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 conducted under the condition of a 14-h light and a 10-h dark photoperiod at 25 &#176;C. Unless otherwise stated, all explants- or bacteria-related operations were performed under aseptic conditions in a laminar flow hood. All the media ingredients for A. rhizogenes and in vitro plant cultures are provided in Table 1. After adjusting the pH, all media were autoclaved at 120 &#176;C for 20 min. Solidified media were prepared by filling 25 mL of medium into a Petri dish of 9-cm diameter and allowing it to solidify.
CAUTION: Deposit all the genetically modified bacteria and plants into the appropriate waste container. Operate all hazardous chemicals in a fume cupboard and deposit them in the hazardous waste container.
1. Preparation of TB explants
<ol>
	<li>Preparation of TB seeds
	<ol>
		<li>Select plump and undamaged TB seeds (Figure 1A1) that have been preserved at a temperature less than&#8722;20 &#176;C for no more than 2 years.</li>
		<li>Soak the seeds in water at 28 &#176;C for approximately 20 min so that the seed coat can be easily peeled off (Figure 1A2). If necessary, use scissors to cut a slot on the seeds to facilitate the peeling.</li>
	</ol>
	</li>
	<li>Sterilization of TB seeds
	<ol>
		<li>Place 100&#8211;200 peeled seeds into a 100 mL sterilized conical flask.</li>
		<li>Disinfect the seeds using 75% ethanol for 30 s.</li>
		<li>Replace the ethanol with 5% sodium hypochlorite and disinfect for 15 min. 
		NOTE: Disinfection using mercury bichloride at a concentration of 1 g/L for 8 min may be used as an alternative sterilizer to replace sodium hypochlorite in any case of inadequate sterilization. 
		CAUTION: Mercury bichloride is hazardous and not an environment-friendly material. Operate it in the fuming cupboard and deposit it into the hazardous waste container, if mercury bichloride is used in any case.</li>
		<li>Pour out the sodium hypochlorite.</li>
		<li>Wash the seeds using sterile deionized water 5 times.</li>
		<li>Blot the seeds dry with a sterile bibulous paper.</li>
	</ol>
	</li>
	<li>Preparation of TB seedlings
	<ol>
		<li>Prepare 50 mL Murashige and Skoog (MS) basal medium (1962) supplemented with 30 g/L sucrose and 7 g/L agar powder (MSSA) (Table 1) in a 300 mL plant tissue culture bottle.</li>
		<li>Adjust the pH to 5.8 before autoclaving.</li>
		<li>Distribute 10 seeds evenly per bottle of MSSA medium.</li>
		<li>Germinate the seeds in a culture room at 25 &#176;C &#177; 1 &#176;C under the light condition for 7&#8211;10 days.</li>
	</ol>
	</li>
	<li>Preparation of sterile explants
	<ol>
		<li>Select robust seedlings of TB (Figure 1C), when the 2 pieces of cotyledons are unfolded.</li>
		<li>Cut off the seedlings from the roots (Figure 1C red-dash arrowheads), avoiding contact with the medium.</li>
		<li>Place them in a sterile Petri dish.</li>
		<li>Cut the hypocotyls into 0.8&#8211;1 cm segments, and shear the cotyledons into approximately 0.5 cm pieces.</li>
		<li>Preculture these explants on MSSA medium under the light condition for 24 h.</li>
		<li>Transfer them into a 100 mL sterilized conical flask, which are now ready for infection.</li>
	</ol>
	</li>
</ol>
2. Preparation of A. rhizogenes for transformation
NOTE: The A. rhizogenes strain ACCC10060 was kindly provided by the Institute of Medicinal Plant Development and preserved at &#8722;80 &#176;C. A. rhizogenes was transformed with the binary vector pK7GWIWG2D (II) that harbors a T-DNA carrying the b4 gene accompanying a GFP as an indicator gene and the Kan resistance gene as a selectable marker. The gene b4 is a member of the transcription factor bHLH family, which has not yet been published. To evaluate the potential of TB hairy roots, A. rhizogenes was transformed with the binary vector pK7WG2D containing the MYB116 gene to investigate its effect on the production of secondary metabolites such as flavonoids at the level of gene expression and by metabolic analyses. Activated A. rhizogenes should be well prepared at the same time with the explants.
<ol>
	<li>Activation of A. rhizogenes
	<ol>
		<li>Thaw A. rhizogenes on ice</li>
		<li>Dip the bacteria and line them evenly onto yeast mannitol medium (YEB) supplemented with 15 g/L agar powder, 50 mg/L rifampicin, and 50 mg/L spectinomycin (YEBARS, pH 7.0).</li>
		<li>Incubate the bacteria at 28 &#176;C for 12&#8211;16 h.</li>
		<li>Pick a monoclonal colony and culture it in another Petri dish in the same above-described manner.</li>
		<li>Select monoclonal colonies and culture them in a 100 mL sterilized conical flask containing 20 mL of YEB medium supplemented with 50 mg/L rifampicin and 50 mg/L spectinomycin (YEBRS, pH 7.0) at 28 &#176;C and 200 rpm for 16&#8211;18 h until the OD600 value reaches 2.0.</li>
		<li>Incubate 2%&#8211;4% of the abovementioned culture in another 100-mL conical flask containing 20 mL of YEBRS medium at 28 &#176;C and 200 rpm for 4-5 h until the OD600 value reaches approximately 0.5 (Figure 1D).</li>
	</ol>
	</li>
</ol>
3. Infection and screening of TB explants
NOTE: The objective of this protocol is to obtain genetically transformed hairy roots. The wild-type roots were used as the negative control to assess the transgenic expression. In this protocol, A. rhizogenes was transformed with binary vector either pK7WG2D carrying the gene of FtMYB116 or pK7GWIWG2D (II) carrying the gene of b4 in advance.
<ol>
	<li>Resuspension of A. rhizogenes
	<ol>
		<li>Transfer the culture obtained in step 2.1.6 into a 50-mL sterilized centrifugal tube.</li>
		<li>Spin at 4,000 x g for 10 min at 20 &#176;C.</li>
		<li>Remove the supernatant and resuspend the bacterial pellet with MS medium supplemented with 30 g/L sucrose and 300 &#956;M acetosyringone (AS) (MSSAS, pH 5.8) to OD600 &#8776; 0.2.</li>
	</ol>
	</li>
	<li>Infection of explants
	<ol>
		<li>Infuse the bacterial suspension obtained in step 3.1.3 into a conical flask containing the explants prepared in step 1.4.6 for 10 min (Figure 1E).</li>
		<li>Take out the explants, and blot them dry using a sterile bibulous paper.</li>
	</ol>
	</li>
</ol>
4. Coculture of explants with A. rhizogenes
<ol>
	<li>Place a sterile 9 cm diameter filter paper on the MS medium, which is solidified using 7 g/L agar powder supplemented with 30 g/L sucrose and 100 &#956;M AS (MSSAAS medium, pH 5.2).</li>
	<li>Overlay the explants on the filter paper at 25 &#176;C for 3 days in the dark (Figure 1F).</li>
</ol>
5. Induction and selective culture
<ol>
	<li>Place approximately 20 infected explants on the MSSA medium supplemented with 500 mg/L cefotaxime and 50 mg/L kanamycin (Kan) (MSSACK, pH 5.8) (Figure 1G).</li>
	<li>Incubate them vertically under the light condition at 25 &#176;C &#177; 1 &#176;C. The hairy roots occur approximately 1 week after incubation (Figure 1H black-dash arrowheads indicate occurrence of hairy roots). 
	NOTE: Replace MSSACK medium every 15 days if necessary.</li>
</ol>
6. Subculturing TB hairy roots
NOTE: This procedure aims to harvest vigorous hairy roots. Regularly observe the growth of hairy roots during propagation, and remove the contaminated and inactivated ones in a timely manner. If necessary, repeat the following steps to propagate more hairy roots. It takes approximately 10&#8211;14 days from subculturing to harvest.
<ol>
	<li>Select the hairy roots showing white appearance and rapid growth.</li>
	<li>Cut them into pieces of 2-3 cm.</li>
	<li>Clearly number them on a clean bench.</li>
	<li>Subculture them in a 100 mL sterilized conical flask containing 5 mL of MS medium supplemented with 30 g/L sucrose and 50 mg/L Kan (MSSK, pH 5.8) at a rotary speed of 80 rpm at 25 &#176;C in the dark until they overspread to the bottom of the flask (Figure 1I).</li>
</ol>
7. Identification of transformed hairy roots and conservation
NOTE: Transformed hairy roots can be identified based on the aspects of morphology and gene level. Identification can also be conducted according to the hairy root genome and resistance, which are not covered in this protocol. This procedure primarily focuses on reporter gene and target gene identification.
<ol>
	<li>Remove tawny and contaminated hairy roots and select those with white appearance.</li>
	<li>Evaluate if there is green fluorescence under a blue/light dual ultraviolet transilluminator.</li>
	<li>Select the hairy roots exhibiting a strong fluorescence signal in the numbered tubes or wrapped using a marked tinfoil after drying them out with an absorbent paper.</li>
	<li>Lyophilize them in liquid nitrogen, followed by storing all the harvest at &#8722;80 &#176;C for further investigation.</li>
	<li>Gene identification
	<ol>
		<li>Triturate 0.1 g of the hairy roots into fine powder in liquid nitrogen.</li>
		<li>Prepare the genomic DNA of independent transgenic lines of TB using the modified cetyltrimethylammonium bromide (CTAB) method25 according to the instruction of the manufacturer of the plant genomic DNA kit.</li>
		<li>Perform polymerase chain reaction (PCR) using 100 ng of genomic DNA template and primers listed in Table 2.</li>
		<li>Perform the amplification cycle as follows: predenaturation at 94 &#176;C for 5 min, denaturation at 94 &#176;C for 30 s, primer annealing at 55 &#176;C for 30 s, and primer extension at 72 &#176;C for 30 s. After 36 cycles and a final extension step at 72 &#176;C for 10 min, analyze the amplification products on 1% agarose gels.</li>
		<li>Stain the gels with nucleic acid staining and visualize them under UV light.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest to disclose.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>MS salts with vitamins</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>M8521</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>S8210</td>
			<td></td>
		</tr>
		<tr>
			<td>Other chemicals unstated</td>
			<td>Beijing Chemical Works, China</td>
			<td></td>
			<td>ethanol, mercury bichloride, etc.</td>
		</tr>
		<tr>
			<td>PHS-3C pH meter</td>
			<td>Shanghai INESA Scientific Instrument Co., Ltd, China</td>
			<td>a008</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>S8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans2K DNA Marker</td>
			<td>TransGen Biotech, Beijing, China</td>
			<td>BM101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Solarbio Life Science, Beijing, China</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
	</tbody>
</table>

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 ...

inducing-hairy-roots-agrobacterium-rhizogenes-mediated-transformation

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Genetics

11.8K Views.  China Academy of Chinese Medical Sciences. 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.

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5&#39; and 3&#39; flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 &#176;C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 &#181;mol m-2s-1 light intensity, at 22 &#176;C to 24 &#176;C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3&#8211;4 transgenic plants per 10,000 T1 seeds propagated.

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula

Agrobacterium-mediated transformation using a floral-dip method can be successfully employed to create stable transgenic lines of the extremophyte model Schrenkiella parvula. We present a protocol modified from that for Arabidopsis thaliana, considering different growth habits and physiological characteristics of the extremophyte.

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to visceral pathology. Deciphering the nature of the vector/parasite interactions is of primary importance for better understanding of Leishmania transmission to their hosts. Among the parameters controlling the sand fly vector competence (i.e. their ability to carry and transmit pathogens), parameters intrinsic to these insects were shown to play a key role. Insect immune response, for example, impacts sand fly vector competence to Leishmania. The study of such parameters has been limited by the lack of methods of gene expression modification adapted for use in these non-model organisms. Gene downregulation by small interfering RNA (siRNA) is possible, but in addition to being technically challenging, the silencing leads to only a partial loss of function, which cannot be transmitted from generation to generation. Targeted mutagenesis by CRISPR/Cas9 technology was recently adapted to the Phlebotomus papatasi sand fly. This technique leads to the generation of transmissible mutations in a specifically chosen locus, allowing to study the genes of interest. The CRISPR/Cas9 system relies on the induction of targeted double-strand DNA breaks, later repaired by either Non-Homologous End Joining (NHEJ) or by Homology Driven Repair (HDR). NHEJ consists of a simple closure of the break and frequently leads to small insertion/deletion events. In contrast, HDR uses the presence of a donor DNA molecule sharing homology with the target DNA as a template for repair. Here, we present a sand fly embryo microinjection method for targeted mutagenesis by CRISPR/Cas9 using NHEJ, which is the only genome modification technique adapted to sand fly vectors to date.

Sand Fly Phlebotomus papatasi Embryo Microinjection for CRISPR/Cas9 Mutagenesis

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

Sand flies are the natural vectors for Leishmania species, protozoan parasites producing a broad spectrum of symptoms ranging from cutaneous lesions to ...

Reusable Single Cell for Iterative Epigenomic Analyses

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. 
In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. 
Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic ...

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...