Hairy root generation in common bean (Phaseolus vulgaris L.) and selection of Agrobacterium rhizogenes clones

Immerse the common bean seeds in 96% volume ethanol for 0h 5m 0s and wash them three times with sterile water.

Immerse the seeds in 2% volume sodium hypochlorite for 0h 5m 0s and wash them three times with sterile water.

Keep the seeds at 4°C in a disinfected container, e.g., sterile , or reused Petri dishes previously sterilized with 96% volume ethanol.

The disinfected seeds are placed, using sterile forceps, in a metal tray on a wet paper towel, previously sterilized in an autoclave, leaving 2 cm between the seeds.

Cover the metal tray with aluminum foil and incubate it in a growth chamber at 28°C for 46h 0m 0s to 48h 0m 0s in the dark.

Note

Position the tray at a slight downward angle to improve seed germination. The hilum should face downward.

Spread 150µL-200µL of the inoculum in Petri dishes containing solid LB medium with the appropriate selection antibiotic.

Incubate the Petri dishes inoculated in the previous step, for approximately 30h 0m 0s at 30°C.

Scratch off this thin layer of dried culture with a sterilized yellow tip or something similar.

Transfer this culture to an and add LB liquid medium.

Centrifugate the 1.5 ml Eppendorf tube 8000rpm and then homogenize the content using a micropipette. The content must be viscous, but liquid enough to be pipetted.

Prepare the inoculum in Eppendorf tubes (preferably 0.6 ml) by mixing an equal volume 50% (v/v)of the liquid content previously obtained, and 80% volume glycerol. Mix tubes by inversion and immediately place them in liquid nitrogen; finally, store the inoculum at -80°C.

Carefully puncture the hypocotyl area of the seeds several times, using a sterile needle tip (0.4 mm).

Apply the inoculum of A. rhizogenes on the wounded zone, taken directly from the plates, using an autoclaved micropipette tip

Place the infected seedlings on the top of plastic tubes, i.e., containing B & D medium. https://link.springer.com/book/10.1007/1-4020-3735-X

Place the plastic tubes inside glass tubes containing autoclaved deionized water and cover the glass tubes with plastic caps to prevent water evaporation.

Place glass tubes on racks and incubate in a growth chamber at 28°C,16h 0m 0s light/8h 0m 0s dark until hairy roots emerge, 10-12 days post-infection (dpi).

Once the hairy roots have emerged, remove the primary root by cutting the stem 1-2 cm below the hairy roots.

Transfer the seedlings to autoclaved glass tubes containing sterile B & D medium and seal the tube hole with parafilm or adhesive plastic.

Note

Make sure the level of the B & D medium is below the hairy root calluses, as covering hairy root calli with B & D medium may retard their growth.

Incubate the seedlings for approximately three days under the same conditions described above to increase the biomass of hairy roots.

Observe the fully developed hairy roots (15 to 16 dpi) using an epifluorescence microscope to remove non-fluorescent roots.

Collect the fluorescent hairy roots carrying the RNAi-silencing or the overexpression vector, and the control vector, at the selected sampling time.

Extract total RNA from hairy roots using an appropriate protocol and perform cDNA synthesis. We recommend using the following protocol dx.doi.org/10.17504/protocols.io.8epv5jq24l1b/v1

Quantify transcript levels of the gene of interest by qPCR using two reference genes.