TITLE: Marchantia agrobacterium transformation of sporelings in multi-well plates (plus materials info)
AUTHORS: Linda Silvestri, Eftychis Frangedakis, Susana Sauret-Gueto, Marius Rebmann, Marta Tomaselli

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A modification of the </span><a href="https://doi.org/10.1093/pcp/pcn085" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ishizaki et al 2008</span></a><span style = ":;font-style:italic;"> </span><span style = "font-style:italic;">Agrobacterium</span><span> mediated Marchantia sporeling transformation protocol is used. </span></div><div class = "text-block">Sterilised spores are grown for 5 days in 0.5x strength Gamborg plates prior to co-cultivation for 2 days with agrobacterium in liquid media in multiwell plates. Sporelings are then spread on media with the appropriate selective antibiotic. In about 7 days, positive transformants start to emerge.</div></div>

[STEPS]
?. [Spores sterilisation and plating]
Day 0Transfer sporangia to a 1.5 mL Eppendorf tube. Use 2 sporangia per construct, e.g. for 6 transformations, use 12 sporangia.
?. [Agrobacterium preparation]
Day 0/1Transform Agrobacterium with your plasmid of interest. On ice, add 50 ng of the plasmid to 50 μL of thawed electro-competent Agrobacterium cells and transfer to a chilled 2 mm gap Electroporation cuvette. Electroporate with a 2.5 kV pulse.
?. [Agro-mediated sporelings transformation]
Day 5Prepare a 50 mL Falcon tube with 4 mL per transformation of 0.5 x Gamborg B5 plus supplements media. Add acetosyringone to a final concentration of 100μM.
?. [Agro-mediated sporelings transformation]
Day 7 After 2 days, plate the transformed sporelings as follows:
?. [Agro-mediated sporelings transformation]
Day 14After 5-7 days successful transformants start to be visible on the plate and can be screened for fluorescence (J in Figure)
?. [Agro-mediated sporelings transformation]
?. [Spores sterilisation and plating]
Use metal tweezers to crush the sporangia, add 0.5 mL of Milton solution per tube, vortex to get a fine pulp.
?. [Spores sterilisation and plating]
Place a 40μM cell strainer on a 50 mL falcon tube, pour the 0.5 mL of crushed sporangia onto the filter, rinse the eppendorf tube with 1 ml of fresh Milton solution and pour onto the filter. Wash filter with 2.5ml of fresh Milton solution.
?. [Spores sterilisation and plating]
Divide the filtrate solution containing the spores (total volume aprox 4 mL) in 4 new 1.5 mL eppendorf tubes. Spores should be sterilised in Milton for 20-30 min, starting from step 1.2. Let tubes stand for the remaining time.
?. [Spores sterilisation and plating]
Spin tubes at 16000g for 2min in a tabletop centrifuge.
?. [Spores sterilisation and plating]
Discard supernatant and pool spores in a single tube, re-suspend pellets in 25μL per transformation planned.
?. [Spores sterilisation and plating]
Plate spores on 0.5 x Gamborg plates using 50 μL per plate (1 plate is used for 2 transformations). Seal plates with micropore tape and grow for 5 days at 21oC under continuous light (A in Figure).
?. [Agrobacterium preparation]
Recover transformed cells by adding 0.5 mL of LB or SOC to the cuvette and incubate 1-2h at 28°C and 150 rpm
?. [Agrobacterium preparation]
Plate 50 μL on LB plates with appropriate antibiotics for selection. Incubate plates for 2-3 days at 30°C
?. [Agrobacterium preparation]
Day 3Inoculate a single colony of Agrobacterium transformed with the plasmid of interest, into 5 mL LB media plus antibiotics, incubate at 28°C with shaking at 150 rpm for 2 days.
?. [Agrobacterium preparation]
Day 5Centrifuge the 5 mL agrobacterium culture for 10 minutes at 3000g. Discard supernatant, re-suspend in 5 mL of 0.5x Gamborg B5 plus supplements. Add acetosyringone to a final concentration of 100 μMIncubate with for 6h at 28°C and 150 rpm.
?. [Agro-mediated sporelings transformation]
Using a sterile scalpel transfer 5 days old Marchantia sporelings from plates to the 50 mL Falcon tube (B and C in Figure) and mix well.
?. [Agro-mediated sporelings transformation]
Using a serological pipette add 4 mL of the spore solution into each well of a 6-well plate (D in Figure).
?. [Agro-mediated sporelings transformation]
Add 100 μL of the Agrobacterium culture to each well.
?. [Agro-mediated sporelings transformation]
Seal the plate with micropore tape and place on a shaker at 120 rpm for 2 days at 21oC under continuous light (E in Figure).
?. [Agro-mediated sporelings transformation]
Wash the sporelings by pouring into the strainer 50 mL of sterile water supplemented with 100 μg/ml cefotaxime to remove excessive Agrobacterium.
?. [Agro-mediated sporelings transformation]
Plate sporelings on 0.5 x Gamborg B5 plus vitamins 1.2% agar plates with 100 μg/ml cefotaxime and selective antibiotics (H and I in Figure)
?. [Agro-mediated sporelings transformation]
Repeat steps 6.1 to 6.3 for all wells.
?. [Agro-mediated sporelings transformation]
Using a pipette transfer the sporelings from the first well to a 70 μm cell strainer placed in a 50 mL Falcon tube (F and G in Figure).