Improved plastid transformation in Marchantia polymorpha

Abstract

Plastids are subcellular organelles which perform photosynthesis in plants and algae. They are descended from free-living photosynthetic bacteria which were engulfed by early eukaryotes ~1.5 billion years ago. Nearly all plastids retain a small genome separate from the nuclear genome of the plants in which they reside, which is termed the plastome (plastid genome). This genome encodes many of the proteins required for photosynthesis, as well as bacteria-like transcriptional and translational machinery. Biotechnological techniques for making precise modifications to the plastome have been available for three decades, nearly as long as the equivalent techniques for the plant nuclear genome. However, while nuclear genetic engineering quickly moved from a laboratory novelty to a tool used for the improvement of crop plants planted on over a billion acres, plastome engineering remains largely confined to research use. The primary reasons for this lack of application are the species restrictions and technical difficulty of the transformation process, which until recently was only possible in a few species.

In 2007, a protocol for plastid transformation was reported for Marchantia polymorpha, a thalloid liverwort classically used as a model species. Marchantia offers rapid generation time, small size, simple genetics, and asexual reproduction by means of gemmae, small disks of tissue which provide a powerful platform for live-tissue microscopy. In this thesis, tools for Marchantia plastid transformation are systematically improved by the generation of the first plastome sequence assembly for a widely used laboratory strain of Marchantia, optimisation of the transformation protocol itself, and a comparison of the in vivo activity of plastid regulatory elements through a fluorescent marker and quantitative microscopy. The highly conserved nature of the land plant plastome suggests the improvements to plastid transformation developed in Marchantia will translate to other species.