Mar 18, 2025

Public workspaceA simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes V.2

PLOS One
Peer-reviewed method
DOI
dx.doi.org/10.17504/protocols.io.261ge3xkjl47/v2
  • Shaun Ferguson1,
  • Nikolaj B. Abel1,
  • Dugald Reid2,
  • Lene H. Madsen1,
  • Thi-Bich Luu1,
  • Emma Birkeskov1,
  • Kasper R. Andersen1,
  • Jens Stougaard1,
  • Simona Radutoiu1
  • 1Department of Molecular Biology and Genetics, Aarhus University, Denmark;
  • 2Department of Animal, Plant and Soil Sciences, School of Agriculture, Biomedicine and Environment, La Trobe University, Melbourne, Australia
Shaun Ferguson
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DOI: dx.doi.org/10.17504/protocols.io.261ge3xkjl47/v2
External link: https://doi.org/10.1371/journal.pone.0291680
Protocol Citation: Shaun Ferguson, Nikolaj B. Abel, Dugald Reid, Lene H. Madsen, Thi-Bich Luu, Emma Birkeskov, Kasper R. Andersen, Jens Stougaard, Simona Radutoiu 2025. A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3xkjl47/v2Version created by Shaun Ferguson
Manuscript citation:
Ferguson S, Abel NB, Reid D, Madsen LH, Luu TB, et al. (2023) A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes. PLOS ONE 18(11): e0291680. https://doi.org/10.1371/journal.pone.0291680
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 25, 2023
Last Modified: March 18, 2025
Protocol Integer ID: 124521
Funders Acknowledgements:
This work was funded by the project Molecular Mechanisms and Dynamics of Plant–microbe interactions at the Root–Soil Interface (InRoot)
Grant ID: NNF19SA0059362
Abstract
For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer.Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.
Guidelines
Whenever the protocol requires working with bacteria, whether picking a clone or streaking it fresh, always start from a single colony and ensure proper streaking technique on fresh media to obtain isolated single colonies.

Make sure your antibiotics and media containing antibiotics are fresh, otherwise selection my be insufficient resulting in false positive transconjugant colonies.

Ensure you wash the cells before and after biparental mating. This ensures adequate removal of both the antibiotics for plasmid selection, and the supplements required by the auxotrophic E. coli.

For this protocol we use E. coli strain ST18 for transformation of our cloned construct and subsequent biparental mating to transfer the construct to R. rhizogenes. Another E. coli strain could be substituted but it must contain the RP4 conjugative machinery for mobilisation of pIV101 and we would recommend that it also contains the lacZΔM15 mutation for blue white/screening as well as an auxotrophy for efficient counterselection (E.g. ΔdapA, or ΔhemA).

The Modular Cloning (MoClo) strategy implemented for pIV101 is based on the publication by Weber et al. 2011.

For standard molecular biology techniques (E.g. PCR, plasmid preparation etc.) please refer to Molecular Cloning: A Laboratory Manual, 4th edition.

Materials
Chemicals for Golden Gate reaction
  • Vector backbone (pIV101)
  • Additional assembly piece(s)
  • 10X NEB BSA (If using BsaI-HF®v2)
  • NEB BsaI-HF®v2
  • NEB BbsI-HF®
  • 10X NEB T4 Ligase Buffer
  • NEB T4 Ligase, 2,000,000 cohesive end units/ml
  • Nuclease-free water

Chemicals for E. coli chemical transformation
  • Filter sterilised diaminopimelic acid (DAP) stock at 30 mM in water (For ΔdapA mutant E. coli strains).
  • Previously prepared chemically competent aliquots of E. coli donor strain.
  • KCM buffer (5X)
  • 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) 1:1000

Media for bacteria
  • LB medium

Material for seed germination and hairy root transformation
  • Sand paper
  • Hypochlorite (12% stock solution)
  • Nuclease-free water
  • Filter paper
  • Gamborg B5 media including vitamins (Duchefa-biochemie)
  • Scalpel for plant cutting
  • Syringe needles (0.4 mm)
  • Parafilm
  • 3M Milipore tape

Antibiotics(Stock)
  • Rifampicin (50 mg/ml DMSO)
  • Streptomycin (50 mg/ml water)
  • Spectinomycin (50 mg/ml water)
  • Ampicillin (100 mg/ml water)

Buffers and stock solutions
KCM Buffer (5X):
  • 0.5M KCl
  • 0.15 M CaCl2
  • 0.25 M MgCl2
  • Filter sterilise through 0.2 uM filter. Store at -20.

5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal):
  • 20 mg/ml in dimethylformamide (2% w/v)

Auxotrophic E. coli supplement

5-aminolevulinic acid (ALA):
  • 50 mg/ml dissolved in water and filter sterlised

Diaminopemelic acid (DAP):
  • 30 mM dissolved in water and filter sterlised

General materials
  • Thermocycler suitable for Golden Gate reactions
  • Tabletop microcentrifuge
  • Spectrophotometer
  • 1.5 ml nuclease-free microcentrifuge tubes
  • Round and square (11 cm2) petri dishes
Safety warnings
Please refer to the manufacturers safety warnings for the individual reagents and chemicals used throughout.
Before start
Ensure you have an appropriate E. coli cloning/donor strain that contains the RP4 conjugative machinery to enable conjugal transfer of pIV101. Some commonly used strains for this include: E. coli S17/ST18, E. coli MFDpir, E. coli WM3064.

If using an auxotrophic E. coli strain such as ST18 or MFDpir make sure you have the required supplement (e.g. 5-aminolevulinic acid (ALA) for ST18 or diaminopimelic acid (DAP) for MFDpir).

pIV101 is available on Addgene and can be found here: https://www.addgene.org/196671/.

You must have a pBR322-derived R. rhizogenes acceptor strain e.g. R. rhizogenes AR1193.
Golden Gate cloning reaction with plasmid pIV101
Golden Gate cloning reaction with plasmid pIV101
Add the following components to a Golden Gate reaction:
  • Plasmid pIV101 DNA (Amount100 ng ) - AmountX µL
  • Insert(s) DNA (Amount100 ng ) - AmountX µL
  • Ligase buffer (10x) - Amount1 µL
  • T4 DNA Ligase - Amount1 µL
  • BpiI (BbsI) (or BsaI for level 1 constructs) - Amount1 µL
  • MilliQ water - up to final volume of Amount10 µL
15m


Incubate reaction in thermocycler with the following steps:
TemperatureTimeCycles
37C00:05:00 30-60 X
16C00:05:00
80C00:10:001

Note
If the Golden Gate reaction is failing to incorporate your desired insert, an additional step that may help:

Following completion of the reaction cycle from step 1.1 - add 1 μl of the restriction enzyme used in the reaction (step 1) to the mix again and incubate for a further 1 - 2hrs at 37C

Because the ligase has already been deactivated, additional active restriction enzyme can help to deplete any remaining plasmids that do not contain the desired insert fragments

4h
Transformation of chemical competent E. coli ST18
Transformation of chemical competent E. coli ST18
Mix in a 1.5 ml tube:
  • Amount20 µL 5X KCM buffer
  • Amount5 µL Golden Gate cloning mix (from step 1.1 following completion of the program)
  • Amount75 µL ddH2O

5m
Incubate for Duration00:02:00 on ice
2m
Thaw a Amount100 µL 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
1m
Incubate the mix for Duration00:20:00 on ice

20m
Incubate for Duration00:10:00 at TemperatureRoom temperature

10m
Add Amount800 µL LB with 50 μg/ml 5-aminolevulinic acid (ALA) and grow for Duration01:00:00 at Temperature37 °C with shaking

1h
Centrifuge to pellet the cells at Centrifigation14000 rcf, 25°C, 00:02:00

2m
Resuspend the pellet in Amount100 µL of LB containing 50 μg/ml ALA

2m
Plate out the Amount100 µL of resuspended pellet onto LB agar containing 100 μg/ml ampicillin, 150 μg/ml spectinomycin, and 50 μg/ml ALA

5m
  • 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 Temperature37 °C DurationOvernight and check for colonies the following day



Note
If you are having difficulties obtaining E. coli transformants then we suggest trying electrocompetent E. coli cells which have a higher transformation efficiency. For transformation by electroporation we recommend first performing a clean up of the Golden Gate reaction mixture DNA to prevent arcing

16h
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

Note
If your insert is large then we recommended sending the construct for whole plasmid sequencing as an alternative to confirmation by standard Sanger sequencing

1d
R. rhizogenes transformation
R. rhizogenes transformation

Note
This section and the next section can be carried out in parallel to save time

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)
10m
Incubate for Duration48:00:00 at Temperature28 °C with shaking

2d
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)
5m
Incubate the LB broth from step 15 DurationOvernight at Temperature37 °C with shaking.

1d
Centrifuge ~100 μl of the overnight broth for the E. coli ST18 culture carrying the construct of interest at Centrifigation14000 rcf, 00:02:00

2m
Resuspend the E. coli pellet in Amount1 mL sterile dH2O and repeat the previous step. This is to wash away the broth culture containing antibiotics

3m
Resuspend the E. coli pellet in Amount50 µL sterile dH2O
2m
Centrifuge 0.3 - 1 ml of the broth culture of R. rhizogenes from step 13-14 at Centrifigation8000 rcf, 00:02:00

Note
Adjust the volume of R. rhizogenes that you spin down so that the pellet is similar in size to E. coli

2m
Resuspend the R. rhizogenes pellet in Amount1 mL sterile dH2O and repeat the previous step. This is to wash away the broth culture containing antibiotics
3m
Resuspend the R. rhizogenes pellet in Amount50 µL sterile dH2O
2m
Perform a biparental mating by mixing the resuspended E.coli ST18 (step 19) and R. rhizogenes (step 22) in Amount100 µL total volume and spot onto plates of LB media supplemented with 50 μg/ml ALA (but no antibiotics) and then wait until the spot is dry

Note
Can be better to aliquot the 100 μL across multiple spots (at least 2 x 50 μl spots)

30m
Grow the biparental mating spot plates DurationOvernight at Temperature28 °C
16h
Scrape the biparental mating spot of the E. coli ST18 clone + R. rhizogenes and resuspend in Amount1 mL sterile dH2O

Note
Be sure to avoid collecting any agar (better off to leave some of the mating colony behind) as the agar contains ALA which we need to avoid

5m
Centrifuge at Centrifigation14000 rcf, 00:02:00 to pellet, and resuspend in Amount1 mL sterile dH2O. This step should wash away any residual supplement from the mating plates that enables E. coli growth

2m
Centrifuge again at Centrifigation14000 rcf, 00:02:00 to pellet, and resuspend in Amount1 mL sterile dH2O (The total volume will be more due to the pellet)
2m
Transfer the resuspended mating mix from the previous step onto LB media plates supplemented with 100 μg/ml ampicillin, 100 μg/ml spectinomycin, and 100 μg/ml rifampicin. (No ALA). Perform spread plating to obtain single colonies, see step 28.1

2m
R. rhizogenes transformation
R. rhizogenes transformation
Plate different volumes of the resuspended mating mix onto separate plates to ensure single colonies (avoid getting a lawn of bacteria). We suggest three plates, each with the following volume:
Plate 1 - 50 μl
Plate 2 - 100 μl
Plate 3 - Pellet the remaining volume and then resuspend in 100 μl of dH2O to spread
Note
It is best to choose single colonies from the plate with the fewest colonies present.

R. rhizogenes transformation
R. rhizogenes transformation
Incubate the plate(s) for Duration48:00:00 at Temperature28 °C
Note
Attention: Colonies should be present within 1 - 2 days, colonies arising after this time may be false positives so regularly check your plates during incubation for first colonies that appear.

2d
Re-streak (at least once) the R. rhizogenes strains carrying the construct of interest on fresh LB agar containing 100 μg/ml ampicillin, 100 μg/ml spectinomycin, and 100 μg/ml rifampicin (ensure single colonies)
Note
Re-streaking the single colonies onto fresh media to obtain new single colonies is critical.

The E. coli should not grow on the initial spread plates (Steps 28 and 29), however it is important to purify your transconjugant R. rhizogenes clones to remove any remaining E. coli cells.

Plasmid from dead E. coli cells may still produce false positive results during PCR confirmations for R. rhizogenes transconjugant clones, so pure single colonies is crucial.

5m
Critical
Incubate the plate(s) for Duration48:00:00 at Temperature28 °C
2d
Preparation of seeds for hairy root transformation (for Lotus japonicus)
Preparation of seeds for hairy root transformation (for Lotus japonicus)
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

2m
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 TemperatureRoom temperature

15m
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
5m
Fill the tube with sterile water and incubate for at least Duration02:00:00 at TemperatureRoom temperature with shaking (alternatively incubate DurationOvernight at Temperature4 °C )

2h
Using sterile forceps, transfer seeds to square petri dishes containing sterile filter paper soaked in sterile dH2O (approximately 15 min per plate)
15m
Incubate the square plates containing the surface sterilised seeds sitting on damp filter paper
from the previous step for Duration72:00:00 at Temperature21 °C
3d
Transfer germinated seeds to a square petri dish that contains solid Gamborg B5 media including vitamins
15m
Grow seedlings for Duration72:00:00 at Temperature21 °C with 16H/8H light/dark cycle, until the root has attached itself to the media
3d
Preparation of R. rhizogenes for hairy root transformation
Preparation of R. rhizogenes for hairy root transformation
Resuspend the R. rhizogenes strain from the agar plate (step 30-31) into sterile dH2O as a thick suspension (OD600 >2). Approximately Amount100 µL 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

Note
The actual concentration is not important, there just needs to be an excess of the R. rhizogenes


10m
Hairy root transformation of Lotus seedlings with R. rhizogenes
Hairy root transformation of Lotus seedlings with R. rhizogenes
Wound seedlings with syringe needle (0.4mm) at the hypocotyl
1m
Add one large drop (~ Amount100 µL ) of the thick suspension of R. rhizogenes (from step 40) on top of the wound

1m
Incubate the seedling for Duration01:00:00 horizontally to let the R. rhizogenes infect the hypocotyl
1h
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)
1m
Grow the infected seedlings for Duration72:00:00 at Temperature21 °C in the dark to enhance infection
3d
Grow the infected seedlings for 3 weeks at Temperature21 °C with 16H/8H light/dark cycle until transformed roots emerge

3w
Selecting hairy roots
Selecting hairy roots
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

Note
The Golden Gate assembly constructs should be designed to include a fluorescent marker to distinguish hairy roots from non-transformed roots. Alternative markers like RUBY can also be used

1h
Transfer plants with transformed roots to new pots or plates and grow plants at Temperature21 °C with 16H/8H light/dark cycle for one week

1w
For rhizobium inoculation of hairy roots (optional)
For rhizobium inoculation of hairy roots (optional)
Resuspend the rhizobium strain (or your bacteria of interest) from a freshly streaked agar plate into sterile dH2O
10m
Adjust the OD600 of the suspension to between 0.01 - 0.05 in a volume that is sufficient to provide Amount100 µL per plant to be inoculated
15m
If plants are on square agar plates: lay the square plate flat. Inoculate the roots of the plants by carefully applying Amount100 µL 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
5m
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
5m
If the plants are in pots: determine the volume that is equal to Amount100 µL X the number of plants present, and distribute this evenly to the plant pot substrate using a pipette
5m
Grow the inoculated plants for 3 weeks post inoculation at Temperature21 °C with 16H/8H light/dark cycle until nodules are formed
3w
Protocol references
Golden Gate cloning:
Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PloS One. 2008;3(11):e3647. doi: https://doi.org/10.1371/journal.pone.0003647.

Modular Cloning (MoClo):
Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PloS One. 2011;6(2):e16765. doi: https://doi.org/10.1371/journal.pone.0016765.

E .coli strains:
Ferrieres L, Hémery G, Nham T, Guérout A-M, Mazel D, Beloin C, et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. Journal ofBacteriology. 2010;192(24):6418-27. doi: https://doi.org/10.1128/JB.00621-10.

Thoma S, Schobert M. An improved Escherichia coli donor strain for diparental mating. FEMS Microbiology Letters. 2009;294(2):127-32. doi: https://doi.org/10.1111/j.1574-6968.2009.01556.x.


R. rhizogenes pBR322-acceptor strains:
Stougaard J, Abildsten D, Marcker KA. The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants. Molecular and General Genetics MGG. 1987;207(2):251-5. doi: https://doi.org/10.1007/BF00331586.

Radutoiu S, Madsen LH, Madsen EB, Nielsen AM, Stougaard J. Agrobacterium rhizogenes pRi TL-DNA integration system: a gene vector for Lotus japonicus transformation.Lotus japonicus Handbook: Springer; 2005. p. 285-7.

Green, M. R., & Sambrook, J. (2012). Molecular cloning. A Laboratory Manual 4th.