TITLE: Anthoceros agrestis  (hornwort) transformation v01
AUTHORS: Eftychis Frangedakis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Anthoceros agrestis</span><span>  (hornwort) transformation</span></div><div class = "text-block">pre-print:</div><div class = "text-block"><a href="https://biorxiv.org/cgi/content/short/2021.01.07.425778v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://biorxiv.org/cgi/content/short/2021.01.07.425778v1</span></a></div><div class = "text-block">This protocol works better for the Oxford strain</div></div>

[STEPS]
?. Tissue preparation: Collect approximately 2 g of thallus tissue grown for 4 weeks under low light intensity (approximately 0.1 g of tissue per petri dish - 20 petri dishes in total) - Figure 4.1Split tissue into three parts, transfer each part into a 50 mL falcon tubes containing 15 mL of sterile water and homogenize using a homogenizer and corresponding dispensing tools (for 5 sec, lowest power 8000 rpm) - Figure 4.2-5Transfer the homogenized tissue from one falcon tube into a 100 μm cell strainer,  wash with 50 mL of sterile water and transfer onto two petri dishes (six plates in total) containing solid KNOP medium add 2-3 mL of sterile water and place at 21°C under 12 hours light and 12 hours dark,  light intensity 3-5 μmol m−2s−1 - Figure 4.6-7After 3-4 weeks transfer the tissue  from the petri dishes into a falcon tube using sterile scalpels - Figure 4.8-9Re-homogenize in 15-20 mL of sterile water and filter using a cell strainer - Figure 4.10-11Spread out again onto 4 petri dishes with solid KNOP medium (if necessary, to facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish) and allow to grow for 2 days at 21°C under continuous light (12 hours light and 12 hours dark also ok) (light intensity 30 μmol m−2s−1) - Figure 4.12Split tissue into three parts, transfer each part into a 50 mL falcon tubes containing 15 mL of sterile water and homogenize using a homogenizer and corresponding dispensing tools (for 5 sec, lowest power 8000 rpm) - Figure 4.2-5Transfer the homogenized tissue from one falcon tube into a 100 μm cell strainer,  wash with 50 mL of sterile water and transfer onto two petri dishes (six plates in total) containing solid KNOP medium add 2-3 mL of sterile water and place at 21°C under 12 hours light and 12 hours dark,  light intensity 3-5 μmol m−2s−1 - Figure 4.6-7After 3-4 weeks transfer the tissue  from the petri dishes into a falcon tube using sterile scalpels - Figure 4.8-9Re-homogenize in 15-20 mL of sterile water and filter using a cell strainer - Figure 4.10-11Spread out again onto 4 petri dishes with solid KNOP medium (if necessary, to facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish) and allow to grow for 2 days at 21°C under continuous light (12 hours light and 12 hours dark also ok) (light intensity 30 μmol m−2s−1) - Figure 4.12Transfer the homogenized tissue from one falcon tube into a 100 μm cell strainer,  wash with 50 mL of sterile water and transfer onto two petri dishes (six plates in total) containing solid KNOP medium add 2-3 mL of sterile water and place at 21°C under 12 hours light and 12 hours dark,  light intensity 3-5 μmol m−2s−1 - Figure 4.6-7After 3-4 weeks transfer the tissue  from the petri dishes into a falcon tube using sterile scalpels - Figure 4.8-9Re-homogenize in 15-20 mL of sterile water and filter using a cell strainer - Figure 4.10-11Spread out again onto 4 petri dishes with solid KNOP medium (if necessary, to facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish) and allow to grow for 2 days at 21°C under continuous light (12 hours light and 12 hours dark also ok) (light intensity 30 μmol m−2s−1) - Figure 4.12After 3-4 weeks transfer the tissue  from the petri dishes into a falcon tube using sterile scalpels - Figure 4.8-9Re-homogenize in 15-20 mL of sterile water and filter using a cell strainer - Figure 4.10-11Spread out again onto 4 petri dishes with solid KNOP medium (if necessary, to facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish) and allow to grow for 2 days at 21°C under continuous light (12 hours light and 12 hours dark also ok) (light intensity 30 μmol m−2s−1) - Figure 4.12Re-homogenize in 15-20 mL of sterile water and filter using a cell strainer - Figure 4.10-11Spread out again onto 4 petri dishes with solid KNOP medium (if necessary, to facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish) and allow to grow for 2 days at 21°C under continuous light (12 hours light and 12 hours dark also ok) (light intensity 30 μmol m−2s−1) - Figure 4.12
?. Agrobacterium culture preparation:  Inoculate 5 mL LB media with a single Agrobacterium colony (AGL1 strain), 10 μg/mL rifampicin, 50 μg/mL carbenicillin and the plasmid-specific selection antibiotic. Incubate the preculture at 28°C for 2 days at 120 rpm.
?. Co-cultivation:  Centrifuge 5 mL of 2 d Agrobacterium culture (OD: 2.5 - 2.7) for 7 min at 1800 xg.Remove supernatant and re-suspend in 5 mL liquid KNOP plus 2% (w/v) sucrose and 100 μM acetosyringone.  Incubate the culture with shaking (120 rpm) at 28°C for 5 hours.  Wash the regenerating thallus using a 100 μm cell strainer.Transfer ½ of the tissue from one plate into a single well of a 6-well plate containing 4 mL of liquid KNOP medium supplemented with 2% (w/v) sucrose - Figure 4.13Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14Remove supernatant and re-suspend in 5 mL liquid KNOP plus 2% (w/v) sucrose and 100 μM acetosyringone.  Incubate the culture with shaking (120 rpm) at 28°C for 5 hours.  Wash the regenerating thallus using a 100 μm cell strainer.Transfer ½ of the tissue from one plate into a single well of a 6-well plate containing 4 mL of liquid KNOP medium supplemented with 2% (w/v) sucrose - Figure 4.13Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14Incubate the culture with shaking (120 rpm) at 28°C for 5 hours.  Wash the regenerating thallus using a 100 μm cell strainer.Transfer ½ of the tissue from one plate into a single well of a 6-well plate containing 4 mL of liquid KNOP medium supplemented with 2% (w/v) sucrose - Figure 4.13Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14Wash the regenerating thallus using a 100 μm cell strainer.Transfer ½ of the tissue from one plate into a single well of a 6-well plate containing 4 mL of liquid KNOP medium supplemented with 2% (w/v) sucrose - Figure 4.13Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14Transfer ½ of the tissue from one plate into a single well of a 6-well plate containing 4 mL of liquid KNOP medium supplemented with 2% (w/v) sucrose - Figure 4.13Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14Add 80  μL of Agrobacterium culture and 100 μM (final concentration) acetosyringone to the medium.Co-cultivate the tissue and Agrobacterium for 3 days with shaking at 110 rpm at 21°C with only ambient light from the room (1-3μmol m−2s−1) - Figure 4.14
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?. 1st selection:Using a sterile plastic pipette transfer the tissue from a single well into a 100 μm cell strainer, drain and then transfer on growth media containing the appropriate antibiotic. To facilitate spreading of the tissue, add 2 mL of sterile water to the petri dish - Figure 4.15-17Plate the tissue on solid KNOP plates supplemented with 100 μg/mL cefotaxime and 10 μg/mL Hygromycin and grow at 21°C  under 12 hours light and 12 hours dark, 35 μmol m−2s−1
?. 2nd selection:After 3 weeks transfer the tissue to fresh growth media containing 100 μg/mL cefotaxime and 10 μg/mL Hygromycin. To facilitate spreading of tissue on the petri dish  add 2 mL of sterile water. Grow at 21°C  under 12 hours light and 12 hours dark, 35 μmol m−2s−1  - Figure 4.18After 6-8 weeks successful transformants are visible on the petri dish (successful transformants can be identified using a microscope after 4 weeks selection based on rhizoid production) - Figure 4.19 and Figure 5 & 6.
?. IMPORTANT: The light intensity used to cultivate A. agrestis tissue is a very critical factor for successful transformation. Tissue should be grown under low light intensity (3-5 μmol m−2s−1) and should have a morphology similar to the tissue in Figure 1 bottom left image.
?. Axenic cultures of A. agrestis  gametophytes can be routinely propagated by monthly sub-culturing as shown in Figure 2.
?. 3rd selection:To eliminate false positives, transfer surviving tissue fragments again on 100 μg/mL cefotaxime and 10 μg/mL Hygromycin containing growth media. Grow at 21°C  under 12 hours light and 12 hours dark, 3-35 μmol m−2s−1 - Figure 7.
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