Choosing a propagation technique
I started collecting and growing Marchantia isolates in 2010, after listening to an inspiring talk from Kimi Ishizaki about his doctoral work with Takayuki Kohchi in Kyoto. I knew of Takayuki's early work, as we'd been post-docs together in Howard Goodman's lab at Harvard about 20 years earlier - but to hear of the intervening developments was a wake-up call. The pioneering work that they had done made it clear that Marchantia had unique benefits of simplicity and experimental speed and access that were ideal for our engineering aspirations. I took to haunting local (poorly maintained) plant nurseries around Cambridge and purchasing plants that had suffered invasion by male and female Marchantia. I isolated and grew the isolates, looking for vigorous vegetative growth and ease of spore production. Those early days were characterised by homemade LED panels, hacking Ikea bookshelves for growth racks and experimenting with simple propagation methods, mainly using Jiffy 7 peat pellets.
As time has gone on, we have explored a wider variety of propagation techniques, which promise faster growth, pod-like growth vessels and less waste. This has been a pet project of mine - as low-cost systems would have major impact on the use of the Marchantia system for day-to-day research, including for education and community labs - and open the prospect of larger-scale production of engineered biomass under contained conditions.
Support membranes for NFT hydroponics
I have tested a number of potential substrates for supporting Marchantia growth and the current favoured solution uses 3D printed trays with 20mm high support pegs to support: (i) a layer of heavy duty 220gsm black PVC mesh (available from Amazon in 2m width, £8.99/m). This provides a layer of support to stop any sagging, used in all of our NFT hydroponic designs. (ii) A dual layer of Henofa Klavermat 300 capillary matting covered with an attached surface layer of 30µm thick micro-perforated black plastic film. The Klavermat 300 + BF is available directly from Henofa, a specialist manufacturer of capillary matting based in The Netherlands (Download a specification sheet).
The Klavermat 300 + BF material has two important features - the capillary matting is black, light-shielding, and is highly efficient at drawing up water. Second, the attached micro perforated film has extensive and very fine (sub-millimetre) perforations. A comparison between this and other available perforated plastics is shown below. Back illuminated photos taken at the same scale are: (Left) Perforated black polythene with 1cm/1.4cm hole spacing, (Middle) Microperforated Poly Film used as a cover for capillary matting with 0.5cm/0.7cm hole spacing. Works well as a growing surface for thallus pieces - not successful for gemmae, and (Right) Henofa perforated black plastic film (taken from Klavermat 300 BF sheet).
Larger 1cm spaced perforations require individual thallus fragments to be placed directly over a perforation to allow proper feeding and subsequent growth of the plants. The 0.5cm spaced holes allow larger thallus pieces to be distributed randomly over the surface with a good chance of successful growth - however this is not successful with smaller gemmae propagules. In contrast, the micro-perforated Henofa film allows random spreading and placement of thallus or gemmae over the surface with good contact with the capillary matting below. The uninterrupted part of the Henofa film is impermeable, but with so many perforations, water flows directly through these. Marchantia gemmae can be harvested, suspended in water and directly pipetted onto the film surface. As the solution flows quickly through the film, most individual gemma are lodged over holes in the film (see image below, left). If the gemmae are left to grow, the majority of gemmae (~90%) establish themselves and grow rapidly to form large assemblies of thallus. The image below (right) shows the same part of a Henofa-covered NFT hydroponics unit after 3.5 days. Most of the gemmae grew well, at the expected rate - while only four of the gemma in the field of view (circled) did not grow well because they had no contact with the underlying nutrient solution.
The fine and closely spaced perforations in the Henofa film make it a reliable surface for planting Marchantia tissue fragments or gemmae. However note that due to the small size of Marchantia spores, placing a suspension of spores on the film result in the majority of them being swept through the microperforations and end up underneath the film - so not recommended unless some form of encapsulation might be used. In practice, the use of the Henofa film greatly helps reduce problems due to algal growth (especially seen with mats without an impermeable covering) - due to effective permeability and light blocking. Any problems are usually due to over-wetting of nutrient solutions, with media being exposed on or across the growing surface.
Day 0: Marchantia gemmae distributed on Henofa Klavermat 300 BF surface
Day 3.5: Growth of gemmalings on Henofa Klavermat 300 BF surface, except those circled, which don't have access to a suitable pore in the plastic film