Soybean Hairy Root Transformation for the Analysis of Gene Function

Our soybean hairy root transformation method is sufficient to simultaneously study the functions of several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches. Soybean hairy root transformation is a useful tool for the analysis of gene function in soybean plants. The technology that are currently used include CRISPR-Cas9 genome editing, RNA interference, transcriptomics and proteomics, and imaging technologies.

The major challenge in the modern era of functional genomics is sufficient analyzing the function of numerous genes. Most trends are polygenic, being achieved by the interaction of many genes. Our hairy root protocol will facilitate gene function analysis with sufficient throughput so that polygenic networks can begin to be understood.

We have established the importance of the Ti plasmid in agrobacterium rhizogene strains. We found that without screening for the Ti plasmid, it increased the risk for their transformed soybean cotyledons to fail to produce any colors. As a result, screening for their plasmid became a vital step before their transformation.

Compared to other gene expression techniques, our hairy root expression system is easy to handle, less time-consuming, and cost-effective. Also, our new protocol has improved hairy root transformation rates up to 50%by checking for the Ti plasmid in agrobacterium prior to cotyledon transformation. By characterizing the function of more than a dozen transcription factor genes, our recent results have paved the way for multi-gene engineering studies aimed at unlocking the biosynthesis of valuable phytoliths and metabolites in plants.

In the future, we will use this hairy root transformation method to dissect the transcription factor gene networks that regulate the biosynthesis of phytoalexins. The approach could be used to enhance the production of these specialized metabolites for pharmaceutical and agricultural industries. To begin, place 16 to 20 pristine round Williams 82 soybean seeds in a 50-milliliter centrifuge tube inside a biosafety cabinet.

Add 30 milliliters of 70%isopropyl alcohol to the tube, and shake for 30 seconds. Decant the alcohol. Next, gently shake the seeds with 30 milliliters of 10%bleach for 10 seconds, and allow the seeds to sit in the solution for five minutes at room temperature.

After five minutes, drain the bleach. Then rinse the seeds three times with 30 milliliters of sterile ultrapure water for one minute per rinse, and discard the water between each rinse. Place the sterilized seeds on filter paper saturated with five milliliters of germination and co-cultivation medium in a sterile Petri dish.

Place the plates in the dark at room temperature for three days before transferring them at 22 degrees Celsius under 16-hour cool white T5 fluorescent lights for four days to allow the seeds to germinate. 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 sterilize to 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 into 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 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, 1/3 to 1/2 of the colonies were negative for the virD2 gene screening. Fluorescence microscopy demonstrates the subcellular localization of GFP-GmJAZ 1-6.

Gene expression analysis confirmed the overexpression of the glycine transcription factor GmHSF6-1 and RNAi silencing of GmMYB29A2 in Williams 82 hairy roots.