Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

The A. rhizogenes transformation protocol was adapted and modified from Horn et al.⁷ and the genotype tested was S. tuberosum ssp. tuberosum (cv. Désirée). The A. tumefaciens transformation protocol was adapted and modified from Banerjee et al.²² and the genotypes tested were S. tuberosum ssp. tuberosum (cv. Désirée) and S. tuberosum ssp. andigena. The main steps of both procedures are summarized in Figure 1 and Figure 2, respectively.

NOTE: In all the steps of the procedure performing in vitro transfers, do so rapidly, and when possible, maintain the plates or pots closed, thus minimizing plant exposure to the air to avoid wilting and contamination. Otherwise stated, all the plant incubations were done in cabinets under the conditions of 12 h of 24 °C light/12 h of 20 °C dark and 67 µmol m^-1 s^-1. Otherwise stated, perform all the bacteria manipulation and in vitro plant transfers in aseptic conditions in a laminar flow hood. All the media recipes for Agrobacterium and in vitro plant cultures are provided in Table S1.

CAUTION: Deposit all the genetically modified bacteria and plants to the appropriated waste container.

1. Agrobacterium cultures used for transformation

NOTE: The strain used for A. rhizogenes transformation was the C58C1:Pri1583⁷ (kindly provided by Dr. Inge Broer) and that for the A. tumefaciens was the GV2260 (kindly provided by Dr. Salomé Prat). A. rhizogenes was transformed with the binary vector PK7GWIWG2_II-RedRoot (VIB-Department of Plant Systems Biology at Universiteit Gent; http://gateway.psb.ugent.be) that contains a T-DNA carrying a transformation marker to monitor the hairy root formation. To compare the transformed roots generated by A. rhizogenes and A. tumefaciens, both were transformed with the binary vector pKGWFS7 which contains a T-DNA carrying the FHT promoter driving the β-glucoronidase (GUS) reporter gene and the Kanamycin resistance gene as a selectable marker¹⁵.

1. Pick a colony of Agrobacterium and grow it overnight (O/N) in 5 mL of YEB medium supplemented with antibiotics (Table S1) in a 50 mL centrifuge tube at 28 °C with shaking at 200 rpm.
2. For A. tumefaciens transformation, measure the optical density, which must be OD₆₀₀ = 0.6-1.0.
   1. If the optical density is higher, make a subculture lowering it to OD₆₀₀ = 0.3 with fresh media and wait until the culture reaches OD₆₀₀ = 0.6-1.
3. Centrifuge 1 mL of Agrobacterium culture at 3,000 x g in a bench-top centrifuge for 10 min at room temperature.
4. Remove the supernatant by pipetting, and resuspend cells in 1 mL of fresh YEB medium without antibiotics. Repeat this step to ensure the complete removal of antibiotics.
   1. For A. tumefaciens transformation, in the last resuspension add the appropriate YEB volume to obtain a final optical density of OD₆₀₀ = 0.8.
5. Keep cells on ice while preparing the plants to be infected.

2. Plant material for transformation

1. Make one or two node stem cuttings either containing the apical or the auxiliary buds from sterile in vitro potato plants (donor plants); grow them in solid 2MS medium in pots for 3 to 4 weeks (Figure 1A and Figure 2A).

3. Plant transformation using A. rhizogenes (Figure 1)

NOTE: This procedure allows the obtaining of transformed hairy roots. To evaluate the transgene expression, a negative control is needed. To prepare the negative control, follow the procedure using an A. rhizogenes strain either untransformed or transformed with the empty vector that includes the transformation marker gene.

1. Use fresh media plates; alternatively, the plates can be kept at 4 °C with the lid side up, tightly sealed with transparent film to avoid media dehydration. To prepare the square media plates, incline them ~15°, fill with 40 mL of MS, and let them solidify. This will minimize the contact of the aerial part of the plant with the medium.
2. Transfer very carefully a donor plant from the 2MS medium to a 120 mm x 120 mm square plate.
3. Inject to one stem internode 3 μL of the A. rhizogenes culture using a surgical needle and repeat it twice per plant in different internodes when possible.
   NOTE: Consider each injection as an independent transformation event (Figure 1B).
4. Transfer immediately the entire plant to a square plate with solid MS medium supplemented with 0.1 mM acetosyringone. Accommodate 2 plants per plate.
   NOTE: The 1 M acetosyringone stock solution is prepared in DMSO and can be stored at -20 °C.
5. Seal the plate using surgical tape and arrange it vertically inside a growth cabinet for 4 days.
6. Transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 µg/mL] to kill A. rhizogenes.
7. After 10-12 days, hairy roots will start to appear (Figure 1C,1D). Then, excise the native roots of the plant, and transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 µg/mL].
   NOTE: Transformed hairy roots can be checked by red fluorescence when using a DsRed transformation marker (Figure 3D). 
8. To obtain a composite plant (Figure 1E), let the transgenic hairy roots grow for 3-4 weeks in MS medium supplemented with cefotaxime sodium [500 μg/mL] (change the medium every week).
9. Depending on the purpose, propagate the transgenic hairy roots and the negative controls as follow.
   1. Transfer the whole composite plant to a hydroponic (Table S2) or soil medium to allow for massive development.
   2. To individually propagate the transformed hairy roots, using a scalpel cut the roots expressing the red fluorescent transformation marker (DsRed protein) when they are 4-8 cm long (Figure 1E) and transfer them into a Petri dish with Gamborg B5 solid medium supplemented with 2% sucrose and cefotaxime sodium [500 μg/mL]. Seal the plates with plastic laboratory film and grow them in the dark at 20 °C.
      NOTE: The roots can be manipulated under a stereomicroscope equipped to detect the fluorescence under sterile conditions (see Table of Materials).
   3. For biomass production (i.e., gene expression analysis), cut a 5 cm long hairy root and propagate it in a 150 mL Erlenmeyer flask with 20 mL of Gamborg B5 liquid medium supplemented with 2% sucrose and cefotaxime sodium [500 µg/mL]. Grow it for 6 weeks in the dark at 20 °C and 60 rpm.

Figure 1: Timeline to obtain potato transgenic hairy roots using A. rhizogenes. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the hairy roots are shown. Representative images of different stages are depicted: the initiation of the process using 3-week-old in vitro plants (A), then infection of the plants by injecting A. rhizogenes (B), the formation of the proliferative tissue (C, arrows) with emerging hairy roots (D), and the developed hairy roots expressing the red fluorescent transformation marker DsRed (E). Please click here to view a larger version of this figure.

4. Plant transformation using A. tumefaciens (Figure 2)

NOTE: This procedure allows the obtaining of transformed plants. To evaluate the transgene’s effect, a negative control is needed. One option is to follow the procedure using an A. tumefaciens transformed with the empty vector. Alternatively, wild type plants can be used.

1. Cut and place a leaf from the 3-4-week-old plants (Figure 2A) in a Petri dish. Using a scalpel exclude the petiole and make transverse cuts (1-3 depending on the leaf size) from the center of the leaf to the edges avoiding cutting them off (Figure 2B).
2. Immediately place the leaf floating on 10 mL of fresh 2MS liquid media in a Petri dish with the abaxial side up and close the plate. Repeat the step accommodating up to 15 leaves for cv. Désirée and 25 leaves for ssp. andigena (depending on the leaf size).
3. Immediately add 80 µL of A. tumefaciens culture at OD₆₀₀ = 0.8 in the liquid media and homogenize the plate manually for 1 min to distribute the bacterial solution.
4. Carefully seal with sealing film, cover with aluminum foil and incubate for 2 days in a chamber at 24 °C to let the transformation occur.
5. Transfer the leaves keeping abaxial side up to CIM medium (Figure 2B) and incubate them for one week in a growth cabinet.
   1. Scrape the CIM medium with the tweezers so that the leaves can be better accommodated on the media.
6. Transfer the leaves keeping abaxial side up to SIM medium (Figure 2C) and incubate them in a growth cabinet, refreshing the medium every 7-10 days, until the shoots are about 2 cm tall.
   1. Scrape the SIM medium with the tweezers so that the leaves can be fully surrounded by the media. When the emerged shoots reach the lid, work with tall Petri dishes (100 x 20 mm, height x diameter).
      NOTE: The callus will form after 2-3 weeks in SIM medium (Figure 2D) and the shoots after 6-7 weeks (Figure 2E). The shoots will be considered as independent transformation events when they emerge from callus formed from independent wounds.
7. Cut three shoots emerged from each callus (considered the same transformation event) (Figure 2E), transfer them to culture flasks with MG medium supplemented with cefotaxime sodium [250 mg/L] to allow rooting, label the subset with a number and incubate in a growth cabinet for 3-4 weeks or until the shoots are vigorous (Figure 2F).
   NOTE: When cutting the shoots, remove well the callus otherwise the root will not form.
   1. Repeat the step as many times as independent lines are needed. Up to 5 different transformation events can be placed in a culture flask with a diameter of 8 cm to work in full confidence that the plants from different events are not mixed.
8. Select the most vigorous plant of each event, cut the apical segment of the shoot with 3-4 internodes and place it in a new culture flask with 2MS medium supplemented with cefotaxime sodium [250 mg/L].
   NOTE: In 3-6 weeks the plant will grow efficiently, developing a vigorous shoot and roots.
   1. Bring back to the chamber the non-selected shoots until the plant selected has fully developed.
9. Cut stem segments from the plant with at least one internode or with the apical bud and transfer them to new 2MS medium supplemented with cefotaxime [250 mg/L]. Incubate them in the growth cabinet.
   1. Replicate every 3-4 weeks to establish the in vitro transformed lines.
      NOTE: The cefotaxime sodium is needed in at least three subsequent transfers to 2MS medium to be sure to kill the A. tumefaciens; afterwards, if A. tumefaciens overgrowth is observed, transfer the plants again to 2MS media supplemented with cefotaxime sodium.
10. To characterize the plant phenotype, transfer plants to soil for their full characterization or to hydroponics culture for root inspection.
    1. Keep the tubers produced in soil to propagate and maintain the established lines.

Figure 2: Timeline to obtain potato transformed plants using A. tumefaciens. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the plants are shown. Representative images of different stages are depicted: the initiation of the process using leaves from 3-week old in vitro plants (A), the transfer of the wounded and infected leaves to the CIM media (B), the leaves when transferred to SIM media (C), the visualization of the callus around the wounded areas after 2-3 weeks in SIM media (D), the shoot formation after 9-11 weeks in SIM media (E), and the shoots after being transferred to MG media (F). Please click here to view a larger version of this figure.

5. Hydroponic culture

1. Prepare the Hoagland’s solution to a half strength (0.5x) (Table S2) in a 10 L bucket.
2. Immerse an aquarium pump to maintain homogeneity and proper oxygen conditions.
3. Cover the walls of the bucket with aluminum foil to grow roots in dark conditions.
4. Avoiding root damage, transfer in vitro plants to hydroponic culture.
   NOTE: Remove any remaining in vitro medium from roots to avoid microorganism proliferation during incubation by shaking carefully the roots immersed in water.
5. Cover the plants with transparent film like a glasshouse to allow adequate acclimatization and incubate in the growing chamber.
6. Make holes in the film after 3 days and remove it completely one week after.
7. Replace with fresh media every 10 days.

6. GUS histochemical reporter gene assay

NOTE: In our case the GUS analysis was performed with roots of 2-3 weeks grown in hydroponics or in vitro.

1. Fix the roots with 90% chilled acetone (v/v) and incubate it for 20 minutes on ice.
2. Perform two washings with distilled water.
3. Add fresh GUS staining solution (Table 1) and apply vacuum (-70 Pa) for 20 min.
4. Incubate at 37 °C in dark to protect the photosensitive GUS for 4 h or until a blue color is visible.
   NOTE: The presence of ferri- and ferrocyanide in GUS solution minimize the diffusion of reaction products and provide more precise localization.
   CAUTION: Use a fume hood and wear protective clothing when handling the toxic cyanide derivatives in GUS solution (the potassium ferricyanide and potassium ferrocyanide). The GUS substrate and the disposal material should be disposed safely.
5. Remove GUS staining solution and discard it in appropriate containers.
6. Perform two washings with ethanol 70% (v/v).
7. Observe under a bright field microscope.
   NOTE: GUS staining is stable for a few weeks; however, during the first week, the GUS signal is clear and diffuses less into neighboring cells. For longer storage, seal the tube and store it at 4 °C.

Table 1: GUS staining solution recipe.