Agrobacterium mediated transformation of Marchantia spores
Grind and sterilise the spore heads: Get ready: At the flow hood.
Prepare eppendorf tubes, and 3 x 50mL falcon tubes: 1) for sterile water, 2) for Milton solution (1 Milton tablet in 25ml sterile water), and 3) for the washed spores heads.
Use two sporangia per transformation.
Check number of sporangia in the spore head (this is variable, ex there may be only 1 sporangia in one spore head, and 5 sporangia in another spore head).
Add the spores heads to an eppendorf tube.
Depending on final number of spore heads needed, you may need to divide them into several eppendorfs tubes.
Add 0.5ml Milton solution to each eppendorf tube.
Use a plastic pestle to crush the sporangia and vortex them until completely re-suspended.
Filter crushed spore heads: Place the 70 μm cell strainer on a 50 ml falcon tube.
Pour onto the filter the solution with ground sporangia from all eppendorfs, pour the 0.5 ml from each eppendorf.
The solution with spores will flush through the cell strainer.
Rinse the Eppendorf(s) and strainer with 0.5 ml of Milton solution for each two sporangia used (ex. 3 ml for 12 sporangia).
Aliquot the solution with spores in eppendorf tubes (0.5 or 1ml per tube), let them sit for 5 minutes and spin for 6-7 minutes at 13000rpm.
Discard the supernatant and resuspend the spores in each tube with 150 μl of sterile water.  
Plate spores: Plate the 150 μl of re-suspended spores on a 1/2 GB plate (no antibiotics).
Use an L-shaped spreader.
If 0.5 ml aliquots made (equivalent to 2 sporangia), use one plate per transformation.
If 1 ml aliquots made (equivalent to 4 sporangia), use half a plate per transformation.
Grow upside down (rhizoids will grow away from the media) for 5-6 days at 21 ºC CL 150μE Sporelings (germinated spores) have red fluoresence from chlorophyll autofluorescence after ~ 3 days. 
Agro transformation: Mix DNA and Agro: For each agro transformation, add 20-50ng of plasmid to 50 μL of thawed electrocompetent Agrobacterium cells (kept on ice).
Electroporate in a chilled 2 mm cuvette with a 2.5 kV pulse.
Recover Agro: Immediately add 1mL of SOC (or LB) and incubate 1-2h at 28 ºC 150 rpm.
Plate Agro: Streak 20-50μL on LB plates + antibiotics (Kan 50, Tet 5, for GV2260 with a pGreen plasmid).
Each Agro transformation in a different plate.
Grow for 3 days at 28 ºC or 30 ºC, incubator. 
Optional Agro colony PCR: all agrobacterium colonies on LB + antibiotics plates should be positive, however if there are some issues with the transformations it may be useful to do a quick screen to check if there are any false positives on the plate.
In that case, do a colony PCR of Agro colonies.
Grow Agro culture:   (~ 15'' – set up sometime in the afternoon).
For each Agro transformation pick a single colony from plate, inoculate 5 ml LB media + antibiotics (Kan 50, Tet 5 for GV2260 with a pGreen plasmid) in a 15 ml falcon tube (unskirted tube).
Incubate at 28 ºC - 150 rpm (wrap the tubes in foil) and let grow for 1.5 days.  
Agro induction with Acetosyringone: (~20'' plus 6 h induction – set up in the morning): Centrifuge the agrobacterium culture(s) 10'' at 3000g.
For each agrobacterium culture, discard the supernatant and re-suspend in 3 ml of 1/2 GB media sup containing 100 μM Acetosyringone.
Add acetosyringone to a final concentration of 100 μM (for 3ml media add 3 μl 100 mM stock solution).
Shake for 6 h at 28 ºC at 150 rpm in the dark (wrap the tubes in foil).
Agro co-Cultivation with sporelings.
Scrape the 6 days old sporelings from the plate using an L shaped spreader, gather the sporelings and move them into a 100-250 ml autoclaved flask.
Use one plate per flask (transformation) if you plated 2 sporangia per plate, or half a plate if you plated 4 sporangia per plate.
Add to each flask 25 ml of 1/2 GB media sup and 25 ul of 100mM Acetosyringone, for a final concentration of 100μM.
To each flask, add 1ml of the appropiate Agrobacterium culture (induced 6 h prior).
Co-cultivate for 2 days (at least 36 h or 1.5 days) on a shaker in the tissue culture room (21 ºC, CL 150-200 uE) Sporelings Collection.
Add cefotaxime 100mg/ml stock solution to sterile water for a final concentration of 100ug/ml, 150-200 ml are needed per flask of co-cultivation.
For each co-cultivation flask (transformation), collect the sporelings  by pouring the flask contents in a 70 μm cell strainer placed on a 1L bottle.
Rinse the sporeling by pouring over them 150-200ml of water + cefo 100 Sporelings plating.
For each co-cultivation flask (transformation), with the help of a tip and a few ml of water + cefo remove the sporelings from the cell strainer and spread them on at least 3 plates (1/2 GB plates with antibiotics), make sure they are well spread, the better the spread the better the transgenic plants will grow.   
Screening for transformants: Positive transformants grow while non-trasnformed sporelings die.
Positive transformants become evident from about 1 week after plating.
Positive transformants with a fluorescent marker in the T-DNA can start to be screened under the microscope after about 5 days.
Once positive transformants have grown, from about a week and a half onwards, they should be transferred to new 1/2 GB plates with antibiotics, so they can grow faster and make gemmae
