Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa

Materials and reagents

Biological materials

1. Camelina sativa cultivar DH55 seeds

2. Agrobacterium strain C58C1 containing constitutive or chemically inducible plasmid expression constructs.

   Plasmids used in this study harbor constructs encoding chloroplast-GFP (CD3-995), peroxisomes-mCherry (CD3-983), Golgi body-mCherry (CD3-967), ER-mCherry (CD3-959) as described in Nelson et al. [9], and Dex-inducible pBAV150 [10] plasmid with DNA encoding the N-terminal (1–32 aa) region of a protein coded by the At3G28580 gene

   
   

Reagents

1. Plastic domes (Hummert International, catalog number: 11-33480) and trays (Hummert International, catalog

   number: 11-33010)

2. Pots 8 cells; width × length × depth: 12.34 × 12.34 × 5.77 cm (T.O. PLASTICS, catalog number: 715352C)

3. Soil (Berger 60, catalog number: BM2; BM6, mixed in 1:1 ratio)

4. Yeast extract (Fisher Scientific, catalog number: BP1422-500)

5. Bacto^TM peptone (Thermo Fisher Scientific, catalog number: 211677)

6. Sucrose (Fisher Scientific, catalog number: S5-500)

7. MgSO₄·7H₂O (Fisher Scientific, catalog number: 10034-99-8)

8. NH₄Cl (Sigma, catalog number: 12125-02-9)

9. KCl (Fisher Scientific, catalog number: P217-500)

10. CaCl₂ (Sigma, catalog number: C2661)

11. FeSO₄·7H₂O (Sigma, catalog number: F-7002)

12. NaH₂PO₄·H₂O (JT Baker, catalog number: JTB-3818-05)

13. Na₂HPO₄ (Fisher Scientific, catalog number: 7558-79-4)

14. Glucose (Sigma, catalog number: G7021)

15. MES (Sigma, catalog number: M8250)

16. MgCl₂·6H₂O (Fisher Scientific, catalog number: BP214-500)

17. Murashige and Skoog medium (Sigma, catalog number: M5519)

18. Bleach (Clorox Concentrated Germicidal Bleach)

19. Ethanol (Decon Labs Inc., catalog number: 2701)

20. Agarose (GOLDBIO, catalog number: 9012-36-6)

21. Silwet L-77 (Vac-In-Stuff, catalog number: VIS-30)

22. Tween^® 20 (Hoefer, catalog number: 9005-64-5, GR128-500)

23. Antibiotics: kanamycin sulfate (Kan) and rifampicin (Rif) (Fisher Scientific, catalog number: BP906-5, 13292-46-1, respectively)

24. Nalgene^TM Rapid-Flow^TM sterile disposable filter units (Thermo Fisher Scientific, catalog number: 568-0020)

25. Dexamethasone (Sigma, catalog number: D1756)

26. Perfluorodecalin (Strem Chemicals, catalog number: 09-5960)

27. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418)

28. Agar (Fisher Scientific, catalog number: BP1423-500)

29. Acetosyringone (Sigma, catalog number: D134406)

    
    

Solutions

1. Washing solution (see Recipes)

2. Infiltration solution (see Recipes)

3. Agrobacterium growth media (see Recipes)

4. 30 μM Dexamethasone (Dex) solution with 0.04% Tween-20 (see Recipes)

5. 70% ethanol (see Recipes)

6. 0.1% agar media (see Recipes)

7. Phosphate buffer (50 mM, 25×, pH 5.5) (see Recipes)

Recipes

Recipes 1, 2, and 3 are adapted from Zhang et al. [1].

1. Washing solution (1 L)

   Reagent	Final Concentration	Volume
   MgCl2 (1 M)	10 mM	10 mL
   Acetosyringone (0.1 M)	100 μM	1 mL
   ddH2O	n/a	989 mL
   Total	n/a	1 L

   
   

2. Infiltration solution (1 L)

   Note: Water should be added in increments of 100 mL. Allow the solution to mix completely before adding any more water. Murashige and Skoog medium and sucrose will take a substantial volume when fully dissolved. Adjust pH to 6.0 using 1 M KOH; use freshly prepared.

   Reagent	Final Concentration	Quantity or Volume
   Murashige and Skoog medium	1/4 × (w/v)	1.1 g
   Sucrose	1% (w/v)	10 g
   Acetosyringone (0.1 M)	100 µM	1 mL
   Silwet L-77	0.01% (v/v)	100 μL
   ddH2O	n/a	see note
   Total	n/a	1 L

   
   

3. Agrobacterium growth media (1 L)

   Note: Adjust the pH to 5.5 using 1 M KOH and autoclave the media. After autoclaving, add the filtered induce buffer components. Acetosyringone is prepared in DMSO.

   Reagent	Final Concentration	Quantity or Volume
   Yeast extract	0.1% (w/v)	1.0 g
   BactoTM peptone	0.5% (w/v)	5.0 g
   Sucrose	0.5% (w/v)	5.0 g
   MgSO4·7H2O	2.03 mM	0.5 g
   Agarose	1% (w/v)	10.0 g
   NH4Cl	18.70 mM	1.0 g
   KCl	2.01 mM	0.15 g
   CaCl2	90.10 μM	0.01 g
   FeSO4·7H2O	8.99 μM	0.0025 g
   ddH2O	n/a	889 mL (see note)
   Induce buffer composition:
   Phosphate buffer (50 mM, 25×, pH 5.5)	1×	40 mL
   Glucose (20%, 20×)	1×	50 mL
   MES (1 M, 50×, pH 5.5)	1×	20 mL
   Acetosyringone (200 mM, 1,000×)	1×	1 mL
   Total	n/a	1 L

   
   

4. 30 μM Dexamethasone (Dex) solution with 0.04% Tween-20 (100 mL)

   Note: Dex is prepared in DMSO.

   Reagent	Final Concentration	Volume
   Dex (30 mM)	30 μM	100 μL (see note)
   Tween-20	0.04%	40 μL
   ddH2O	n/a	99.86 mL
   Total	n/a	100 mL

   
   

5. 70% ethanol (100 mL)

   Reagent	Final Concentration	Volume
   Ethanol (95%)	70%	73.7 mL
   ddH2O	n/a	26.3 mL
   Total	n/a	100 mL

   
   

6. 0.1% agar media (100 mL)

   Note: Autoclave the media and stir before it cools.

   Reagent	Final Concentration	Volume
   Agar	0.1%	100 mg
   ddH2O	n/a	100 mL
   Total	n/a	100 mL (see note)

   
   

7. Phosphate buffer (50 mM, 25×, pH 5.5; 100 mL)

   Note: Adjust the pH to 5.5 using 1 M KOH.

   Reagent	Final Concentration	Volume
   NaHPO.HO	48.02 mM	662.7 mg
   NaHPO	1.98 mM	28.05 mg
   ddH2O	n/a	100 mL
   Total	n/a	100 mL (see note)

   
   

Laboratory supplies

1. Laboratory glassware

2. Pipette tips: 10 μL, 200 μL, and 1,000 μL (USA Scientific, catalog number: 1111-3700, 1111-1700, 1112-1720)

3. Black marker (Sharpie Permanent Markers, catalog number: 33861PP)

4. Kimwipes (Fisher Scientific, catalog number: 06-666A)

5. 15 mL and 50 mL centrifuge tubes (Fisher Scientific, catalog numbers: 14-959-53A, 14-432-22)

6. 9 cm round Petri dishes (Fisher Scientific, catalog number: FB0875712)

7. 1.5 mL microcentrifuge tubes (Fisher Scientific, catalog number: 01-549-746)

8. 1 mL tuberculin syringes without needle (BD Biosciences, catalog number: 309659)

9. Damp paper towel (Georgia-Pacific, catalog number: P200)

10. Sterile double-distilled water

11. Micropipettes 20 μL, 200 μL, and 1 mL (Gilson, catalog number: F144056M, F144058M, F144059M, respectively)

12. 1 L beaker (Pyrex, catalog number: 1000)

Equipment

1. Cork borer 4 mm diameter

2. Autoclave (Primus Sterilizer Co. Inc. 1317, catalog number: 09415.1256)

3. Fisher vortex Genie 2 (Fisher Scientific, catalog number: 12-812)

4. Plant growth chamber or walk-in growth room with humidity maintained at ≥ 50%. Light intensity should be 135–145 μmols^-1·m^-2 at soil level

5. Freezer (-80 °C) (Panasonic VIP Plus, model: MDF-V76VC-PA)

6. Balance (Mettler Toledo, model: PB1501)

7. Spectrophotometer (Bio-Mini, model: SHIMADZU)

8. Laminar flow hood (SterilGARD, model: 3 Advance)

9. Incubator at 28 °C (VWR, model: 3020)

10. Analytical balance for weighing chemicals

11. pH meter (SevenCompact, serial number: B408309625)

12. Confocal microscope (Zeiss, model: LSM 800)

Software and datasets

1. Fiji (ImageJ, version 2.15.0, 10/12/2023)

2. Photoshop (Adobe PS, 22.4.3, 07/19/2021)

3. Prism v10.1 (GraphPad, 07/01/2023)

4. BioRender.com online tool

Procedure

1. Plant growth and maintenance

   1. Soak non-sterilized, dry Camelina seeds (~100–150) in 4–6 mL of 0.1% sterile agar in 15 mL falcon tubes and keep at 4 °C in the dark for 3–7 days. The Camelina seeds utilized in this study were multiplied and bulked inside the greenhouse chamber.

   2. Sow approximately 18–20 seeds in individual soil mix–containing pots (12.34 × 12.34 × 5.77 cm) with the help of a Pasteur pipette and cover with a transparent plastic lid to maintain moisture. Keep pots in a plant growth chamber or growth room chamber under 12 h light and 12 h dark conditions at 22 ± 2 °C day/night temperature regime and light intensity of ~135–145 μmols^-1·m^-2. We use a mix of 50/50 sodium and metal halide light (but other lights might be suitable) at 50%–70% relative humidity.

   3. After 4–5 days, thin seedlings to nine plants per pot and remove the lid. Select the seedlings that look visibly similar and discard the rest (Figure S1).

   4. Allow seedlings to establish for the next 5–7 days until the first true leaves are completely opened (Figure 1).

      
      

      
      Figure 1. Representative image depicting Agrobacterium-infiltrated plants of Camelina sativa DH55 cultivar. Ten-to-twelve-day-old plants with opened true leaves were infiltrated with Agrobacterium strain C58C1 carrying desired constructs. White arrows indicate representative leaves imaged immediately after infiltration. The abaxial part of the leaves will have a wet appearance until the infused liquid containing Agrobacteria evaporates. Scale bar is in centimeters.

      
      

2. Agrobacterium strain suspension preparation for infiltration

   1. Agrobacterium suspension preparation

      1. Streak an Agrobacterium strain C58C1 stock with the desired constructs on a sterile plate of Agrobacterium growth media containing appropriate antibiotics and 200 μM acetosyringone inside a laminar airflow cabinet.

         Keep plates with bacteria at 28 °C for 24–36 h. In this work, marker plasmids of plastid-GFP (CD3-995), peroxisomes-mCherry (CD3-983), Golgi body-mCherry (CD3-967), ER-mCherry (CD3-959), and the N-terminal ER signal (1–32 aa) region of the At3G28580-encoded protein were used, and plates contained Rif (34 mg/mL) + Kan (50 mg/mL).

      2. Scrape a thin layer of Agrobacterial cells from each plate using a 200 μL pipette tip and resuspend bacteria in 1 mL of washing solution (see Recipes) in a 1.5 mL microcentrifuge tube by vortexing.

      3. Dilute an aliquot of 100 μL of resuspended Agrobacteria 10× in order to measure the OD_600. A value equal to or greater than 0.6 is desired in the final infiltration solution. Multiply this OD value by 10 to determine the OD₆₀₀ of the original undiluted resuspended Agrobacteria. This is done to obtain enough inoculum, sufficient to infiltrate all the true leaves per pot for each construct. After that, adjust the original suspension of Agrobacteria to an OD₆₀₀ of 0.6 using the infiltration solution (see Recipes). For colocalization studies, mix strains in a 1:1 ratio using an OD₆₀₀ of 0.6 for each strain and immediately infiltrate the leaves.

   2. Agrobacterium infiltration in Camelina

      1. Infiltrate the Agrobacterium in the infiltration solution into the abaxial surface of Camelina leaves (2 true leaves per plant) with the help of a 1 mL needleless plastic syringe. Use four or more plants per construct for infiltrations. Keep plants in the light for 1 h to allow the water-soaked leaves to dry before placing plants in the dark for 24 h at room temperature using plastic trays.

      2. Return the Agrobacterium-infiltrated plants to a growth chamber or growth room for another 3–5 days before observing leaves using confocal microscopy. If using an inducible promoter, spray the inducer over leaves 21 h before examination, as described in Banday et al. [11], using a confocal microscope (Figure 2A and 2B). In the example in Figure 2A, 30 μM Dex with 0.04% Tween-20 was applied.

         
         

         
         Figure 2. Agrobacterium-mediated transient subcellular localization in Camelina sativa DH55 cultivar. A. Upper panel: Confocal micrographs showing localization of Chloroplast-GFP (CD3-995), Peroxisome-mCherry (CD3-983), and Golgi-mCherry (CD3-967). Lower panel: Confocal micrographs of ER-mCherry (CD3-959), N-Term-ER-GFP (Dex: At3G28580; 1-32 aa region), and colocalization of N-Term-ER-GFP (Dex: At3G28580) with ER-mCherry (CD3-959; shown in magenta color). For localization and colocalization studies, images were taken 3 and 5 days after transfer to the light conditions, respectively. Chloroplast autofluorescence is shown in blue. Yellow arrowheads indicate chloroplast autofluorescence (shown in insets), magenta arrowheads indicate chloroplast-GFP overlap with chloroplast autofluorescence, and white arrows indicate colocalization of N-Term-ER-GFP (Dex: At3G28580) and ER-mCherry (CD3-959). Scale bars, 100 μm. B. Details of the separate channels of individual confocal micrographs: chloroplast autofluorescence (Blue), GFP signal (Green), and mCherry signal (Red). For colocalization, the mCherry signal is shown in magenta color. Scale bars, 100 μm. C. Efficiency of each transiently expressed construct. Data represent the average ± SE (n = 8) in percentage (%) obtained from experiments performed on two different days. The four images from each independent experiment were used for the calculations. See Table S1 for details.

Data analysis

The confocal microscope method was adapted from Banday et al. [11]. Zeiss LSM 800 was used to visualize GFP and mCherry fluorescence (Argon laser/excitation: 488 nm; emission collection: 505–530 nm for GFP; Argon laser/excitation: 594 nm; emission collection: 610–630 nm for mCherry) and chlorophyll autofluorescence (He-Ne laser/excitation: 633nm; emission collection: 650–750 nm or 645–700 nm). Images of abaxial leaf surface were captured using EC Plan-Neofluar 40/1.3 Oil DIC M27 objectives, pinhole at 1 AU for each channel, and photomultipliers master gain between 450 and 700. The images were optical sections captured at 1,024 × 1,024 pixels scanning resolution in maximum speed mode. Fluorescence of GFP, mCherry, and chlorophyll was acquired in sequential acquisition mode. Plant leaves were mounted in perfluorodecalin for optical enhancement [12]. ImageJ (Fiji) and Adobe PS were used to process the images. Each C. sativa imaging experiment included four independent samples per construct, and the experiments were done twice. Four images from different plant leaves from each experiment were used to calculate the efficiency of the transformation of cells per image. Using this protocol, all infiltrated leaves showed expression of the respective constructs. However, the efficiency of cells transformed/image was lower in the leaves of older plants (Table S1).

For calculation of efficiency analysis, the formula was adapted from Zhang et al. [1]. The graph was plotted using Prism software (Figure 2C and Table S1).

Efficiency (%) = Number of transiently transformed cells *100/Total number of cells

Validation of protocol

1. This protocol was adapted from Zhang et al. [1] and optimized for the Camelina crop. The protocol was validated using five different constitutive or chemically inducible plasmid expression constructs.

2. Each Camelina imaging experiment included four independent samples per construct, and the experiments were done twice. Four images from different plant leaves from each experiment were used to calculate the efficiency of the transformation of cells per image.

3. The student’s t-test shows that the efficiency of cells transformed/image was significantly lower in leaves of older plants (Table S1).

General notes and troubleshooting

1. Plants' age is very important. Leaves of older Camelina plants require needle punctures for successful infiltration and the efficiency of transformation declines (Table S1).

2. Pots must be well watered during the experiment to maintain humidity.

3. Keep only nine plants in one pot; plant density affects the growth of plants. A higher number of plants results in slower growth. Conversely, a lower number of plants per pot hastens the growth of plants.

4. Do not keep bacterial cultures for more than 36 h.

5. For better infiltration, the infiltration solution should be introduced into the abaxial surfaces of leaves.

6. Take confocal images of the abaxial surface to obtain a greater number of cells in the same focal plane.

7. Use perfluorodecalin to prevent the leaves from drying and for better imaging.

8. Clean the workbench before making an Agrobacterial suspension with the help of 70% ethanol. Later on, after completion of infiltration in plants, the remaining bacterial suspension must be treated with bleach for 24 h before being discarded into the sink.

Acknowledgments

This work was supported by a Dropkin Postdoctoral Fellowship awarded to PK and an Undergraduate fellowship from Arnold and Mabel Beckman Foundation to JLR. This protocol was adapted from Zhang et al. [1] and modified for the Camelina crop. We thank Dr. Isobel Parkin (Agriculture and Agri-Food Canada) for providing Camelina seeds. We thank Dr. Joanna Jelenska for valuable suggestions.

Competing interests

The authors declare that they have no conflicts of interest.

References

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3. Waraich, E. A., Ahmed, Z. I., Ahmad, R., Ashraf, M. Y., Saifullah, Naeem, M. and Rengel, Z. (2013). Camelina sativa, a climate proof crop, has high nutritive value and multiple uses: A review. Aust. J. Crop Sci. 7(10):1551–1559. https://www.cropj.com/waraich_7_10_2013_1551_1559.pdf
4. Neupane, D., Lohaus, R. H., Solomon, J. K. Q. and Cushman, J. C. (2022). Realizing the Potential of Camelina sativa as a Bioenergy Crop for a Changing Global Climate. Plants 11(6): 772. https://doi.org/10.3390/plants11060772
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Supplementary information

The following supporting information can be downloaded here:

1. Figure S1: Visible appearance of 5-days-old seedling.
2. Table S1: Details of efficiency analysis in percentage.