Seedling Flood Assay for Screening Bacterial Resistance in Tomatoes

Abstract

Source: Hassan, J. A., et al. High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay. J. Vis. Exp. (2020).

This video demonstrates a method to screen for bacterial resistance in different tomato strains using ten-day-old seedlings inoculated with a bacterial suspension. It explains how bacteria invade through stomata, deliver effector proteins to suppress plant immunity, and how plant responses differ between susceptible and resistant strains. The outcome is assessed by observing visible growth differences, with resistant plants remaining healthy and susceptible ones showing tissue damage.

Protocol

1. Tomato seedling flood method

1. Take the plates with the 10-day-old seedlings out of the growth chamber and put in the biosafety cabinet to prepare the plates for flooding.
2. Remove the surgical tape from two plates.
3. Set a timer for 3 min. Measure 6 mL of the final inoculum of P. syringae pv. tomato (Pst) (Pst19 OD₆₀₀ = 0.0075 or PstDC3000 OD₆₀₀ = 0.005) and transfer 6 mL of inoculum to each plate with the 10-day-old seedlings.
4. Gently push the seedlings down into the inoculum with a sterile pipette tip. Start the timer.
5. Hold one plate in each hand. Tilt the front of the plate down to accumulate inoculum and mainly submerge the cotyledons and leaves of the seedlings.
6. Swish side to side 5–7x and then tip the plates back to cover the roots and the whole plate.
7. Tilt the plates down again to submerge the cotyledons and leaves, and repeat for a total of 3 min.
8. Pour the inoculum off the plates, set the plates down on a flat surface, and then pour off any residual inoculum a second time.
9. Rewrap the plates with surgical tape and repeat steps 1.2–1.8 for any remaining plates.
10. Re-incubate the plates in the growth chamber after all plates have been flooded.

Phenotype after 7–10 days for PstDC3000 or 10–14 days for Pst19.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biosafety cabinet, class II type A2
BRAND Disposable Plastic Cuvettes, Polystyrene	VWR International	47744-642
Chenille Kraft Flat Wood Toothpicks	VWR International	500029-808
Glass Alcohol Burner Wick	Fisher Scientific	S41898A / No. W-125
Glass Alcohol Burners	Fisher Scientific	S41898 / No. BO125
Glycerol ACS reagent	VWR International	EMGX0185-5
Kimberly-Clark™ Kimtech Science™ Kimwipes™ Delicate Task Wipers	Fisher Scientific	06-666-A
Magnesium chloride, ACS grade	VWR International	97061-356
Magnesium sulfate heptahydrate, ACS grade	VWR International	97062-130
Microcentrifuge tubes, 1.5 mL
Microcentrifuge tubes, 2.2 mL
Murashige & Skoog, Basal Salts	Caisson Laboratories, Inc.	MSP01-50LT
Pipet-Lite XLS LTS 8-CH Pipet 20-200uL	Rainin	L8-200XLS
Pipet-Lite XLS LTS 8-CH Pipet 2-20uL	Rainin	L8-20XLS
Polystyrene 100mm x 25mm sterile petri dish	VWR International	89107-632
Polystyrene 150mm x 15mm sterile petri dish	Fisher Scientific	FB08-757-14
Polystyrene 150x15mm sterile petri dish	Fisher Scientific	08-757-148
Tips LTS 20 μL 960/10 GPS-L10	Rainin	17005091
Tips LTS 250 μL 960/10 GPS-L250	Rainin	17005093