Improved G-AgarTrap: A highly efficient transformation method for intact gemmalings of the liverwort Marchantia polymorpha

Abstract

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.

Transformation of Riccia fluitans, an Amphibious Liverwort Dynamically Responding to Environmental Changes

“…At first, a method based on M. polymorpha transformation protocols [ 18 ] was tested using chopped R. fluitans 001TC LF thallus pieces, which failed to produce lines surviving the hygromycin selection. Several modifications such as injuring thallus pieces in a bead ruptor, enzymatic digestion of cell walls and testing different Agrobacterium strains (EHA105, LBA4404, GV3101) as well as co-cultivation on solid medium and in the dark, reported to increase M. polymorpha transformation efficiencies [ 33 ], also failed to generate hygromycin-resistant R. fluitans lines expressing mCherry.…”

Extensive N4 Cytosine Methylation is Essential forMarchantiaSperm Function

“…Gemmae (Mpdnmt3b and Mpcmta knockdown mutants) or F1 spores (Mpdn4mt1 CRISPR/Cas9 deletion mutants and proMpDN4MT1a/b:MpDN4MT1a/b-Citrine lines) were transformed with the above constructs using agrobacteria transformation (strain GV6620) and positive transformants were selected on hygromycin (Mpdnmt3b and Mpcmta knockdown mutants, and Mpdn4mt1 knockout mutant) or gentamycin (proMpDN4MT1a/b:MpDN4MT1a/b-Citrine lines), as described (45,46). The knockdown lines were checked by qPCR using antheridiophores as previously described (14), with primers JW778 and JW779 for Mpcmta knockdown, primers JW448 and JW449 for Mpdnmt3b knockdown, and primers JW438 and JW439 (Table S5) to compare expression to MpEF1α (Mp3g23400).…”

Genome Editing in Plants: Exploration of Technological Advancements and Challenges

“…[187]. Many other novel methods—such as magnetofaction using iron nanoparticles for pollen transformation [188], agar-trap method for liverwort transformation [189], mesoporous silica nanoparticles [190,191], carbon nano-fibres [192], and fluorescently labeled starch nano-particles [193]—have been developed which majorly use particle bombardment but can be used on a very wide platform and with much higher efficiency. Of these advancements, pollen magnetofaction is particularly interesting in that it provides an opportunity to generate transformed seeds directly without regeneration, and is genotype independent as well as culture-free.…”