Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

NOTE: Steps 1.4-1.8, 2.3-2.5, 3.3-3.6, 4.1-4.6, 5.1-5.7, and 6.1-6.3 must be performed in aseptic conditions, using a clean hood to prevent contamination.

1. Plant material propagation for transformation

1. Place commercially available wild-type (WT) Plantago lanceolata seeds (see Table of Materials) in a 50 mL centrifuge tube up to the 5 mL line, depending on the number of plants desired.
   NOTE: Alternatively, a 2 mL microcentrifuge tube can be used when a small number of seeds are needed, but it should be filled to a volume no greater than 0.1 mL, as too many seeds can reduce the efficiency of sterilization.
2. Immerse the seeds in 75% ethanol for 60 s.
3. Discard the ethanol, then immerse the seeds in 20% sodium hypochlorite (20% NaClO, 80% sterile water) for 40 min, gently inverting the tube so that all the seeds come into contact with the solution.
   NOTE: The sodium hypochlorite solution must be freshly made for optimal results.
4. Under a laminar flow hood, discard the sodium hypochlorite solution, then wash the seeds with distilled water (five times). Add a small volume of water to the seeds after the final rinse, as this can help aid the movement of the plants onto plates.
5. Using sterilized forceps, transfer the seeds onto pre-prepared 95 mm x 100 mm Petri dishes with solid MS medium (Table 1). Spread the seeds evenly across the surface of the plate, with approximately 1 cm between each seed to prevent overcrowding of the germinated seedlings (Figure 1A) .
6. Seal the plates with two layers of paraffin film to prevent contamination, then incubate under a cool white grow light (see Table of Materials) at room temperature (22 °C with 50 µmol m^-2s^-1, 12 h days). The seeds typically germinate within 5-6 days.
7. When the seedlings have germinated and are large enough to transfer (Figure 1B), typically 2 or 3 days after germination, use sterilized forceps to transfer the seedlings into sterile boxes with the 50-100 mL of MS medium (Table 1). Ideally, plant only five seedlings per box to obtain the best quality roots.
8. Seal the boxes with surgical tape, then allow the plants to grow under a cool white grow light (see Table of Materials), in the same conditions mentioned in step 1.6. The plants should be ready for transformation in about 3-4 weeks, or when the main roots have grown about 2 cm in length and the lateral roots appear white.
   NOTE: Medium preparation recipes and vitamin stocks are included in Table 1 and Table 2.

2. Plasmid construction and E. Coli transformation

NOTE: The exact plasmid construction procedure varies depending on the gene of interest. In this procedure, the restriction enzymes HindIII and SalII were used to insert the 1.5 kb AtPP2 promoter into the binary plasmid pBI101 (see Table of Materials) with GUS, using the standard cloning procedure²⁰. AtPP2 (phloem protein 2) is a gene that is specifically expressed in phloem²¹.

1. Clone the promoter using primer pairs 5'-AGTCAAGCTTCAAGTCCCTGTGGCTACTGAAC-3' (Forward) and 5'-AGTCGTCGACAAACCAGTATGATGTATTTATTTTTG-3' (Reverse) from Arabidopsis. Figure 2 shows a diagram of the binary plasmid vector with the AtPP2:GUS insert.
2. After plasmid construction, transform the plasmids into DH5a E. coli (see Table of Materials) competent cells using the heat shock method²², and then incubate for 1.5 h at 37 °C with shaking (150 rpm).
3. Take 150 µL of each transformation culture, plate onto LB agar media plates (Table 1) with the appropriate selection (50 mg/L kanamycin for the strain used in this protocol; see Table of Materials), and then incubate the plates for 16-24 h at 37 °C.
   NOTE: The bacterial strain used in this study is A. tumefaciens GV3101.
4. Next, use colony PCR to screen the colonies for positive recombinants²³.
   NOTE: In this protocol, the following primers were used to amplify the targeted gene; 5'-ATGTTACGTCCTGTAGAAACCCCAA-3'(Forward) and 5'-TCATTGTTTGCCTCCCTGCTGC-3' (Reverse).
   1. Run the reaction in a thermocycler (see Table of Materials) with cycling conditions of 3 min at 95 °C, followed by 35 cycles of: 30 s at 95 °C, 30 s at 55 °C, 2 min at 72 °C, and a final 10 min elongation step at 72 °C.
   2. Inoculate the positive colonies in 6 mL of LB broth (Table 1) with the appropriate antibiotic (50 mg/L kanamycin) and grow at 37 °C overnight, at 200-250 rpm.
5. After overnight growth, extract the plasmids from the bacteria using standard procedures²⁴.

3. A. tumefaciens transformation with plasmid

1. After plasmid extraction, use electroporation to transform the modified plasmid into the desired strain of competent cells. In this procedure, A. tumefaciens strain GV3101 was used. Follow standardized methods for electroporation techniques²⁵.
2. After electroporation, resuspend the competent cells in 1 mL of LB broth and then incubate for 2-4 h at 28 °C at 100 rpm.
3. Collect the cells through centrifugation at 6,800 x g for 3 min in a tabletop microcentrifuge (see Table of Materials) at room temperature (22 °C), then spread 50-100 µL onto an LB agar plate with an appropriate selection agent (50 mg/L kanamycin for the plasmid used in this protocol).
4. After the cells have incubated for 2 days at 28 °C, identify positive colonies containing the gene of interest through the use of colony PCR. In this protocol, use the primers and conditions mentioned in step 2.4.
5. Next, use the positive colonies to streak a stock plate with LB agar media + selection. The plate can be stored at 4 °C for up to 1 month.
6. Alternatively, for long-term storage, inoculate a positive colony with a small volume of LB with appropriate selection. Shake the inoculated culture overnight at 28 °C at 200 rpm, then prepare a glycerol stock (50% w/v glycerol in a 50:50 mix of bacteria and glycerol), which can be stored at -80 °C for up to 10 years.

4. A. tumefaciens preparation

1. Streak A. tumefaciens containing the desired plasmid onto prepared 95 mm x 100 mm solid LB plates with the appropriate selection agent. In this protocol, the bacterial strain GV3101 with the plasmid insert AtPP2:GUS was used, with 50 mg/L kanamycin added for the selection.
2. Seal the plates with paraffin film, then incubate at 28 °C for up to 48 h, or until the bacteria grows large enough to pick.
3. Use a pipette tip to pick a bacteria colony 2 days before transformation, and inoculate it in a 15 mL round bottom tube containing 6 mL of liquid LB with appropriate selection. Shake at 200 rpm in a 28 °C tabletop shaker overnight, until OD₆₀₀ reaches 0.6-0.7.
   NOTE: Plates and the 6 mL bacterial inoculation can be stored at 4 °C for up to 1 month.
4. When the bacteria reach the correct OD₆₀₀, use a pipette to transfer A. tumefaciens to a sterile flask containing 100 mL of liquid LB with a selection agent. Typically, 200 µL of bacteria per 100 mL LB is appropriate for propagation. Shake at 200 rpm at 28 °C overnight, until OD₆₀₀ reaches 0.6-0.7.
5. Transfer the bacteria into 50 mL sterile centrifuge tubes, and centrifuge at 2,200 xg for 10 min at room temperature (22 °C) in a tabletop centrifuge to collect the bacteria.
6. Discard the supernatant using a pipette. Resuspend the bacterial pellet in 5 mL of room temperature (22 °C) liquid suspension solution (SS) (Table 1) by pipetting, then add up to 50 mL of SS and invert several times to mix. The bacteria is now ready for transformation.
   NOTE: The liquid SS must be prepared freshly, within 1 week of transformation.
   ​CAUTION: All material that comes into contact with A. tumefaciens needs to be discarded in a biohazard waste bin. Leftover liquids from bacterial cultures can be sterilized with sodium hypochlorite (bleach) at a concentration of 20% or higher.

5. Transformation of Plantago roots

1. When the plants reach the ideal stage for transformation (seedlings are 3 weeks old) (Figure 1C), use sterile forceps and scissors to separate the roots from the rest of the plant (Figure 3A). Discard the leaf and stem material.
2. Immediately after cutting, transfer the root pieces to sterile boxes containing sterile water using sterile forceps. This step allows the roots to stay hydrated while all tissue is collected.
3. When all the roots are cut, pour the A. tumefaciens/SS suspension into sterile 150 mm x 15 mm disposable Petri dishes. Transfer the roots into the A. tumefaciens culture and inoculate for at least 20 min (Figure 3B).
4. During incubation, use a sterile scalpel with a sharp blade to cut the roots into 1 cm fragments, separating the primary roots from the lateral roots. Make thin, shallow cuts on the surface of the roots to allow the bacteria to infect the plant.
   NOTE: If dealing with a large number of plants, transfer the root pieces into the bacterial culture in batches, to ensure that all the roots are immersed during inoculation.
5. After incubation, use the sterile forceps to transfer the root pieces to sterile paper towels to remove excess bacteria. Avoid drying the roots for more than 60 s, as this can cause dehydration and damage the root tissue. Ideally, 10-15 roots can be dried simultaneously (Figure 3C).
6. Transfer the dried roots to prepared 95 mm x 15 mm Petri dishes with solid co-culture media (Table 1), around 10-20 roots per plate, depending on the size of the roots (Figure 3D).
7. Seal the plates with two layers of transparent plastic film, then cover with aluminum foil. Incubate at room temperature (22 °C) for 3 days. This step provides time for the bacteria to infect the roots without the presence of an antibiotic selection (Figure 3E).

6. Selection and whole plant regeneration

1. After incubation in co-culture media, transfer the root pieces to prepared 95 mm x 15 mm Petri dishes with solid shoot induction media (SIM) (Table 1) with Timentin (500 mg/L; see Table of Materials) and appropriate antibiotic selection. In this protocol, we used kanamycin (100 mg/L).
   NOTE: The bottom of the roots must come into complete contact with the medium. Roots that do not touch the medium's surface are too long and must be cut to prevent the tissue from escaping selection.
2. Seal the plates with two layers of transparent plastic film, then grow under a grow light for 1 month (see step 1.6 for appropriate conditions), or until the shoots begin to emerge.
   NOTE: Typically, shoot initials can be observed after 2 weeks of growth, and the shoots are typically visible after 1 month.
3. When the plantlets are 1.5-2.0 cm long (Figure 1D), transfer them into prepared sterile boxes with solid root induction media (Table 1).
4. Grow the plants under a grow light (see step 1.6 for conditions) for several weeks, until roots form. Roots can typically first be seen after 1 week.
   ​NOTE: It is recommended to allow the roots to grow for several weeks before moving to the soil, as plants with larger root systems tend to have a higher survival rate in soil.

7. Soil transfer

1. When the root systems have become large enough to transfer (Figure 1E), typically after 1 month of growth, transfer the plants into 3.5 in square pots containing pre-wetted all-purpose soil (BM7). In this protocol, BM7 bark mix was used (see Table of Materials).
   1. Remove any medium that sticks to the roots by washing them gently in water.
      NOTE: The plants can be grown to maturity in a greenhouse at 800 to 1400 µmol photons m^-2 s^-2 using 600 W high sodium pressure lights (see Table of Materials), or in a growth chamber at room temperature (22 °C) with cool white lights at 50 µmol m^-2s^-1, 12 h days.
2. Cover the plants with a plastic potting cover, then cover them with a clear plastic bag. This step allows the plants to remain in a humid environment as they adapt to the soil.
3. After about 3-5 days, remove the plastic bag, then slowly remove the lid to allow acclimation to the outside environment.
   NOTE: Depending on the time of the year and the environment to which the plants are transferred, the time that the plants need to adapt can vary. Checking the plants daily and adding water to the pots as needed is recommended.
4. Water the plants regularly and add fertilizer as needed. Plants can also be transferred to larger pots for further growth (Figure 1F).

8. β-glucuronidase (GUS) histochemical staining

1. Prepare β-glucuronidase (GUS) staining solution, according to published protocols¹⁵.
2. When the shoot initials are about 0.5-1 cm long, remove a small tip of a young, fully expanded leaf (<5 mm in length is usually sufficient) and immediately transfer to 0.5-1 mL of GUS staining solution in a 1.5 or 2 mL microcentrifuge tube. The solution should fully cover the plant tissue.
3. Place the opened tubes in a vacuum desiccator and vacuum at 20-25 kPa for 5-10 min. Small bubbles should be visible in the solution during the vacuum procedure. This allows the solution to enter the cells of the plant.
4. Allow air to filter back into the vacuum desiccator. Close the tubes and incubate at 37 °C overnight (12 h), or until the blue color is visible.
   NOTE: In this study, GUS activity was localized to the phloem, meaning that in positively transformed plants, blue staining should only be visible in the phloem tissue. Plants without the transgene do not experience staining (Figure 4).
5. To better visualize the stain, transfer the plants to 100% ethanol to remove chlorophyll. To increase the efficiency of the chlorophyll removal process, incubate the tubes at 60 °C for 10 min.
   NOTE: The ethanol may need to be changed several times before all chlorophyll is removed, depending on the size of the leaf being stained.