An optimised transformation protocol forAnthoceros agrestisand three more hornwort species

Abstract

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.

What can hornworts teach us?

“…Both A. agrestis isolates are derived from a single spore and can be routinely grown axenically. High quality genome assemblies are available ( Li et al., 2020 ) and an efficient Agrobacterium -mediated transformation method, using regenerating thallus fragments, has been developed ( Frangedakis et al., 2021b ; Waller et al., 2022 ). Up to 100 and 40 stable transgenic lines can be obtained from 0.2 g of tissue for the Oxford and Bonn isolate, respectively.…”

“…In addition to the widely used CaMV 35S promoter, two native constitutive promoters have been developed for A. agrestis , the promoter regions of the Elongation Factor 1 Alpha (Ef1a) and the Gamma Tonoplast Intrinsic Protein 1;1 (Tip1;1) genes ( Frangedakis et al., 2021b ; Waller et al., 2022 ). Hygromycin is used as an antibiotic selection marker and chlorsulfuron as a herbicide selection marker.…”

“…Four fluorescent proteins (eGFP, mVenus, mTurquoise2 and mScarlet) can be expressed successfully in A. agrestis without toxic effects. A set of targeting peptides for subcellular localization into the chloroplast, mitochondria, Golgi, Endoplasmic reticulum, peroxisomes, nucleus, cytoskeleton and the plasma membrane are also available ( Waller et al., 2022 ). The palette of hornwort species that can be genetically modified was recently expanded by the addition of Anthoceros punctatus , which has been used as a model for plant symbiosis with cyanobacteria, Leiosporoceros dussii , which is the sister to all other hornworts and Phaeoceros carolinianus ( Waller et al., 2022 ).…”

“…Pyrenoids have repeatedly been gained and lost over the course of hornwort evolution ( Figure 3B ) enabling comparative analyses of pyrenoid-bearing and pyrenoid-absent species ( Villarreal and Renner, 2012 ). This framework, combined with the currently established transformation techniques, provides an ideal opportunity to answer key questions related to pyrenoids in hornworts as well as to reveal conserved and divergent features with C. reinhardtii ( Li et al., 2017 ; Frangedakis et al., 2021b ; Waller et al., 2022 ). In particular:…”

“…This can eventually provide a more accurate insight into major events of land plant evolution ( Dolan, 2009 ; Donoghue et al., 2021 ; Frangedakis et al., 2021a ). Importantly, the development of a transformation technique for A. agrestis , and for three additional species ( Waller et al., 2022 ), paves the way for detailed molecular and genetic studies to elucidate hornwort biology. Furthermore, it opens the way to experimentally study the enigmatic features of hornworts that are absent or rarely occur in other land plants, such as the single algal-like chloroplast per cell or the basal sporophyte meristem ( Villarreal and Renzaglia, 2015 ; Frangedakis et al., 2021a ).…”