TITLE: Hornwort (Anthoceros agrestis) biolistics-mediated transformation protocol
AUTHORS: Declan Lafferty, Fay-Wei Li

[DESCRIPTION]
Here we report an efficient biolistics method for generating transient-expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (± 268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48 hours. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from A. fusiformis.

[STEPS]
3. For selection of transformed tissue, use AG medium with 0.2% sucrose and 10 mg/L hygromycin (for hygromycin selection) or other selection agent(s). 
SECTION: Growth media
1. For growing and maintaining gametophyte tissue, use AG medium (modified Hatcher’s medium containing additional 0.2% sucrose, 0.4 g ammonium nitrate, 100 mg/l activated charcoal, 5 mM MES buffering at pH 6.5 and 10 nM BA).  

SECTION: Growth media
2. For preparing tissue for transformation, use AG medium with high sucrose (2% sucrose). 
SECTION: Tissue maintenance
4. Hornwort thallus tissue is maintained on 0.2% sucrose AG medium. 3-6 g of thallus tissue is homogenized and sub-cultured every 4-6 weeks
SECTION: Tissue maintenance
5. For homogenization, tissue is placed in a sterile 50 mL falcon tube with approx. 30 mL sterile water, supplemented with 30 µl of 300 mg/mL timentin. 
SECTION: Tissue maintenance
7. Filter the tissue through a 50 µM cell strainer to remove excess mucilage and water. Rinse the tissue with 50 mL sterile filtered water and remove excess mucilage and water using the cell strainer.
SECTION: Tissue preparation for transformation
10. Hornwort thallus tissue is prepared 7 days prior to transformation. 2 g of healthy thallus tissue is homogenized, following the same method as described above. Healthy tissues are green with expanded thalli and minimal rhizoid growth, as in Fig 1. Poor quality tissues are yellowish green with many rhizoids observed growing upwards. 

SECTION: DNA/gold preparation (10 replicates)
25. Vortex 100 µl of gold particles (0.6 – 1.0 µm size, 50 mg/ml concentration in 1.5 ml tube) at maximum vortex speed for at least 30 sec
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
34. Start the Biorad PDS-1000 and the vacuum pump. Ensure helium pressure line is set at ~600 psi
SECTION: Selection of transformants
42. If using fluorescent markers regulated by strong promoters (eg. 35S or EF1a promoters), transient gene expression can be observed after 24 hr post bombardment, and can continue until more than 3 weeks post bombardment (Fig. 5). Most transient expression will disappear within 2 weeks post bombardment.
 

SECTION: Tissue preparation for transformation
12. Filter the tissue through a 50 µM cell strainer to remove excess mucilage and water. Rinse the tissue with 50 mL sterile filtered water and remove excess mucilage and water using the cell strainer.
SECTION: Tissue preparation for transformation
13. Plate tissue on 2% sucrose AG medium. If there is excess water or condensation on the plate, leave it open in a laminar flow hood to dry (approx. 20 min), as excess tissue moisture can negatively affect results. Draw a 2.5cm circle in the middle of the underside of the plate (I use the inside of a roll of micropore tape). 
Plate approximately 0.15 g of wet-weight, homogenized tissue within the 2.5 cm bullseye mark (Fig. 2).
 

SECTION: Tissue preparation for transformation
14. Using a cell scraper (179693, ThermoFisher Scientific, USA), plate the tissue as thinly as possible and leave open for another 10 mins to dry the tissue, to remove excess water. High moisture will decrease theefficiency of transformation. Don’t want to dry out the tissue too much though, as this will damage the tissue. 
SECTION: Protocol overview
49. 
 

SECTION: DNA/gold preparation (10 replicates)
26. Add 20 µl of 1 µg/µl DNA and vortex for 20 sec
SECTION: DNA/gold preparation (10 replicates)
27. Simultaneously add 100 µl of 2.5 M CaCl2, and 40 µl of 0.1 M spermidine free base and vortex for 1 min
SECTION: DNA/gold preparation (10 replicates)
28. Spin down the mixture at 6000 rpm for 8 sec. Carefully discard the supernatant by pipetting.
SECTION: DNA/gold preparation (10 replicates)
29. Add 180 µl of 70% ethanol to the gold particles in the tube. Dislodge the gold particles from the tube using a pipet tip and vortex at max speed for 20 sec.
SECTION: DNA/gold preparation (10 replicates)
30. Remove the supernatant and add 200 µl of 100% ethanol. Dislodge the gold particles from the tube using a pipet tip and vortex at max speed for 20 sec.
SECTION: DNA/gold preparation (10 replicates)
32. Quickly spread about 5 µl of the gold particle suspension onto each flying disk that was previously sterilized and mounted on the flying disk carrier under laminar flow hood, mixing the gold suspension well in between the spreading, using the sonicator. 10 flying disks macrocarriers may be prepared this way. Let it dry for at least 30 min prior to bombardment.
SECTION: DNA/gold preparation (10 replicates)
31. Spin down the gold particles at 6000 rpm for 8 sec. Remove about 145 µl of supernatant. Resuspend the gold particles in the remaining ethanol by vortexing at max speed for 20 sec. 
SECTION: DNA/gold preparation (10 replicates)
32.2. I use a sonicator to keep the gold/DNA mix homogenous. Can also use a vortex. 
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
35. Shoot a blank shot under 30 psi vacuum to clear the helium line. 
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
36. Clean the bombardment chamber using 70% ethanol and sterilized paper towels.
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
37. Place the 2.5 cm diameter homogenized tissue inside the bombardment chamber at the lowest position. Remove the petri dish lid.
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
38. Follow the Biorad PDS-1000 protocol for assembling the cartridge and rupture disc retaining cap and shooting using 450 psi rupture disk (Fig. 4).
 

SECTION: Biolistics-mediated transformation of hornwort thallus tissue
40. If different DNA constructs are bombarded for different samples, clean the bombardment chamber using 70% ethanol and sterilized paper towels prior to shooting. Ensure the chamber is fully dried prior to resuming bombardments.
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
39. After shooting, immediately place the petri dish lid back and then remove from the chamber.
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
38.1. Let the chamber vacuum pressure reach approximately 29 inHg before engaging the gun.   
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
34.1. We have our Biorad PDS-1000 within a laminar flow hood, to reduce potential contamination  
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
41. Once all samples have been shot, seal the edges of the closed petri dish lid using double layer of parafilm.
SECTION: Selection of transformants
43. For stable transformation, tissue is recovered for 7 days post-bombardment. Transformed tissue is then pooled and homogenized, with a maximum of 5 replicates per homogenization (or else there is too much tissue for the homogenizer). 
SECTION: Selection of transformants
43.1. To homogenize the tissue, follow the same method as described in steps 11 and 12. It is very important not to homogenize for too long, only until the main tissue clumps are broken up, about 3-5 seconds. 
SECTION: Selection of transformants
46. Continue to monitor tissue for contaminants. The tissue will begin to brown and die after approximately three weeks (Fig. 7). 
 

SECTION: Selection of transformants
43.2. Filter the tissue well, removing all mucilage and rinsing the tissue until the flow through is clear
SECTION: Selection of transformants
45. Three days post homogenization, transfer the filter paper to 0.2% AG + 10 mg/L hygromycin solid media. Grow in the same conditions described in step 9.
SECTION: Selection of transformants
44. Using a cell scraper, place a small amount (0.15-0.3 g) of tissue onto 0.2% AG solid media, without selection, overlaid with sterilised 70 mm diameter Whatman filter paper (WHA1001070, Sigma-Aldrich, USA) (Fig. 6). Grow in the same conditions described in step 9.
 

SECTION: Selection of transformants
44.1. I tend to double the plates i.e if I homogenized transformed tissue from five plates, then I will prep ten plates for the filter paper step
SECTION: Selection of transformants
44.2. Can add ~1 mL of sterile MQ water to the tissue to help it spread out nicely, the aim is to avoid clumps of tissue. 
SECTION: Selection of transformants
46.1. When using other selectable markers it may be necessary to transfer to fresh medium after 5 weeks on selection. This is not required for hygromycin selection.
SECTION: Selection of transformants
47. Regenerating thallus tissue will become visible with the naked eye after 8-10 weeks on selection (Fig. 8). 
 



SECTION: Selection of transformants
47.1. Transfer the regenerating tissue to new plates to separate it from non-transformed, dying tissue. 
SECTION: Selection of transformants
48. If using a fluorescent marker, the presence of the transgene can be confirmed using fluorescent microscopy (Fig. 9). Otherwise, continue to sub-culture the stably transformed lines until there is sufficient tissue for DNA extraction. Presence of the transgene can be confirmed via PCR or DNA sequencing.

 

SECTION: Tissue maintenance
6. Tissue is homogenized with a T18 digital ULTRA TURRAX homogenizer (IKA, Germany) for 5 - 10 s at 4000 rpm
SECTION: Tissue preparation for transformation
11. Homogenize tissue with a T18 digital ULTRA TURRAX homogenizer (IKA, Germany) for 5 - 10 s at 4000 rpm
SECTION: Tissue preparation for transformation
11.1. When homogenizing tissue for transformation, be careful not to homogenize too much. This will damage the tissue and unhealthy tissue leads to poor transformation. Homogenize just enough to break up the tissue into small pieces, no clumps, approximately 5 seconds.
SECTION: Tissue preparation for transformation
15. Tissue is left to recover for seven days. Tissue is grown using the same growth conditions as described in step 9.    
SECTION: Tissue maintenance
9. Hornwort thallus tissue is grown at 22°C under a 16/8 h light/dark cycle with 6-25 µmol/m²/s light intensity provided by Ecolux XL Starcoat F32T8 XL SP30 ECO paired with F32T8 XL SP41 ECO fluorescence bulbs (General Electric, USA).
SECTION: Gold particle preparation (10 preps)
16.  Weigh out 50 mg of gold particles (0.6 µm size, ideally from Biorad) in a 1.5 ml tube.
SECTION: DNA/gold preparation (10 replicates)
23. The macrocarriers (#1652335, Bio-Rad, USA), 450 psi rupture discs (#1652326, Bio-Rad, USA), stopping screens (#1652336, Bio-Rad, USA) and macrocarreir holders are sterilized by soaking in 70% ethanol for at least 20 mins in a laminar flow hood, followed by drying until no ethanol remains.
SECTION: Gold particle preparation (10 preps)
17. Add 1 ml of 70% ethanol and vortex vigorously for 3-5 minutes.
SECTION: Gold particle preparation (10 preps)
18. Allow the particles to soak in the liquid for 15 minutes.
SECTION: Gold particle preparation (10 preps)
19.  Pellet the particles for 30 sec at 6000 rpm. Increase vortex time if the liquid is still cloudy.
SECTION: Gold particle preparation (10 preps)
20. Carefully discard the supernatant.
SECTION: Gold particle preparation (10 preps)
21.  Repeat the following wash steps three times: Add 1 ml of sterile water, vortex for 1 min, allow particles to settle for 1 min, pellet the particles by briefly spinning them down and discard the supernatant.
SECTION: Gold particle preparation (10 preps)
22. Add 1 ml of sterile 50% glycerol, vortex vigorously for 1 min and aliquot into 100 µl portions in 1.5 ml tubes. Store at -20°C.
SECTION: DNA/gold preparation (10 replicates)
27.1. Use fresh spermidine and store at -20°C for a maximum of four months. 
SECTION: DNA/gold preparation (10 replicates)
32.1. It is important that the gold/ethanol mix spreads evenly over the macrocarrier (Fig. 3). Clumping of gold particles will negatively impact the efficiency of transformation.

 



SECTION: DNA/gold preparation (10 replicates)
24. Insert the sterilised macrocarrier in the the macrocarrier holder using sterilised forceps.
SECTION: Biolistics-mediated transformation of hornwort thallus tissue
33. While the gold/DNA mix drys, sterilise the Biorad PDS-1000 (#1652257, Bio-Rad, USA) chamber and the laminar flow hood with 3% bleach, followed by 70% ethanol. Allow equipment to dry. 
SECTION: Tissue maintenance
8. Using a Nunc Cell scraper (179693, ThermoFisher Scientific, USA), plate on AG medium with 0.2% sucrose. Plate the tissue thinly and cover the majority (~80%) of the plate, as shown in Fig. 1.