Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Plantago species can be used as model plants for plant vascular biology and stress physiology. The establishment of a transformation protocol enables in-depth studies on these plants to explore the relationships between genes and their associated phenotype. The main advantage of this technique is that it uses foods as explants to transform narrowleaf plantain with high efficiency.

To begin, place commercially available wild type Plantago lanceolata seeds in a 50 milliliter centrifuge tube up to the 5 milliliter mark, depending on the number of plants desired. Immerse the seeds in 75%ethanol for 60 seconds. After discarding the ethanol, immerse the seeds in freshly prepared 20%sodium hypochlorite for 40 minutes by gently inverting the tube.

Under a laminate flow hood, discard the sodium hypochlorite solution, then wash the seeds five times with distilled water. 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. Using sterile forceps, transfer the seeds onto a pre-prepared Petri dish with solid MS medium by spreading the seeds evenly across the surface of the plate, approximately 1 centimeter between each seed to prevent overcrowding of the germinating seedlings.

Seal the plate with two layers of paraffin film to prevent contamination and incubate under a cool white grow light at room temperature. Once the seedlings germinate and are large enough to transfer, typically after two or three days of germination, use sterile forceps and transfer the seedlings into sterile boxes containing 50 to 100 milliliters of MS Medium. Seal the boxes with surgical tape, and allow the plants to grow under the cool white grow light for about three to four weeks.

Streak Agrobacterium tumefaciens containing the desired plasmid onto a prepared solid LB plate with the appropriate selection agent. Incubate the paraffin sealed plate at 28 degrees Celsius for up to 48 hours, or until the bacteria grows large enough to pick. After incubation, Pick a bacteria colony using a pipette tip two days before transformation, and inoculate it in a 15 milliliter round bottom tube containing six milliliters of liquid LB with the appropriate selection agent.

Shake at 200 RPM in a 28 degree Celsius tabletop shaker, overnight, until the OD 600 reaches 0.6 to 0.7. Once the bacteria reach the required OD, transfer 200 microliters of the bacterial culture to a sterile flask containing 100 milliliters of LB with the selection agent. Shake at 200 RPM at 28 degrees Celsius, overnight.

Then transfer the bacterial culture into 50 milliliters sterile centrifuge tubes, and centrifuge at 2, 200G for 10 minutes at room temperature to collect the bacteria. Discard the supernatant and resuspend the bacterial pellet in five milliliters of freshly prepared suspension solution or SS at room temperature by pipetting. Then add more SS up to the 50 milliliter mark and invert to mix several times.

Once the plants reach the ideal stage for transformation, in about three weeks, use sterile forceps and scissors to separate the roots from the plant and discard the leaf and stem material. Immediately after cutting, aseptically transfer the root pieces to boxes containing sterile water using forceps to keep them hydrated. Next, pour the SS suspended Agrobacterium tumefaciens bacterial culture into sterile 150 by 15 millimeter disposable Petri dishes.

Transfer the cut roots into the bacterial culture and incubate for at least 20 minutes. During incubation, use a sterile scalpel with a sharp blade to cut the roots into one centimeter fragments separating the primary roots from the lateral roots. Then make thin shallow cuts on the surface of the roots to allow the bacteria to infect the plant.

After incubation, transfer the root pieces to sterile paper towels to remove excess bacteria. Then transfer the dried roots to the prepared Petri dishes containing solid co-culture media around 10 to 20 roots per plate depending on the size of the roots. Seal the plates with two layers of transparent plastic film and then with aluminum foil to incubate at room temperature for three days.

After three days of incubation. In the co-culture media, transfer the root pieces to prepared Petri dishes with solid shoot induction media or SIM with 500 milligrams per liter of temetin and appropriate antibiotic selection. Incubate the transparent plastic film sealed plates under a grow light for one month or until the shoots begin to emerge.

Once the plantlets grow 1.5 to 2.0 centimeters long. Transfer them into prepared sterile boxes. With solid root induction media.

Grow the plants under a grow light for several weeks until roots form. Typically, after one week, when the root systems become large enough to transfer typically after one month of growth take the plant and gently wash the roots with water to remove any medium that sticks to the roots. Then transfer the plants into 3.5 inch square pots containing pre wetted all-purpose soil BM seven cover the plants with a plastic potting cover and then with a clear plastic bag to ensure the plants remain in a humid environment.

This method was tested on the root leaf and petty old tissue. In the preliminary experiment, although callous could be induced in all tissue types, only the root tissue produced shoot initials after one month in SIM, the leaf and pedal turned brown and died. The progression of shoot initials emerging from transformed tissue was observed from the first day.

The roots were placed on the SIM to when the shoots were ready to be rooted. After one week, the root tissue formed a callous in the beginnings of shoot initials could be observed. Shoots continued to emerge during weeks two and three and after four weeks, the shoots were ready to be transferred to the root induction medium.

Identification of the punitive transgenic plants was conducted using beta glucuronidase GUS Histochemical assay using the leaf segments taken once the shoots were around 0.5 centimeters long. The wild type in transform with empty vectors show no staining pattern. Due to the absence of the GUS gene, the transform with the beta glucuronidase GUS gene showed a clear blue staining pattern in the veins confirming that the plants are transgenic Roots should always be kept hydrated to prevent drying out during the procedure transfer the roots approximately 10 at a time to prevent this.