To infect soybean cotyledons with Agrobacterium rhizogenes, design primers to detect the TI plasmid gene virD2 using the sequence of the Agrobacterium rhizogenes PRI2659 plasmid. Following transformation, test the Agrobacterium colonies for the retention of virD2 by PCR using a PCR kit. After selecting the Agrobacterium rhizogenes colonies containing both virD2 and the gene of interest, streak some colonies onto the LB plates containing 100 milligrams per liter of spectinomycin for the plasmid of interest.
The following day, using a P200 pipette tip, scrape off a 1.5 centimeter length of the Agrobacterium from the LB plate and resuspend it in one milliliter of phosphate buffer. Dilute the resuspended cell suspension and sterilized ultrapure water in acetosyringone. Measure the absorbance using a cuvette tube at an optical density of 600 nanometers.
Next, in a biosafety cabinet, dip a sterilized scalpel in the Agrobacterium solution and make a one millimeter deep cut along the inner surface of the cotyledon. Place six to eight cotyledons cut side down on a Petri dish containing filter paper saturated with germination and cultivation medium with acetosyringone. Incubate the plates at room temperature for three days under a 16 hour photo period.
After three days, transfer the infected cotyledons to hairy root growth, or HRG plates. Incubate the plates in the growth chambers set to 22 degrees Celsius and a light intensity of 100 micromoles on a 16 hour photo period until primary roots with secondary roots two to three centimeters in length are observed. After three to four weeks, harvest primary roots that grow from the callus and contain secondary roots using a sterile scalpel and forceps.
Transfer the roots to selection HRG plates containing appropriate antibiotics and allow them to grow for an additional five days. On day five, harvest transgenic hairy roots with secondary roots that are three to six centimeters in length. If observing fluorescent proteins, ensure that the secondary roots have little autofluorescence.
Next, to perform elicitor or chemical treatments, cut the secondary roots into one centimeter pieces and place approximately 100 milligrams on HRG agar in a pile. Then saturate the pile with 80 microliters of the appropriate treatment solution and allow the plate to incubate at room temperature. After 24 hours, for RNA extraction, rapidly dab the roots dry on a sterilized paper towel and harvest them directly into a two milliliter microcentrifuge tube.
Immediately seal the top of the tube using parafilm and make two small holes using pointed forceps. The colony PCR results of the transformed Agrobacterium are shown. The positive colonies in PCR indicated the gene of interest.
However, one third to one half of the colonies were negative for the virD2 gene screening. Fluorescence microscopy demonstrates the subcellular localization of GFP-GmJAZ1-6. Gene expression analysis confirmed the overexpression of the glyceollin transcription factor GmHSF6-1 and RNAi silencing of GmMYB2912 in Williams 82 harry roots.
