The liverwort Marchantia polymorpha L. is being developed as an emerging model plant, and several transformation techniques were recently reported. Examples are biolistic- and Agrobacterium-mediated transformation methods. Here, we report a simplified method for Agrobacterium-mediated transformation of sporelings, and it is termed Agar-utilized Transformation with Pouring Solutions (AgarTrap). The procedure of the AgarTrap was carried out by simply exchanging appropriate solutions in a Petri dish, and completed within a week, successfully yielding sufficient numbers of independent transformants for molecular analysis (e.g. characterization of gene/protein function) in a single experiment. The AgarTrap method will promote future molecular biological study in M. polymorpha.

Changes in the subcellular localisation of chloroplasts help optimise photosynthetic activity under different environmental conditions. In many plants, this movement is mediated by the blue-light photoreceptor phototropin. A model organism with simple phototropin signalling that allows clear observation of chloroplasts would facilitate the study of chloroplast relocation movement. Here, we examined this process in the simple thalloid liverwort Apopellia endiviifolia. Transverse sections of the thallus tissue showed uniformly developed chloroplasts and no air chambers; these characteristics enable clear observation of chloroplasts and analysis of their movements under a fluorescence stereomicroscope. At 22 C, the chloroplasts moved to the anticlinal walls of cells next to the neighbouring cells in the dark (dark-positioning response), whereas they moved towards weak light (accumulation response) and away from strong light (avoidance response). When the temperature was reduced to 5 C, the chloroplasts moved away from weak light (cold-avoidance response). Hence, both light-and temperature-dependent chloroplast relocation movements occur in A. endiviifolia. Notably, the accumulation, avoidance and cold-avoidance responses were induced under blue-light but not under red-light. These results suggest that phototropin is responsible for chloroplast relocation movement in A. endiviifolia and that the characteristics are similar to those in the model liverwort Marchantia polymorpha. RNA sequencing and Southern blot analysis identified a single copy of the PHOTOTROPIN gene in A. endiviifolia, indicating that a simple phototropin signalling pathway functions in A. endiviifolia. We conclude that A. endiviifolia has great potential as a model system for elucidating the mechanisms of chloroplast relocation movement. | INTRODUCTIONChloroplasts are organelles that perform photosynthesis to sustain plant growth and development. The intracellular locations of chloroplasts change in response to the ambient environments, a process that is important for the optimisation of light utilisation for photosynthesis.Various types of chloroplast relocation movement in response to environmental conditions (e.g., light and temperature) have been reported in model plants such as the thale cress Arabidopsis thaliana, the fern Adiantum capillus-veneris, the moss Physcomitrella patens and the

Liverworts are key species for studies of plant evolution, occupying a basal position among the land plants. Marchantia polymorpha has emerged as a highly studied model liverwort, and many relevant techniques, including genetic transformation, have been established for this species. Agrobacterium-mediated transformation is widely used in many plant species because of its low cost. Recently, we developed a simplified Agrobacterium-mediated method for transforming M. polymorpha, known as AgarTrap (agar-utilized transformation with pouring solutions). The AgarTrap procedure, which involves culturing the liverwort tissue in various solutions on a single solid medium, yields up to a hundred independent transformants. AgarTrap is a simple procedure, requiring minimal expertise, cost, and time. Here, we investigated four factors that influence AgarTrap transformation efficiency: (1) humidity, (2) surfactant in the transformation buffer, (3) Agrobacterium strain, and (4) light/dark condition. We adapted the AgarTrap protocol for transforming intact gemmalings, achieving an exceptionally high transformation efficiency of 97%. The improved AgarTrap method will enhance the molecular biological study of M. polymorpha. Furthermore, this method provides new possibilities for improving transformation techniques for a variety of plant species.

Agrobacterium-mediated genetic transformation is a powerful technique in plant biology. We recently developed a simplified Agrobacterium-mediated genetic transformation method for the liverwort Marchantia polymorpha, named AgarTrap (agar-utilized transformation with pouring solutions). AgarTrap is easy to perform; all procedures can be completed within a week using a single plate of solid medium, and basic operations involve simply pouring the appropriate solutions onto the solid medium. Thus far, we have developed three types of AgarTrap methods (S-AgarTrap, G-AgarTrap, and T-AgarTrap) using three different M. polymorpha tissues: sporelings, intact gemmalings, and mature thallus pieces, respectively. Each AgarTrap method can be used to transform tissues at high efficiency, thereby producing sufficient numbers of transformants for study. The ease and efficiency of these AgarTrap methods will likely prompt widespread molecular biological analyses of M. polymorpha. In this review, we describe the basic characteristics of the three AgarTrap methods and present the detailed protocols used in our laboratory.