TITLE: Hairy root generation in common bean (Phaseolus vulgaris L.) and selection of Agrobacterium rhizogenes clones
AUTHORS: Ronal Pacheco, Georgina Estrada-Navarrete, Noreide Nava, Jorge Solismiranda, carmen.quinto

[DESCRIPTION]
The common bean (Phaseolus vulgaris L.) is one of the legumes used to study the molecular mechanisms that regulate mycorrhizal and rhizobial symbioses. To study these mechanisms, the generation of transgenic hairy roots is a very advantageous method for applying genetic approaches. However, the generation of hairy roots is a difficult task that requires a lot of skill and experience. Here we show an optimized protocol for the generation of hairy roots in common bean.
 


[STEPS]
SECTION: Seeds disinfection (when necessary)
1. Immerse the common bean seeds in 96 % volume ethanol for 5 min  and wash them three times with sterile water.

SECTION: Seeds disinfection (when necessary)
2. Immerse the seeds in 2 % volume  sodium hypochlorite for 5 min and wash them three times with sterile water.
 



SECTION: Seeds disinfection (when necessary)
3. Keep the seeds at 4 °C in a disinfected container, e.g., sterile  , or reused Petri dishes previously sterilized with 96 % volume ethanol.




SECTION: Seeds germination (1st day)
4. 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.

 
 
  

SECTION: Seeds germination (1st day)
5. Cover the metal tray with aluminum foil and incubate it in a growth chamber at 28 °C  for 2760 min to 2880 min in the dark. 

 

SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
6. Spread 150 µL-200 µL of the inoculum in Petri dishes containing solid LB medium with the appropriate selection antibiotic. 
 



SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
7. Incubate the Petri dishes inoculated in the previous step, for approximately 1800 min at 30 °C. 
 

SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
7.1. Scratch off this thin layer of dried culture with a sterilized yellow tip or something similar.


SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
7.2. Transfer this culture to an   and add LB liquid medium.
SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
7.3. Centrifugate the 1.5 ml Eppendorf tube 8000 rpm, 1 min  and then homogenize the content using a micropipette. The content must be viscous, but liquid enough to be pipetted.

  
SECTION: Preparation of the inoculum of A. rhizogenes K599 (2nd day)
7.4. 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.
 




SECTION: Seedling transformation by A. rhizogenes K599 (3rd day)
8. Carefully puncture the hypocotyl area of the seeds several times, using a sterile needle tip (0.4 mm). 
 
      
 


SECTION: Seedling transformation by A. rhizogenes K599 (3rd day)
9. Apply the inoculum of A. rhizogeneson the wounded zone, taken directly from the plates, using an autoclaved micropipette tip 
SECTION: Generation of hairy roots
10. 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 
SECTION: Generation of hairy roots
11. Place the plastic tubes inside glass tubes   containing autoclaved deionized water and cover the glass tubes with plastic caps to prevent water evaporation.

SECTION: Generation of hairy roots
12. Place glass tubes on racks and incubate in a growth chamber at 28 °C,960 min light/480 min dark until hairy roots emerge, 10-12 days post-infection (dpi). 
 


SECTION: Generation of hairy roots
13. Once the hairy roots have emerged, remove the primary root by cutting the stem 1-2 cm below the hairy roots. 
SECTION: Generation of hairy roots
14. Transfer the seedlings to autoclaved glass tubes containing sterile B & D medium and seal the tube hole with parafilm or adhesive plastic. 
 
 


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


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

SECTION: Selection of A. rhizogenes clones
17. Collect the fluorescent hairy roots carrying the RNAi-silencing or the overexpression vector, and the control vector, at the selected sampling time. 
 

SECTION: Selection of A. rhizogenes clones
18. 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


SECTION: Selection of A. rhizogenes clones
19. Quantify transcript levels of the gene of interest by qPCR using two reference genes.