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Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)