina. The protocol encompasses instructions for both DNA transfection and assessing protoplast viability test through fluorescein diacetate. The result demonstrated high-yield isolation, successful transfection, and transient expression of protoplast. Our efforts extended to devising a CRISPR/Cas evaluation system based on protoplasts, utilizing a dual-fluorescence marker for broad application. We systematically examined multiple factors influencing transfection by utilizing Nicotiana benthamiana protoplasts. Target construct containing target sequence flanked between dual-fluorescence and CRISPR/Cas construct were co-transfected. The dual-fluorescence system successfully demonstrated the targeted mutation efficiency by CRISPR/Cas construct. This dissertation holds significant potential for advancing plant genetic transformation and genome editing practices, particularly for recalcitrant crops. Our contributions encompass not only methodological improvements but also innovative approaches that pave the way for more efficient, precise, and DNA-free genome editing in plant systems.