A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes

Add the following components to a Golden Gate reaction:

• Plasmid pIV101 DNA (100ng) - XµL
• Insert(s) DNA (100ng) - XµL
• Ligase buffer (10x) - 1µL
• T4 DNA Ligase - 1µL
• BpiI (BbsI) (or BsaI for level 1 constructs) - 1µL
• MilliQ water - up to final volume of 10µL

Incubate reaction in thermocycler with the following steps:

A	B	C
37C	00:05:00	30-60 X
16C	00:05:00
80C	00:10:00	1

Mix in a 1.5 ml tube:

• 20µL 5X KCM buffer
• 5µL Golden Gate cloning mix (from step 1.1 following completion of the program)
• 75µL ddH₂O

Incubate for 0h 2m 0s on ice

Thaw a 100µL aliquot of chemically competent E. coli ST18 cells on ice

Mix together the chemical competent E. coli cells and the reaction mixture from step 2 by pipetting

Incubate the mix for 0h 20m 0s on ice

Incubate for 0h 10m 0s at Room temperature

Add 800µL LB with 5 μg/ml 5-aminolevulinic acid (ALA) and grow for 1h 0m 0s at 37°C with shaking

Centrifuge to pellet the cells at 14000rcf,25°C

Resuspend the pellet in 100µL of LB containing 50 μg/ml ALA

Plate out the 100µL of resuspended pellet onto LB agar containing 100 μg/ml ampicillin, 150 μg/ml spectinomycin, and 50 μg/ml ALA

If using blue/white selection then also add 2% (w/v) 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) to the media

Grow at 37°C and check for colonies the following day

If using X-gal for selection avoid blue colonies as pIV101 contains the lacZα fragment in the GGA cloning site

In parallel with the next step (step 13), select colonies from the transformation (step 12) and confirm the construct from the Golden Gate assembly. This can be carried out by several standard approaches:

• Perform colony PCR to amplify the region of the plasmid that contains the GGA cloning site to ensure that the cloning site contains the expected insert size
• This product can be sent for further confirmation by Sanger sequencing
• Additionally, perform a plasmid preparation (miniprep) from the E. coli clone which can then be used as template for PCR amplification or for whole plasmid sequencing

Start liquid culture of the wild-type Agrobacterium rhizogenes (now Rhizobium rhizogenes ) from a single colony in LB media with 100 μg/ml rifampicin (a 5 ml broth is sufficient)

Incubate for 48h 0m 0s at 28°C with shaking

Inoculate an LB broth containing 100 μg/ml ampicillin, 150 μg/ml spectinomycin, and 50 μg/ml ALA with a single colony for an ST18 clone carrying the construct of interest (from the previous section)

Incubate the LB broth from step 15 24h 0m 0s at 37°C with shaking.

Centrifuge 1 ml of the overnight broth for the E. coli ST18 culture carrying the construct of interest at 14000rcf

Resuspend the E. coli pellet in 1mL sterile dH₂O and repeat the previous step. This is to wash away the broth culture containing antibiotics

Resuspend the E. coli pellet in 50µL sterile dH₂O

Centrifuge 1 ml of the broth culture of R. rhizogenes from step 13-14 at 8000rcf

Resuspend the R. rhizogenes pellet in 1mL sterile dH₂O and repeat the previous step. This is to wash away the broth culture containing antibiotics

Resuspend the R. rhizogenes pellet in 50µL sterile dH₂O

Perform a biparental mating by mixing the resuspended E.coli ST18 (step 19) and R. rhizogenes (step 22) in 100µL and spot onto plates of LB media supplemented with 50 μg/ml ALA (but no antibiotics) and then wait until the spot is dry

Grow the biparental mating spot plates at 28°C

Scrape the biparental mating spot of the E. coli ST18 clone + R. rhizogenes and resuspend in 1mL sterile dH₂O

Centrifuge at 14000rcf to pellet, and resuspend in 1mL sterile dH₂O. This step should wash away any residual supplement from the mating plates that enables E. coli growth

Centrifuge again at 14000rcf to pellet, and resuspend in 100µL sterile dH₂O (The total volume will be more due to the pellet)

Transfer the resuspended mix from the previous step onto LB media plates supplemented with 100 μg/ml ampicillin, 50 μg/ml spectinomycin, and 100 μg/ml rifampicin. (No ALA). Plate out for single colonies

Incubate the plate(s) for 48h 0m 0s at 28°C

Re-streak the R. rhizogenes strains carrying the construct of interest on LB agar containing 100 μg/ml ampicillin, 50 μg/ml spectinomycin, and 100 μg/ml rifampicin to ensure single colonies

Incubate the plate(s) for 48h 0m 0s at 28°C

To scarify the seeds, transfer the required number of seeds to a mortar and rub them with sand paper until they become white on the ends

Transfer the seeds to a sterile tube (at least 15 ml capacity) and sterilise the seeds by immersing them in a 1% hypochlorite solution, and incubate in this solution for 15 min at Room temperature

Remove the hypochlorite solution and discard appropriately. Add sterile water and invert the tube several times. Repeat this 5 times to wash the seeds and remove any residual hypochlorite

Fill the tube with sterile water and incubate for at least 2h 0m 0s at Room temperature with shaking (alternatively incubate at 4°C )

Using sterile forceps, transfer seeds to square petri dishes containing sterile filter paper soaked in sterile dH₂O (approximately 15 min per plate)

Incubate the square plates containing the surface sterilised seeds sitting on damp filter paper

from the previous step for 72h 0m 0s at 21°C

Transfer germinated seeds to a square petri dish that contains solid Gamborg B5 media including vitamins

Grow seedlings for 72h 0m 0s at 21°C with 16H/8H light/dark cycle, until the root has attached itself to the media

Resuspend the R. rhizogenes strain from the agar plate (step 30-31) into sterile dH₂O as a thick suspension (OD₆₀₀ >2). Approximately 100µL is needed per plant, so make sure the volume of water for the suspension is sufficient for the number of plants that will be transformed

Wound seedlings with syringe needle (0.4mm) at the hypocotyl

Add one large drop (~ 100µL) of the thick suspension of R. rhizogenes (from step 40) on top of the wound

Incubate the seedling for 1h 0m 0s horizontally to let the R. rhizogenes infect the hypocotyl

Seal the plates containing the now infected seedlings with parafilm on the sides and bottom (prevents dehydration). Seal top with micropore tape on the top edge (Keeps the plates sealed but allows gas exchange)

Grow the infected seedlings for 72h 0m 0s at 21°C in the dark to enhance infection

Grow the infected seedlings for 3 weeks at 21°C with 16H/8H light/dark cycle until transformed roots emerge

Hairy roots will emerge and develop from the infected wound sites

Place the plates containing the transformed plants on a transilluminator. Remove the non-fluorescently labelled roots (untransformed roots) using a scalpel

Transfer plants with transformed roots to new pots or plates and grow plants at 21°C with 16H/8H light/dark cycle for one week

Resuspend the rhizobium strain (or your bacteria of interest) from a freshly streaked agar plate into sterile dH₂O

Adjust the OD₆₀₀ of the suspension to between 0.01 - 0.05 in a volume that is sufficient to provide 100µL per plant to be inoculated

If plants are on square agar plates: lay the square plate flat. Inoculate the roots of the plants by carefully applying 100µL of the suspension from the previous step using a pipette. Ensure that you apply the inoculum evenly to as much of the root as possible

Leave inoculated plant plate(s) flat for a short period so that the bacterial suspension can spread evenly across the plate and ensure contact with the roots

If the plants are in pots: determine the volume that is equal to 100µL X the number of plants present, and distribute this evenly to the plant pot substrate using a pipette

Grow the inoculated plants for 3 weeks post inoculation at 21°C with 16H/8H light/dark cycle until nodules are formed