Aug 10, 2022
Anthoceros agrestis Bonn (hornwort) transformation v02
- 1University of Cambridge;
- 2University of Zürich
Protocol Citation: Eftychis Frangedakis, Manuel Waller 2022. Anthoceros agrestis Bonn (hornwort) transformation v02. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzjydv8j/v1
Manuscript citation:
https://doi.org/10.1101/2022.08.10.503456
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 03, 2022
Last Modified: August 10, 2022
Protocol Integer ID: 68156
Abstract
Anthoceros agrestis Bonn (hornwort) transformation v02
Materials
KNOP recipe:
Stock 1
25g/L KH2PO4
Stock 2
25g/L KCl
Stock 3
25g/L MgSO4 7H2O
Stock 4
100g/L Ca(NO3)2 4H2O
autoclave and store at RT or 4°C
KNOP solid working solution:
In 600 mL of water add:
10ml Stock 1
10ml Stock 2
10ml Stock 3
10ml Stock 4
12.5mg FeSO47H2O
pH to 5.8 with KOH
top up water to 1L after adjusting pH
add 7 gr of Gelzan - G1910 - CAS Number71010-52-1
KNOP liquid working solution:
In 600 mL of water add:
10ml Stock 1
10ml Stock 2
10ml Stock 3
10ml Stock 4
12.5mg FeSO47H2O
10 gr of sucrose (1% w/v final concentration, 2% also fine)
40mM MES (very important)
pH to 5.5 with KOH
top up water to 1L after adjusting pH
Filter sterile (do not autoclave), aliquot into 50mL falcon tubes and store at -20°C.
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Sterile disposable scalpels (#0501, Swann Morton)
Razor blades (#11904325, Fisher Scientific)
100 µm cell strainer (#352360, CORNING),
6-well plate (#140675, ThermoFisher)
3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone) (#115540050, Acros Organics, dissolved in dimethyl sulfoxide (DMSO) (#D8418, SIGMA))
Cefotaxime (#BIC0111, Apollo Scientific)
Hygromycin (#10687010, Invitrogen)
Corning Disposable Vacuum Filter/Storage Systems (#430767)
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
Figure 1:
Top: 4 week old A. agrestis Bonn thallus
Bottom: 7 week old A. agrestis Bonn thallus (this tissue is also good for transformation)
Axenic cultures of A. agrestis gametophytes can be routinely propagated by monthly sub-culturing as shown in Figure 2.
Figure 2: A. agrestis Bonn tissue culturing
For sub-culturing, a small piece of thallus tissue is cut using sterile disposable scalpels and placed on plates containing fresh growth medium. Scale bars: 2 mm. Petri dish dimensions: 92 x16 mm.
Tissue similar to the bottom images is optimal for transformation
Figure 3: Transformation method outline. A) Thallus tissue is routinely propagated on a monthly basis under low light. 4-6 week old tissue is fragmented with the aid of a razor blade, transferred to a cell strainer, and washed thoroughly with sterile water. B) The tissue is then co-cultivated with Agrobacterium for three days (under low light) and C) spread on antibiotic-containing growth medium. After approximately 4-6 weeks, putative transformants are visible. A final round of selection is recommended to eliminate false-positive transformants.
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
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.
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
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
Figure 4
Figure 5: The emergence of rhizoids is an indication of successful transformation (yellow arrows: transformed thallus fragment, blue arrow: dying thallus fragment).
Video 1, Example of tissue fragmentation for A. agrestis Bonn.