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 eppendfors , 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 daysOnce 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
