Name: inducing hairy roots by agrobacterium rhizogenes-mediated transformation in tartary buckwheat (fagopyrum tataricum)
Uploaded: 2020-03-11T14:00:00
Description: Watch this Scientific Journal Video about Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum at JoVE.com

This work was supported by the Fundamental Research Funds for the Central public welfare research institutes ZXKT17002.

The TB used in this study was named as BT18, which originated from the breed of &#34;JinQiao No.2&#34; cultivated by the Research Center of Small Miscellaneous Grain of Shanxi Academy of Agricultural Science. The primary steps of this protocol are illustrated in Figure 1.
NOTE: Operate explants-related manipulation rapidly, and when possible, keep the Petri dishes closed to avoid wilting and contamination. Unless otherwise stated, all the explant incubations were ...

TB has been used in several studies related to secondary metabolites at genetic and metabolic levels1,2,5,27,28. Hairy root culture, as a unique source for metabolite production, plays a pivotal role in metabolic engineering29 and can be used to alter metabolic pathways by inserting the related genes. Kim et al.2...

Tartary buckwheat (TB) (Fagopyrum tataricum (L.) Gaertn) is a type of dicotyledon belonging to the genus Fagopyrum and the family Polygonaceae1. As a type of Chinese medicine homologous food, TB has been receiving considerable interest owing to its distinctive chemical composition and diverse bioactivities against diseases. TB is primarily rich in carbohydrates, proteins, vitamins, and carotenoids as well as in polyphenols such as phenolic acids and flavonoids1. Various biological and pharmacological activities of flavonoids, including antioxidative, antihypertensive2, and anti-inflammatory as well as anticancer and antidiabetic properties, have been demonstrated3.
Agrobacterium rhizogenes is a soil bacterium that contributes to the development of hairy root disease in several higher plants, especially dicotyledons, by infecting wound sites4,5. This process is initiated by the transfer of the T-DNA in the root-inducing (Ri) plasmid5,6 and is commonly accompanied by the integration and expression of an exogenous gene from the Ri plasmid and the subsequent steps of generating the hairy root phenotype7. A. rhizogenes-mediated transgenic hairy roots, as a powerful tool in the field of plant biotechnology, have been most widely used owing to their stable and high productivity and easy obtainment in a short period. Moreover, hairy roots induced by A. rhizogenes are efficiently distinguished by their plagiotropic root development and highly branching growth in a hormone-free medium8. They can be used in several fields of research, including artificial seed production, root nodule research, and in studying the interactions with other organisms such as mycorrhizal fungi, nematodes, and root pathogens7,9. In addition, hairy root transformation cultures have been extensively used as an experimental system to investigate the biochemical pathways and chemical signaling and to produce plant secondary metabolites that are used as pharmaceuticals, cosmetics, and food additives8,10. The valuable secondary metabolites, including indole alkaloids, aconites, tropane alkaloids, terpenoids, and flavonoids, synthesized in wild-type hairy roots have been investigated for several decades in numerous species, such as ginsenoside in Panax ginseng11, coumarine in Ammi majus12, and phenolic compounds in TB2,13.
Hairy roots have been produced using A. rhizogenes in 79 plant species from 27 families14. For instance, A. rhizogenes-mediated hairy root transformation has been reported in soybean15,16, Salvia17, Plumbago indica18, Lotus japonicus19, and chicory (Cichorium intybus L.)20. TB hairy root transformation has also been investigated2. Few detailed protocols are available regarding the development of hairy roots mediated by A. rhizogenes either carrying a binary vector or not. For instance, Sandra et al.21 introduced a method of producing transgenic potato hairy roots sustained in wild-type shoots. The fully developed hairy roots could be visualized 5-6 weeks after the injection of A. rhizogenes carrying the gus reporter gene into the stem internodes of potato plants. Another study had also reported a transgenic hairy root system induced by A. rhizogenes harboring the gusA reporter gene in jute (Corchorus capsularis L.)22. Furthermore, Supaart et al.23 obtained transgenic tobacco hairy roots using A. rhizogenes transformed with the expression vector pBI121 carrying the gene of &#916;1-tetrahydrocannabinolic acid (THCA) synthase to produce THCA.
However, a step-by-step process for an effective generation of hairy root transformation, especially in TB, has been relatively less demonstrated. In this study, we have described a detailed protocol using A. rhizogenes carrying the reporter gene (GFP), a selective marker (Kan), and a gene of interest (b4, an identified from our group but unpublished gene from basic helix-loop-helix (bHLH) family) to generate hairy root genetic transformation in TB. The experiment lasted for 5-6 weeks, from the inoculation of seeds to generation of hairy roots, involving the explant preparation, infection, coculturing, subculturing, and subsequent propagation. Furthermore, A. rhizogenes containing a binary plasmid carrying the TB transgene of myeloblastosis transcription factor 116 (FtMYB116) was used to determine whether FtMYB116 can promote accumulation of flavonoids, particularly rutin, in TB at the gene and metabolic level through the TB hairy root transformation. FtMYB116, which is a light-induced transcriptional factor, regulates the synthesis of rutin under different light conditions5. Chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), flavonoid-3&#39;-hydroxylase (F3&#39;H), and flavonol synthase (FLS)24 are key enzymes involved in the metabolic pathway of rutin biosynthesis. Therefore, this study demonstrates the overexpression of FtMYB116 in TB hairy roots and the expression of key enzyme genes as well as the content of rutin and other flavonoids such as quercetin.

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			<td>2*Taq PCR MasterMix</td>
			<td>Aidlab, China</td>
			<td>PC0901</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>A8190</td>
			<td></td>
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			<td>Applied Biosystems 2720 thermo cycler</td>
			<td>ThermoFisher Scientific, US</td>
			<td>A37834</td>
			<td></td>
		</tr>
		<tr>
			<td>AS</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>A8110</td>
			<td>Diluted in DMSO, 100 mM</td>
		</tr>
		<tr>
			<td>binary vectors</td>
			<td>ThermoFisher Scientific (invitrogen), US</td>
			<td>/</td>
			<td>pK7WG2D/pK7GWIWG2D (II)</td>
		</tr>
		<tr>
			<td>Cefotaxime,sodium</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>C8240</td>
			<td>Diluted in Water, 200 mg/mL</td>
		</tr>
		<tr>
			<td>CF15RXII high-speed micro</td>
			<td>Hitachi, Japan</td>
			<td>No. 90560201</td>
			<td></td>
		</tr>
		<tr>
			<td>Diposable Petri-dish</td>
			<td>Guanghua medical instrument factory, Yangzhou, China</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>DYY-6C electrophoresis apparatus</td>
			<td>Bjliuyi, Beijing China</td>
			<td>ECS002301</td>
			<td></td>
		</tr>
		<tr>
			<td>EASYspin Plus Plant RNA Kit</td>
			<td>Aidlab, China</td>
			<td>RN38</td>
			<td></td>
		</tr>
		<tr>
			<td>ELGA purelab untra bioscience</td>
			<td>ELGA LabWater, UK</td>
			<td>82665JK1819</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoch Microplate Spectrophotometer</td>
			<td>biotek, US</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Gateway BP/LR reaction enzyme</td>
			<td>ThermoFisher Scientific (invitrogen), US</td>
			<td>11789100/11791110</td>
			<td></td>
		</tr>
		<tr>
			<td>HYG-C multiple-function shaker</td>
			<td>Suzhou Peiying Experimental Equipment Co., Ltd. China</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Kan</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>K8020</td>
			<td>Diluted in Water, 100 mg/mL</td>
		</tr>
		<tr>
			<td>MLS-3750 Autoclave sterilizer</td>
			<td>Sanyo, Japan</td>
			<td>/</td>
			<td></td>
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			<td>MS salts with vitamins</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>M8521</td>
			<td></td>
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			<td>NaCl</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>S8210</td>
			<td></td>
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			<td>Other chemicals unstated</td>
			<td>Beijing Chemical Works, China</td>
			<td></td>
			<td>ethanol, mercury bichloride, etc.</td>
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			<td>PHS-3C pH meter</td>
			<td>Shanghai INESA Scientific Instrument Co., Ltd, China</td>
			<td>a008</td>
			<td></td>
		</tr>
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			<td>Plant Genomic DNA Kit</td>
			<td>TIANGEN BIOTECH (BEIJING) CO., LTD</td>
			<td>DP305</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampin</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>R8010</td>
			<td>Diluted in DMSO, 50 mg/mL</td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>S8040</td>
			<td>Diluted in Water, 100 mg/mL</td>
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			<td>Sucrose</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>S8270</td>
			<td></td>
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			<td>Trans2K DNA Marker</td>
			<td>TransGen Biotech, Beijing, China</td>
			<td>BM101-01</td>
			<td></td>
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			<td>Tryptone</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>LP0042</td>
			<td></td>
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			<td>Whatman diameter 9 cm Filter paper</td>
			<td>Hangzhou wohua Filter Paper Co., Ltd</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract powder</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>LP0021</td>
			<td></td>
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inducing hairy roots by agrobacterium rhizogenes-mediated transformation in tartary buckwheat (fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant secondary metabolites such as flavonoids, especially rutin. Agrobacterium rhizogenes have been gradually used worldwide to induce hairy roots in medicinal plants to investigate gene functions and increase the yield of secondary metabolites. In this study, we have described a detailed method to generate A. rhizogenes-mediated hairy roots in TB. Cotyledons and hypocotyledonary axis at 7&#8211;10 days were selected as explants and infected with A. rhizogenes carrying a binary vector, which induced adventitious hairy roots that appeared after 1 week. The generated hairy root transformation was identified based on morphology, resistance selection (kanamycin), and reporter gene expression (green fluorescent protein). Subsequently, the transformed hairy roots were self-propagated as required. Meanwhile, a myeloblastosis (MYB) transcription factor, FtMYB116, was transformed into the TB genome using the A. rhizogenes-mediated hairy roots to verify the role of FtMYB116 in synthesizing flavonoids. The results showed that the expression of flavonoid-related genes and the yield of flavonoid compounds (rutin and quercetin) were significantly (p &#60; 0.01) promoted by FtMYB116, indicating that A. rhizogenes-mediated hairy roots can be used as an effective alternative tool to investigate gene functions and the production of secondary metabolites. The detailed step-by-step protocol described in this study for generating hairy roots can be adopted for any genetic transformation or other medicinal plants after adjustment.

Agrobacterium rhizogenes-mediated TB hairy root transformation 
This study describes the step-by-step protocol that was established to obtain genetically transformed hairy roots using A. rhizogenes. It took approximately 5-6 weeks from the inoculation of TB seeds to the harvesting of the identified hairy roots, and some key steps are depicted in Figure 1 (A-H). Briefly, sterilized shelled seeds were inoculated (Fig...

Watch this Scientific Journal Video about Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum at JoVE.com

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat Fagopyrum tataricum

We describe a method of inducing hairy roots by Agrobacterium rhizogenes-mediated transformation in Tartary buckwheat (Fagopyrum tataricum). This can be used to investigate gene functions and production of secondary metabolites in Tartary buckwheat, be adopted for any genetic transformation, or used for other medicinal plants after improvement.

Inducing Hairy Roots by Agrobacterium rhizogenes-Mediated Transformation in Tartary Buckwheat (Fagopyrum tataricum)

Tartary buckwheat (TB) [Fagopyrum tataricum (L.) Gaertn] possesses various biological and pharmacological activities because it contains abundant ...

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.

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