Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants
2Department of Plant Sciences, Sheridan Research and Extension Center, University of Wyoming, Sheridan, WY 82801, USA
- Bleach (Clorox®)
- Ethanol (Thermo Fisher Scientific, cat # S25309B)
- Tween 20 (Thermo Fisher Scientific, cat # BP337-100)
- Murashige and Skoog (1962) salts and vitamins (PhytoTechnology Laboratories, cat # M519)
- Sucrose (PhytoTechnology Laboratories, cat # S391)
- BAP (PhytoTechnology Laboratories, cat # B800)
- NAA (PhytoTechnology Laboratories, cat # N600)
- TC agar (PhytoTechnology Laboratories, cat # A111)
- Sodium hydroxide (Thermo Fisher Scientific, cat # S318-3)
- YEP (PhytoTechnology Laboratories, cat # Y8575)
- LB (PhytoTechnology Laboratories, cat # L475)
- Bactoagar (Thermo Fisher Scientific, cat # DF0140-01-0)
- Mannitol (Thermo Fisher Scientific, cat # M120-500)
- L-Glutamate (Thermo Fisher Scientific, cat # ICN19467790)
- Tryptone (Thermo Fisher Scientific, cat # BP9726-500)
- Yeast extract (Thermo Fisher Scientific, cat # AAH2676922)
- NaCl (Thermo Fisher Scientific, cat # S271-500)
- KH2PO4 (Thermo Fisher Scientific, cat # P286-1)
- MgSO4.7H2O (Thermo Fisher Scientific, cat # AC423905000)
- Fe-EDTA (Thermo Fisher Scientific, cat # AC304680051)
- Pro Mix BX potting mix (Premier Horticulture, Inc., cat # 10281RG)
- DNeasy Plant Mini kit (QIAGEN, cat # 69104)
- Primers (Integrated DNA Technologies)
- Agarose (Thermo Fisher Scientific, cat # BP160-100)
- Gel Red nucleic acid stain (Phenix Research, cat # RGB4103)
- TAE (Thermo Fisher Scientific, cat # BP13321)
- Biosafety cabinet class II type B2 (Nuaire, model cat # Nu-430-600) 100 × 15 mm plastic
- Petri dishes (Genesee Scientific, cat # 32-107)
- 50 ml centrifuge tube (Thermo Fisher Scientific, cat # 14-432-22)
- 125 ml conical flasks (Pyrex, cat # 4980)
- GA7 Magenta vessel (Thermo Fisher Scientific, cat # NC9357464)
- Scalpels (PhytoTechnology Laboratories, cat # S963)
- Scalpel Blade (PhytoTechnology Laboratories, cat # S970)
- Forceps (PhytoTechnology Laboratories, cat # F639)
- Sterile Whatman 3MM filter paper (Thermo Fisher Scientific, cat # 09-820A)
- Incubator shaker (VWR, model #1575)
- Centrifuge (Beckman Coulter Inc., model #J-251)
- Glass bead sterilizer (Sigma-Aldrich, model Steri-250)
- Micropipettors (Eppendorf)
- Micropipette tips (Genesee Scientific, cat # 24-120RL, 24-150RL, 24-165RS)
- 1.7 ml centrifuge tubes (Genesee Scientific, cat # 24-282)
- Conviron Growth chamber (Controlled Environments Inc., model # CMP6010)
- Zeiss Stemi SV11 microscope (Carl Zeiss AG., cat # TLB 3.1) with an X-Cite 120 fluorescence illumination system (X-Cite, model # XI-120)
- C1000 Touch Thermal PCR (Bio-Rad, model # 1851148)
- Camelina regeneration (CR) medium, pH 5.8: Murashige and Skoog (1962) salts and vitamins, 30 g/L sucrose, 6.6 μM BAP, 2.6 μM NAA, 7.0 g/L TC Agar.
- Liquid co-cultivation medium (Liquid CR), pH 5.8: Murashige and Skoog (1962) salts and vitamins, 30 g/L sucrose, 6.6 μM BAP, 2.6 μM NAA.
- Selection medium (Sel), pH 5.8: Murashige and Skoog (1962) salts and vitamins, 30 g/L sucrose, 6.6 μM BAP, 2.6 μM NAA, 7.0 g/L TC Agar, 50 mg/L cefotaxime, and 40 mg/L kanamycin.
- Solid Agrobacterium culture medium: yeast extract peptone (YEP) medium (10 g/L peptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0) supplemented with 15 g/L Bactoagar.
- Liquid MG/L medium, pH 7.0: 5.0 g/L mannitol, 1.0 g/L L-Glutamate, 5.0 g/L tryptone, 2.5 g/L yeast extract, 5.0 g/L NaCl, 150.0 mg/L KH2PO4, 100.0 mg/L MgSO4.7H2O, 2.5 mg/L Fe-EDTA.
- Liquid MS medium, pH 5.8: Murashige and Skoog (1962) salts and vitamins.
- Camelina rooting medium: Murashige and Skoog (1962) salts and vitamins, 30 g/L sucrose, 2.1 μM NAA, 7.0 g/L TC Agar.
- Camelina sativa cvs. Pl650159 and Pl650161
Antibiotic stock solutions
- Rifampicin: 20 mg of antibiotic dissolved in 500 µl of methanol.
- Kanamycin sulfate: 100 mg of antibiotic dissolved in 1 ml of water and sterilized using a 0.2 µm filter membrane.
- Cefotaxime: 100 mg of each antibiotic dissolved in 1 ml of water and filter sterilized.
- Binary vector containing an egfp/nptII fusion gene under the control of a cauliflower mosaic virus 35S (CaMV 35S) promoter.
- Agrobacterium culture stock containing the binary vector (stored in glycerol at −80°C).
1.Initiation of micropropagation cultures from seeds
1.1.Immerse seeds in 70% ethanol for 1 min.
1.2.Transfer seeds to 25% commercial bleach solution containing one drop 100% Tween 20 (added using 1 ml micropipette) and surface-sterilize for 15 min with constant agitation.
1.3.Rinse twice for 5 min with sterile distilled water.
1.4.Blot dry seeds on filter paper and transfer to CR medium.
1.5.Cover Petri dishes in aluminum foil and incubate in darkness at 25°C for 2 d.
1.6.Transfer Petri dishes containing the germinated seedlings to cool white fluorescent light (75 mm m-2 s-1 and 16 h photoperiod) at 25°C for 5 d.
1.7.Excise meristems from germinated seedlings and transfer to fresh CR plates under conditions described in the previous step 1.6.
1.8.Proliferate cultures by subculture to fresh CR medium at two week intervals.
2.Initiation of Agrobacterium culture
2.1.Thaw Agrobacterium culture containing the binary plasmid at room temperature.
2.2.Spread approximately 20 µl of bacterial culture on a Petri dish containing solid YEP medium with 20 mg/L rifampicin and 100 mg/L kanamycin.
2.3.Incubate dishes in the dark at 26°C for 3 d.
2.4.Isolate a single colony growing on YEP medium and transfer it to a 125 ml conical flask containing 30 ml MG/L medium with 20 mg/L rifampicin and 100 mg/L kanamycin.
2.5.Incubate on a rotary shaker at 180 rpm at 26°C for 16–20 h. The bacterial culture should appear cloudy at the end of the culture period.
2.6.Transfer the culture to a 50 ml centrifuge tube and spin at 6000 rpm for 8 min at room temperature. Discard the supernatant and resuspend the pellet in 30 ml liquid MS medium. Adjust optical density at 600 nm (OD600) value to 0.2 using liquid MS medium.
2.7.Transfer the contents of the tube to a 125 ml conical flask and incubate for additional 4 h in a rotary shaker under the same conditions as above. Use this culture for co-cultivation.
3.Transformation of C. sativa explants
3.1.Transfer shoots meristems to sterile Petri dishes.
3.2.Add 5.0 ml Agrobacterium culture to explants and mix thoroughly by swirling. Incubate for 7 min. Blot explants dry on filter paper to remove the excess bacteria.
3.3.Transfer blot-dried explants to solid CR medium.
3.4.Seal the Petri dish with Parafilm® and co-cultivate in darkness at 26°C for 2 d.
3.5.After 2 d observe explants for transient GFP expression using microscope equipped with fluorescence illumination system.
3.6.Transfer co-cultivated explants to petri dishes containing solid Sel medium.
3.7.Transgenic cultures can be identified on the basis of GFP fluorescence and kanamycin resistance and can be used to separate transgenic lines from non-transformed cultures. Designate each line as an independent event and transfer to Sel medium.
3.8.Transfer shoots to Magenta GA7 vessels containing 30 ml Camelina rooting medium. Place vessels under conditions previously mentioned in step 1.6.
3.9.Transfer plants to 7 cm plastic pots containing Pro Mix BX potting mix and acclimatize in a growth room for two weeks before transfer to a greenhouse.
4.Molecular analyses of transgenic plants
4.1.Total genomic DNA can be isolated using the QIAGEN DNeasy Plant Mini Kit.
4.2.The presence of the transgenes can be confirmed by PCR using gene specific primers for GFP (5’- ATGGTGAGCAAGGGCGAGG AGCTGT-3’ and 5’- CTTGTACAGCTCGTCCATGCCGAGA-3’) and NPT II (5’- CGGCCG CTTGGGTGGAGAGG CTATT-3’ and 5’- TCAGAAGAACTC GTCAAGAAGGCGA -3’). PCR reactions are carried out under the following conditions: 1 cycle at 95°C for 4 min, 40 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min and a final cycle at 72°C for 4 min.
4.3.PCR products can be visualized by agarose gel electrophoresis.
|1.1–1.4||Seed not germinating||
|1.6, 1.7||Meristem cultures not proliferating||
|2.5||Liquid culture not growing or contaminated||
|3.5||No GFP expression observed||
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