TITLE: Anthoceros agrestis Bonn  (hornwort) transformation v02
AUTHORS: Eftychis Frangedakis, Manuel Waller

[DESCRIPTION]
Anthoceros agrestis Bonn (hornwort) transformation v02





[STEPS]
6. 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 4.1

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 4.2-3

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 4.4-6




7. 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.  
8. 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 4.7

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 4.8-11

After 4-6 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 4.12

The emergence of rhizoids is an indication of successful transformation (yellow arrow: transformed thallus fragment, blue arrow: dying thallus fragment).  Figure 5

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9. 
2nd selection (optional):

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 






1. 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
3. Axenic cultures of A. agrestis  gametophytes can be routinely propagated by monthly sub-culturing as shown in Figure 2.
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13. 

 
 Video 1, Example of tissue fragmentation for A. agrestis Bonn. 
 

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