TITLE: Anthoceros punctatus (hornwort) transformation
AUTHORS: Eftychis Frangedakis, Manuel Waller

[DESCRIPTION]
Anthoceros punctatus  (hornwort) transformation 





[STEPS]
4. Tissue preparation: 

Collect approximately 1 g of thallus tissue grown for 4-6 weeks under low light intensity (approximately 0.1 g of tissue per petri dish - 10 petri dishes in total). Figure1 and Figure 3.1

Transfer the tissue into an empty petri dish,  add sterile water until the tissue is covered and fragment using a razor blade (it takes approximately 5 mins, similar to Video 1). Figure 3.2-3

Transfer the tissue  from the petri dish into a cell strainer positioned on a falcon tube using sterile scalpels and wash the tissue using ~100 ml of sterile water or until the flow through was clear. Figure 3.4-6




5. Agrobacterium culture preparation:  

Inoculate 5 mL LB media with 3-4 Agrobacterium colonies (AGL1: 15 μg/mL rifampicin, 50 μg/mL carbenicillin) (GV3101: 50 μg/mL rifampicin, 25 μg/mL gentamicin) and the plasmid-specific selection antibiotic. 

Incubate the preculture at 28°C for 2 days at 110 rpm.

Centrifuge 5 mL of 2 d Agrobacterium culture (no need to measure OD) for 7 min at 2000 xg.

Remove supernatant and re-suspend in 5 mL liquid KNOP plus 1% (w/v) sucrose and 100 μM acetosyringone.  

Incubate the culture with shaking (120 rpm) at 28°C for 3-5 hours.  
6. Co-cultivation:  

Transferred the fragmented thallus tissue into a 6-well plate (transfer 1⁄6 of the 1 g tissue into a single well) with 5 mL of liquid KNOP medium supplemented with 1% (w/v) sucrose and 30-40 mM MES (VERY IMPORTANT), pH 5.5, 80 μL of Agrobacterium culture and acetosyringone at final concentration of 100 μM.  Figure 3.7

Co-cultivate the tissue with the Agrobacterium for 3 days on a shaker at 110 rpm, with only ambient light.

Using a sterile plastic pipette transfer the tissue of one well  into a cell strainer, drain and then transfer on growth media containing the appropriate antibiotic (onto 1 petri dish from one well).To facilitate spreading of the tissue, 1-2 mL of sterile water is added to the petri dish.  Figure 3.8-11

After 6-7 weeks successful transformants are visible on the petri dish (successful transformants can be identified using a dissecting scope after 4 weeks  selection (sometimes as early as 2 weeks) based on rhizoid production and/or fluorescence if such a marker is present on the construct).  Figure 3.12

The emergence of rhizoids is an indication of successful transformation (yellow arrow: transformed thallus fragment, blue arrow: dying thallus fragment).  Figure 4

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7. 
2nd selection (necessary):

To eliminate false positives, after 4 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 . Sometimes a 3rd round of selection is necessary.






1. Axenic cultures of A. punctatus  gametophytes can be routinely propagated by monthly sub-culturing 
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 Video 1, Example of tissue fragmentation for A. agrestis Bonn. The method is the same for A. punctatus .
 

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