Small Molecule Inhibitors of Human LRRK2 Enhance in Vitro Embryogenesis and Microcallus Formation for Plant Regeneration of Crop and Model Species

In vitro plant embryogenesis and microcallus formation are systems which are required for plant regeneration, a process during which cell reprogramming and proliferation are critical. These systems offer many advantages in breeding programmes, such as doubled-haploid production, clonal propagation of selected genotypes, and recovery of successfully gene-edited or transformed plants. However, the low proportion of reprogrammed cells in many plant species makes these processes highly inefficient. Here we report a new strategy to improve in vitro plant cell reprogramming using small molecule inhibitors of mammalian leucine rich repeat kinase 2 (LRRK2), which are used in pharmaceutical applications for cell reprogramming, but never used in plants before. LRRK2 inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of oilseed rape and barley, and somatic embryogenesis in cork oak. These inhibitors also promoted plant cell reprogramming and proliferation in Arabidopsis protoplast cultures. The benzothiazole derivative JZ1.24, a representative compound of the tested molecules, modified the expression of the brassinosteroid (BR)-related genes BIN2, CPD, and BAS1, correlating with an activation of BR signaling. Additionally, the LRRK2 inhibitor JZ1.24 induced the expression of the embryogenesis marker gene SERK1-like. The results suggest that the use of small molecules from the pharmaceutical field could be extended to promote in vitro reprogramming of plant cells towards embryogenesis or microcallus formation in a wider range of plant species and in vitro systems. This technological innovation would help to develop new strategies to improve the efficiency of in vitro plant regeneration, a major bottleneck in plant breeding.