Using this protocol, we will demonstrate the step by step process for inducing hairy roots in tartary buckwheat for the first time. This technique is a easy and inexpensive for starting to genomics and microbiomics for tartary buckwheat and other plants. Although this detailed method is set up for tartary buckwheat, it can be applied to other dicot plants with some modifications.
Although this method is easy to perform, it has many detailed steps. Visual demonstration can help researchers to achieve a more efficient hairy root induction. Demonstrating the procedure will be Guangtao Qian, a Masters student from my lab.
Begin by selecting plump and undamaged tartary buckwheat seeds and soaking the seeds in 28 degree Celsius water for approximately 20 minutes. When the seed coat can be easily removed, place 100 to 200 peeled seeds into a sterilized 100 milliliter conical flask containing 75%ethanol for 30 seconds. At the end of the sterilization, replace the ethanol with 5%sodium hypochlorite.
After 15 minutes, decant the sodium hypochlorite and wash the seeds with sterile deionized water five times. Then blot the seeds dry with a piece of sterile bibulous paper. To obtain tartary buckwheat seedlings, add 10 seeds per bottle to individual 300 milliliter plant tissue culture bottles containing 50 milliliters of MSSA medium.
Then, germinate the seeds in a culture room at 25, plus or minus 1, degrees Celsius under light conditions. After 7 to 10 days, select robust seedlings with two pieces of unfolded cotyledons and cut the seedlings from the roots. Place the seedlings in a sterile petri dish and cut the hypocotyls into 0.8 to 1 centimeter segments.
Sheer the cotyledons into approximately 0.5 centimeter pieces and pre-culture the resulting explants under light conditions for 24 hours. The next day, transfer the explants into a sterilized 100 milliliter conical flask. To activate A.rhizogenes, thaw the culture on ice before evenly plating the bacteria onto YEB solid medium with antibiotics for a 12 to 16 hour incubation at 28 degrees Celsius.
The next morning, pick a monoclonal colony for culture in a new petri dish as just demonstrated. After another 12 to 16 hour incubation, inoculate the resulting monoclonal colonies in a sterilized 100 milliliter conical flask containing 20 milliliters of YEB solid medium with antibiotics at 28 degree Celsius and 200 revolutions per minute for 16 to 18 hours until the optical density at 600 reaches 2.0. Then transfer 2-4%of the bacterial cell culture into a new 100 milliliter conical flask containing 20 milliliters of fresh YEB solid medium with antibiotics at 28 degrees Celsius and 200 revolutions per minute for 4 to 5 hours until the optical density at 600 nanometers reaches approximately 0.5.
To induce hairy root transformation, transfer the A.rhizogenes culture into a sterile 50 milliliter conical tube and collect the bacteria by centrifugation. Resuspend the pellet in MSSAS medium to an optical density of approximately 0.2 and infuse the suspension into the flask of explants. After 10 minutes, remove the explants from the flask and blot them with a piece of sterile bibulous paper.
Then place a sterile 9 centimeter diameter piece of filter paper onto a dish of MSSAAS medium and overlay the explants on the filter paper for a three day incubation at 25 degrees Celsius in the dark. At the end of the incubation, transfer approximately 20 infected explants onto MSSACK medium and vertically incubate the plants under light conditions at 25, plus or minus 1, degrees Celsius. After 10 to 14 days of culture, select the hairy roots demonstrating a white appearance and rapid growth and cut the roots into two to three centimeter pieces.
Clearly number the roots on a clean bench. Subculture the root pieces in a sterilized 100 milliliter conical flask containing MSSK medium at 25 degree Celsius and 80 revolutions per minute in the dark until they overspread to the bottom of the flask. To identify the hairy roots, remove any tawny and contaminated hairy roots and select those with a white appearance.
If a reporter gene was used, check the roots for fluorescence under a blue or light dual ultra violet transilluminator and select the hairy roots that exhibit a strong fluorescent signal. Then dry the roots with absorbent paper and wrap them in marked aluminum foil for immediate analysis. For long term storage, lyophilize the roots in liquid nitrogen and place the roots at minus 80 degrees Celsius until their investigation.
After genetic transformation and culture, as demonstrated, the hairy roots will exhibit a fluffy white color and a plagiotropic manner in the wound sites of the explants and form a highly branched and interlocked matrix. A reporter gene can be used to facilitate differentiation of the transgenic hairy roots under a blue or light dual ultraviolet transillumination, or the identification of a target gene of interest. Here, the relative expression of a light-induced transcription factor in the transgenic lines of tartary buckwheat hairy roots is shown.
Notably, the biosynthesis of rutin and quercetin are also promoted at the metabolic level in response to the induced transcription factor overexpression. Further, the relative gene expression of flavenoid synthesis pathway genes in all three transgenic lines, are remarkably higher than those measured in the control group, indicating a successful hairy root transformation in tartary buckwheat explants. That is infection of the seeds, selection of the explants, and the concentration of the bacteria directly affect the success of the hairy root induction.
We can assess the gene expression, DNA protein, or protein protein reactions or mass production of secondary metabolites to study transcriptional regulation secondary metabolites. The engineered bacteria are hazardous to humans and his environment. Take care to handle, operate, and dispose of the bacteria properly.
