Principle

Why it works

In Arabidopsis, Agrobacterium tumefaciens applied to developing flowers transforms the female gametophyte, the ovule cells that will become the next generation. Each transformed ovule gives rise to a seed carrying the T-DNA from a binary vector, so transformants are recovered by selecting among seed, with no tissue culture and no regeneration.

Clough and Bent (1998) showed that a simple dip in Agrobacterium suspended in 5% sucrose with a surfactant gives transformation rates comparable to vacuum infiltration. The surfactant, Silwet L-77, is what lets the bacteria reach the ovules; sucrose supports the bacteria and the plant during the hours after dipping.

Safety and biosafety

Before you start

Work with recombinant Agrobacterium and genetically modified plants must be covered by your institution's biosafety approval and done in the containment it specifies. Autoclave all cultures, dipping solution and plant waste before disposal. Silwet L-77 is an eye and skin irritant; wear gloves and eye protection when handling the concentrate.

Materials

Plants and bacteria

Arabidopsis plants, 4–6 per pot, grown under long days (16 h light) until bolting
Agrobacterium tumefaciens strain such as GV3101 (pMP90) carrying your binary vector, confirmed by colony PCR or restriction digest

Reagents

Reagent	Use
LB or YEP medium	Agrobacterium culture
Antibiotics for strain and vector	For GV3101 (pMP90): rifampicin and gentamicin, plus the vector's selection (for example kanamycin or spectinomycin)
Sucrose	Dipping solution
Silwet L-77	Surfactant
½ MS medium with 0.8% agar, plus plant selection agent	T1 selection (for example kanamycin 50 µg/mL or hygromycin 15–25 µg/mL), or glufosinate spray on soil

Recipe: dipping solution (1 L)

Component	Final	Amount for 1 L
Sucrose	5% (w/v)	50 g
Silwet L-77, added just before dipping	0.05% (v/v)	500 µL
Water		to 1 L

Make fresh on the day. No MS salts or pH adjustment are needed.

Procedure

When the primary inflorescences are a few centimetres tall, cut them off at the base.Clipping releases apical dominance and gives many secondary bolts, so more buds are at the receptive stage on the dipping day.
4–6 days laterDip when the plants carry many unopened flower buds and few siliques.Water the plants the day before so they are not stressed.
Streak Agrobacterium from a glycerol stock onto selective LB plates and grow at 28 °C for 2 days.
Inoculate a 5 mL selective starter culture and grow at 28 °C with shaking for about 24 hours.
Dilute the starter 1 : 100 into 200–500 mL of selective medium and grow at 28 °C with shaking for 16–24 hours, to late log phase.About 200 mL is enough for two or three standard pots.
Pellet the cells at 4,000–5,000 × g for 10–15 minutes at room temperature.
Resuspend the pellet in 5% sucrose to an OD600 of about 0.8.Densities from about 0.5 to 2 work; exact OD is not critical.
CriticalImmediately before dipping, add Silwet L-77 to 0.05% (500 µL per litre) and mix gently.Do not exceed about 0.05%: more surfactant damages the plants without improving efficiency.
Optional: remove existing siliques and open flowers.This is not required, but it means nearly every seed you harvest came from a bud that was dipped.
Invert each pot and dip all above-ground parts into the suspension for 2–3 seconds with gentle agitation.A film of liquid should coat the buds.
16–24 hLay the pots on their sides in a tray, cover with a plastic dome or film to keep the humidity high, and keep them in low light.
Set the pots upright and return them to normal growth conditions.
Optional: dip a second time 7 days later to catch buds that were not yet receptive.
When siliques begin to yellow, stop watering, bag the plants and collect dry seed 3–4 weeks after dipping.Keep seed from each pot separate if you want independent lines with certainty.
Pause pointDry the seed for at least a week at room temperature before selection.Dry seed stores for years.
Surface-sterilise the T1 seed (chlorine gas, or 70% ethanol followed by dilute bleach) and sow densely on ½ MS plates containing the selection agent.Alternatively sow on soil and spray with glufosinate for bar-marked vectors, or screen seed under a fluorescence microscope for vectors with a seed-fluorescence marker.
Stratify at 4 °C for 2–3 days, then grow under long days for 7–10 days.
CriticalScore transformants as seedlings with green true leaves and long roots on selection.Non-transformants germinate and can stay green for several days on kanamycin; wait for true leaves before transferring.
Transfer resistant seedlings to soil and confirm each by PCR for the transgene.In the T2, a 3 : 1 resistant-to-sensitive ratio usually indicates a single insertion locus.

Expected results

Transformants are usually a small fraction of the T1 seed, commonly somewhere between a few per thousand and a few per hundred seeds depending on the construct, strain and plant condition. A single well-dipped pot normally gives enough independent transformants for most purposes. Each T1 plant that survives selection is an independent event, except where several seedlings come from the same transformed seed lineage.

Troubleshooting

Problem	Likely cause	What to change
No transformants	Plants dipped past the receptive stage; Agrobacterium lost the vector; Silwet omitted	Dip when most buds are still closed; confirm the vector in the culture you dip with; add Silwet fresh
Plants collapse after dipping	Too much Silwet; strong light or heat while covered	Keep Silwet at 0.05% or below; keep plants in low light while humid
Many false positives on selection	Scored too early; selection too weak; dense sowing shields seedlings	Wait for true leaves; check the antibiotic stock; sow less densely
Few seeds after dipping	Bud damage or water stress	Dip gently; water the day before; keep plants well watered after
T2 ratios far from 3 : 1	Multiple insertions, or transgene silencing	Select lines with single-locus segregation and confirm expression

References

Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16(6), 735–743. doi:10.1046/j.1365-313x.1998.00343.x
Zhang, X., Henriques, R., Lin, S.-S., Niu, Q.-W., & Chua, N.-H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 1(2), 641–646. doi:10.1038/nprot.2006.97

Change log

1.0 (25 October 2026). First version.

How to cite this protocol

Kumar A. Floral dip transformation of Arabidopsis thaliana. plantbiomethods.com, version 1.0, updated 25 October 2026. https://www.plantbiomethods.com/protocols/arabidopsis-floral-dip-transformation.html