This work was supported by the Ministerio de Innovaci&#243;n y Ciencia (AGL2009-13745, FPI grant to PB), the Ministerio de Econom&#237;a y Competitividad and FEDER funding (AGL2012-36725, AGL2015-67495-C2-1-R), and the University of Girona (PhD grant to SF, and grant SING11/1). The authors are grateful to Dr. Inge Broer (Institute for Land Use, University of Rostock, Rostock, Germany) and Dr. Salom&#233; Prat (Centro Nacional de Biotecnolog&#237;a, Madrid, Spain) for providing the A. rhizogenes and the A. tumefaciens strain, respectively, and Dr. Mar&#231;al Soler and Dr. Anna Plasencia for the help and support received in initiating the A. rhizogenes transformation experiments (Toulouse III Paul Sabatier University&#8212;CNRS, Plant Research Laboratory (LRSV), Castanet Tolosan, France). The authors thank Sara G&#243;mez (Departament de Biologia, UdG, Girona) for her valuable assistance in carrying out the laboratory work and taking care of plants, and Ferran Fontdecaba and Carla S&#225;nchez who assisted with some of the experiments while they were doing their final degree projects.&#160;

The A. rhizogenes transformation protocol was adapted and modified from Horn et al.7 and the genotype tested was S. tuberosum ssp. tuberosum (cv. D&#233;sir&#233;e). The A. tumefaciens transformation protocol was adapted and modified from Banerjee et al.22 and the genotypes tested were S. tuberosum ssp. tuberosum (cv. D&#233;sir&#233;e) and S. tuberosum ssp. andigena. The main steps of both procedures are summarized in Figure 1 and Figure 2, respectively.
NOTE: In all the steps of the procedure performing in vitro transfers, do so rapidly, and when possible, maintain the plates or pots closed, thus minimizing plant exposure to the air to avoid wilting and contamination. Otherwise stated, all the plant incubations were done in cabinets under the conditions of 12 h of 24 &#176;C light/12 h of 20 &#176;C dark and 67 &#181;mol m-1 s-1. Otherwise stated, perform all the bacteria manipulation and in vitro plant transfers in aseptic conditions in a laminar flow hood. All the media recipes for Agrobacterium and in vitro plant cultures are provided in Table S1.
CAUTION: Deposit all the genetically modified bacteria and&#160;plants to the appropriated waste container.
1. Agrobacterium cultures used for transformation
NOTE: The strain used for A. rhizogenes transformation was the C58C1:Pri15837 (kindly provided by Dr. Inge Broer) and that for the A. tumefaciens was the GV2260 (kindly provided by Dr. Salom&#233; Prat). A. rhizogenes was transformed with the binary vector PK7GWIWG2_II-RedRoot (VIB-Department of Plant Systems Biology at Universiteit Gent; http://gateway.psb.ugent.be) that contains a T-DNA carrying a transformation marker to monitor the hairy root formation. To compare the transformed roots generated by A. rhizogenes and A. tumefaciens, both were transformed with the binary vector pKGWFS7 which contains a T-DNA carrying the FHT promoter driving the &#946;-glucoronidase (GUS) reporter gene and the Kanamycin resistance gene as a selectable marker15.
<ol>
	<li>Pick a colony of Agrobacterium and grow it overnight (O/N) in 5 mL of YEB medium supplemented with antibiotics (Table S1) in a 50 mL centrifuge tube at 28 &#176;C with shaking at 200 rpm.</li>
	<li>For A. tumefaciens transformation, measure the optical density, which must be OD600 = 0.6-1.0.
	<ol>
		<li>If the optical density is higher, make a subculture lowering it to OD600 = 0.3 with fresh media and wait until the culture reaches OD600 = 0.6-1.</li>
	</ol>
	</li>
	<li>Centrifuge 1 mL of Agrobacterium culture at 3,000 x g in a bench-top centrifuge for 10 min at room temperature.</li>
	<li>Remove the supernatant by pipetting, and resuspend cells in 1 mL of fresh YEB medium without antibiotics. Repeat this step&#160;to ensure the complete removal of antibiotics.
	<ol>
		<li>For A. tumefaciens transformation, in the last resuspension add the appropriate YEB volume to obtain a final optical density of OD600 = 0.8.</li>
	</ol>
	</li>
	<li>Keep cells on ice while preparing the plants to be infected.</li>
</ol>
2. Plant material for transformation
<ol>
	<li>Make one or two node stem cuttings either containing the apical or the auxiliary buds from sterile in vitro potato plants (donor plants); grow them in solid 2MS medium in pots for 3 to 4 weeks (Figure 1A and Figure 2A).</li>
</ol>
3. Plant transformation using A. rhizogenes (Figure 1)
NOTE: This procedure allows the obtaining of transformed hairy roots. To evaluate the transgene expression, a negative control is needed. To prepare the negative control, follow the procedure using an A. rhizogenes strain either untransformed or transformed with the empty vector that includes the transformation marker gene.
<ol>
	<li>Use fresh media plates; alternatively, the plates can be kept at 4 &#176;C with the lid side up, tightly sealed with transparent film to avoid media dehydration. To prepare the square media plates, incline them ~15&#176;, fill with 40 mL of MS, and let them solidify. This will minimize the contact of the aerial part of the plant&#160;with the medium.</li>
	<li>Transfer very carefully a donor plant from the 2MS medium to a 120 mm x 120 mm square plate.</li>
	<li>Inject to one stem internode 3 &#956;L of the A. rhizogenes culture using a surgical needle and repeat it twice per plant in different internodes when possible. 
	NOTE: Consider each injection as an independent transformation event (Figure 1B).</li>
	<li>Transfer immediately the entire plant to a square plate with solid MS medium supplemented with 0.1 mM acetosyringone. Accommodate 2 plants per plate. 
	NOTE: The 1 M acetosyringone stock solution is prepared in DMSO and can be stored at -20 &#176;C.</li>
	<li>Seal the plate using surgical tape and arrange it vertically inside a growth cabinet for 4 days.</li>
	<li>Transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 &#181;g/mL] to kill A. rhizogenes.</li>
	<li>After 10-12 days, hairy roots will start to appear (Figure 1C,1D). Then, excise the native roots of the plant, and transfer the plant to a new square plate with MS medium supplemented with cefotaxime sodium [500 &#181;g/mL]. 
	NOTE: Transformed hairy roots can be checked by red fluorescence when using a DsRed transformation marker (Figure 3D).&#160;</li>
	<li>To obtain a composite plant (Figure 1E), let the transgenic hairy roots grow for 3-4 weeks in MS medium supplemented with cefotaxime sodium [500 &#956;g/mL] (change the medium every week).</li>
	<li>Depending on the purpose, propagate the transgenic hairy roots and the negative controls as follow.
	<ol>
		<li>Transfer the whole composite plant to a hydroponic (Table S2) or soil medium to allow for massive development.</li>
		<li>To individually propagate the transformed hairy roots, using a scalpel cut the roots expressing the red fluorescent transformation marker (DsRed protein) when they are 4-8 cm long (Figure 1E) and transfer them into a Petri dish with Gamborg B5 solid medium supplemented with 2% sucrose and cefotaxime sodium [500 &#956;g/mL]. Seal the plates with plastic laboratory film and grow them in the dark at 20 &#176;C. 
		NOTE: The roots can be manipulated under a stereomicroscope equipped to detect the fluorescence under sterile conditions&#160;(see Table of Materials).</li>
		<li>For biomass production (i.e., gene expression analysis), cut a 5 cm long hairy root and propagate it in a 150 mL Erlenmeyer flask with 20 mL of Gamborg B5 liquid medium supplemented with 2% sucrose and cefotaxime sodium [500 &#181;g/mL]. Grow it for 6 weeks in the dark at 20 &#176;C and 60 rpm.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59119/59119fig1v3.jpg" /> 
Figure 1: Timeline to obtain potato transgenic hairy roots using A. rhizogenes. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the hairy roots are shown. Representative images of different stages are depicted: the initiation of the process using 3-week-old in vitro plants (A), then infection of the plants by injecting A. rhizogenes (B), the formation of the proliferative tissue (C, arrows) with emerging hairy roots (D), and the developed hairy roots expressing the red fluorescent transformation marker DsRed (E). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59119/59119fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Plant transformation using A. tumefaciens (Figure 2)
NOTE: This procedure allows the obtaining of transformed plants. To evaluate the transgene&#8217;s effect, a negative control is needed. One option is to follow the procedure using an A. tumefaciens transformed with the empty vector. Alternatively, wild type plants can be used.
<ol>
	<li>Cut and place a leaf from the 3-4-week-old plants (Figure 2A) in a Petri dish. Using a scalpel exclude the petiole and make transverse cuts (1-3 depending on the leaf size) from the center of the leaf to the edges avoiding cutting them off (Figure 2B).</li>
	<li>Immediately place the leaf floating on 10 mL of fresh 2MS liquid media in a Petri dish with the abaxial side up and close the plate. Repeat the step accommodating up to 15 leaves for cv. D&#233;sir&#233;e and 25 leaves for ssp. andigena (depending on the leaf size).</li>
	<li>Immediately add 80 &#181;L of A. tumefaciens culture at OD600 = 0.8 in the liquid media and homogenize the plate manually for 1 min to distribute the bacterial solution.</li>
	<li>Carefully seal with sealing film, cover with aluminum foil and incubate for 2 days in a&#160;chamber at 24 &#176;C to let the transformation occur.</li>
	<li>Transfer the leaves keeping abaxial side up to CIM medium (Figure 2B) and incubate them for one week in a growth cabinet.
	<ol>
		<li>Scrape the CIM medium with the tweezers so that the leaves can be better accommodated on the media.</li>
	</ol>
	</li>
	<li>Transfer the leaves keeping abaxial side up to SIM medium (Figure 2C) and incubate them in a growth cabinet, refreshing the medium every 7-10 days, until the shoots are about 2 cm tall.
	<ol>
		<li>Scrape the SIM medium with the tweezers so that the leaves can be fully surrounded by the media. When the emerged shoots reach the lid, work with tall Petri dishes (100 x 20 mm, height x diameter). 
		NOTE: The callus will form after 2-3 weeks in SIM medium (Figure 2D) and the shoots after 6-7 weeks (Figure 2E). The shoots will be considered as independent transformation events when they emerge from callus formed from independent wounds.</li>
	</ol>
	</li>
	<li>Cut three shoots emerged from each callus (considered the same transformation event) (Figure 2E), transfer them to culture flasks with MG medium supplemented with cefotaxime sodium [250 mg/L] to allow rooting, label the subset with a number and incubate in a growth cabinet for 3-4 weeks or until the shoots are vigorous (Figure 2F). 
	NOTE: When cutting the shoots, remove well the callus otherwise the root will not form.
	<ol>
		<li>Repeat the step as many times as independent lines are needed. Up to 5 different transformation events can be placed in a culture flask with a diameter of 8 cm to work in full confidence that the plants from different events are not mixed.</li>
	</ol>
	</li>
	<li>Select the most vigorous plant of each event, cut the apical segment of the shoot with 3-4 internodes and place it in a new culture flask with 2MS medium supplemented with cefotaxime sodium [250 mg/L]. 
	NOTE: In 3-6 weeks the plant will grow efficiently, developing a vigorous shoot and roots.
	<ol>
		<li>Bring back to the chamber the non-selected shoots until the plant selected has fully developed.</li>
	</ol>
	</li>
	<li>Cut stem segments from the plant with at least one internode or with the apical bud and transfer them to new 2MS medium supplemented with cefotaxime [250 mg/L]. Incubate them in the growth cabinet.
	<ol>
		<li>Replicate every 3-4 weeks to establish the in vitro transformed lines. 
		NOTE: The cefotaxime sodium is needed in at least three subsequent transfers to 2MS medium to be sure to kill the A. tumefaciens; afterwards, if A. tumefaciens overgrowth is observed, transfer the plants again to 2MS media supplemented with cefotaxime sodium.</li>
	</ol>
	</li>
	<li>To characterize the plant phenotype, transfer plants to soil for their full characterization or to hydroponics culture for root inspection.
	<ol>
		<li>Keep the tubers produced in soil to propagate and maintain the established lines.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59119/59119fig2.jpg" /> 
Figure 2: Timeline to obtain potato transformed plants using A. tumefaciens. The cumulative weeks to reach each stage of the transformation process and the subsequent steps to grow the plants are shown. Representative images of different stages are depicted: the initiation of the process using leaves from 3-week old in vitro plants (A), the transfer of the wounded and infected leaves to the CIM media (B), the leaves when transferred to SIM media (C), the visualization of the callus around the wounded areas after 2-3 weeks in SIM media (D), the shoot formation after 9-11 weeks in SIM media (E), and the shoots after being transferred to MG media (F). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59119/59119fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Hydroponic culture
<ol>
	<li>Prepare the Hoagland&#8217;s solution to a half strength (0.5x) (Table S2) in a 10 L bucket.</li>
	<li>Immerse an aquarium pump to maintain homogeneity and proper oxygen conditions.</li>
	<li>Cover the walls of the bucket with aluminum foil to grow roots in dark conditions.</li>
	<li>Avoiding root damage, transfer in vitro plants to hydroponic culture. 
	NOTE: Remove any remaining in vitro medium from roots to avoid microorganism proliferation during incubation by shaking carefully the roots immersed in water.</li>
	<li>Cover the plants with transparent film like a glasshouse to allow adequate acclimatization and incubate in the growing chamber.</li>
	<li>Make holes in the film after 3 days and remove it completely one week after.</li>
	<li>Replace with fresh media every 10 days.</li>
</ol>
6. GUS histochemical reporter gene assay
NOTE: In our case the GUS analysis was performed with roots of 2-3 weeks grown in hydroponics or in vitro.
<ol>
	<li>Fix the roots with 90% chilled acetone (v/v) and incubate it for 20 minutes on ice.</li>
	<li>Perform two washings with distilled water.</li>
	<li>Add fresh GUS staining solution (Table 1) and apply vacuum (-70 Pa) for 20 min.</li>
	<li>Incubate at 37 &#176;C in dark to protect the photosensitive GUS for 4 h or until a blue color is visible. 
	NOTE: The presence of ferri- and ferrocyanide in GUS solution minimize the diffusion of reaction products and provide more precise localization. 
	CAUTION: Use a fume hood and wear protective clothing when handling the toxic cyanide derivatives in GUS solution (the potassium ferricyanide and potassium ferrocyanide). The GUS substrate and the disposal material should be disposed safely.</li>
	<li>Remove GUS staining solution and discard it in appropriate containers.</li>
	<li>Perform two washings with ethanol 70% (v/v).</li>
	<li>Observe under a bright field microscope. 
	NOTE: GUS staining is stable for a few weeks; however, during the first week, the GUS signal is clear and diffuses less into neighboring cells. For longer storage, seal the tube and store it at 4 &#176;C.</li>
</ol>
<img alt="Table 1" src="/files/ftp_upload/59119/59119tbl1.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Table 1: GUS staining solution recipe.

The authors have no conflicts of interest to disclose.

In potato, the most common system to obtain stable complete transgenic plants uses the transformation by Agrobacterium tumefaciens strains that require organogenesis using exogenous phytohormones. Although the Agrobacterium based protocols has the potential to integrate non-T-DNA vector sequence25, this methodology is still the easiest and less expensive available to transform potato plants. During last years, the interest in A. rhizogenes-mediated transformation has got...

Aside from economic interest, the generation of transgenic plants has its own relevance in research to demonstrate the ultimate function of genes and to better understand plant physiology and development. The most widely used method for plant DNA insertion is Agrobacterium-mediated transformation. Agrobacterium tumefaciens is able to generate crown galls in the infected tissue of many plant species by the action of its tumour-inducing (Ti) plasmid. The plasmid contains a T-DNA region with a set of genes that will be integrated in the plant genome and induce tissue dedifferentiation1,2. The exchange of these genes within the T-DNA by the transgene has allowed the generation of specific plant modifications avoiding phenotypic effects3. To facilitate&#160;the transgene cloning into the T-DNA, the T-DNA region has been excised in an independent plasmid called a binary plasmid, while the rest of the genes of the Ti plasmid (the virulence genes that allow the T-DNA transfer and insertion mechanisms) have been placed in a helper plasmid. For plant biotechnology research, transformation by A. tumefaciens has several advantages: it does not need expensive devices, is able to generate both stable and transient plant transformation, and&#160;low numbers of gene copies are integrated into the chromosome4. However, for most plants, but not Arabidopsis, the generation of stable transformants requires plant regeneration from a single or a few cells using exogenous phytohormones, making this process laborious and time consuming. A. rhizogenes is also able to modify the plant genome, producing hairy roots or adventitious roots at the infection sites due to the expression of rol (root loci) genes encoded in the root-inducing (Ri) plasmid5. Although less studied than A. tumefaciens, A. rhizogenes is also used for obtaining transgenic roots. In this case, the A. rhizogenes contains the original T-DNA in the Ri plasmid and a binary plasmid with a second T-DNA carrying the transgene. When the infection site is in stems or hypocotyls, a composite plant can be obtained, with new hairy transgenic roots emerging from wild type shoots. Alternatively, hairy transformed roots can grow autonomously in vitro in media with carbon source inputs. The use of A. rhizogenes instead of A. tumefaciens to produce transgenic tissue is gaining relevance when the root is the target organ, because plant regeneration is not required and hence it is faster and less costly. Previous studies have demonstrated this methodology appropriated for the phenotypic characterization of root specific genes6,7,8,9.
The potato (Solanum tuberosum) is the fourth most important crop in the world according to the Food and Agriculture Organization of the United Nations (FAO) since the tuber has nutritional relevance for human consumption for being a good source of vitamins and minerals. For that reason, potato has been placed in the spotlight of agricultural biotechnology and also is considered as a good biological model for genetic and developmental studies10,11. Potato transformation significantly contributed to the understanding of molecular mechanisms underlying suberized tissues through the characterization of genes involved in suberin and wax biosynthesis12,13,14,15,16,17, suberin monomer transport18 and transcription regulation19. The suberin feruloyl transferase gene, FHT, is one of these characterized biosynthetic genes; its downregulation gives rise to a strong impairment of the periderm protection, which is correlated with a strong decrease in ferulate esters of suberin and waxes in potato tubers14. Concomitantly, in roots and seeds of Arabidopsis, the knockout of its putative orthologue (ASFT/RWP1) also demonstrated its role in producing alkyl ferulates in suberin20,21. In potato, the FHT transcriptional reporter line and the FHT antibody showed respectively that the promoter activity and the protein are located in the exodermis, the endodermis, the phellogen-derivatives and in wounded tissues15.
In this work, we detail a protocol using A. rhizogenes to produce transgenic hairy roots that are maintained in a wild type shoot, generating composite potato plants, or excised to grow autonomously in vitro. We also provide the protocol using A. tumefaciens to obtain complete transgenic potato plants. As a case study, A. rhizogenes and A. tumefaciens transformed with the same binary vector are used to obtain roots with the FHT promoter driving GUS reporter gene expression. The results are reported and compared.

<table><tbody><tr><td> &lt;p&gt;Acetone&lt;/p&gt; </td><td> &lt;p&gt;Panreac&lt;/p&gt; </td><td> &lt;p&gt;1.310.071.21&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Acetosyringone&lt;/p&gt; </td><td> &lt;p&gt;Acros&lt;/p&gt; </td><td> &lt;p&gt;115540050&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Aquarium pump&lt;/p&gt; </td><td> &lt;p&gt;Prodac&lt;/p&gt; </td><td> &lt;p&gt;MP350&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Autoclave&lt;/p&gt; </td><td> &lt;p&gt;Ragpa Strelimatic&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Bacteriological agar&lt;/p&gt; </td><td> &lt;p&gt;Lab Conda&lt;/p&gt; </td><td> &lt;p&gt;1800&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;BAP&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;B0904&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Beef extract&lt;/p&gt; </td><td> &lt;p&gt;Lab Conda&lt;/p&gt; </td><td> &lt;p&gt;1700&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Plant growing cabinet&lt;/p&gt; </td><td> &lt;p&gt;Nuaire&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Carbenicillin&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;C0109&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Cefotaxime sodium&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;C0111&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;DMSO&lt;/p&gt; </td><td> &lt;p&gt;Merck&lt;/p&gt; </td><td> &lt;p&gt;1029310161&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ecotron infors&lt;/p&gt; </td><td> &lt;p&gt;HT&lt;/p&gt; </td><td> &lt;p&gt;29378&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ethanol&lt;/p&gt; </td><td> &lt;p&gt;Merck&lt;/p&gt; </td><td> &lt;p&gt;1,009,831,011&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Falcon tube&lt;/p&gt; </td><td> &lt;p&gt;Control tecnica&lt;/p&gt; </td><td> &lt;p&gt;CFT011500&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ferricyanate&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;101001081&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ferrocyanate&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;100979088&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Flask (8.06 cm diameter and 11.3 cm height) and plastic lid for &lt;em&gt;in vitro&lt;/em&gt; culture&lt;/p&gt; </td><td> &lt;p&gt;Apiglass&lt;/p&gt; </td><td> &lt;p&gt;ref16&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;GA3&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;G7645&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Gamborg B5 media&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;G0210&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Gelrite&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;G1101&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Glucosa&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;G5767&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Kanamycin&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;K1377&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Leukopor tape&lt;/p&gt; </td><td> &lt;p&gt;BSN Leukopor&lt;/p&gt; </td><td> &lt;p&gt;BDF47467&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Lupe&lt;/p&gt; </td><td> &lt;p&gt;Wild-Heerbrugg&lt;/p&gt; </td><td> &lt;p&gt;M420&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Magnetic shaker&lt;/p&gt; </td><td> &lt;p&gt;Agimatic&lt;/p&gt; </td><td> &lt;p&gt;7000243&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;MES hydrate&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;M2933-25G&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt;</td><td> &lt;p&gt;Panreac&lt;/p&gt; </td><td> &lt;p&gt;131404&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Microscope&lt;/p&gt; </td><td> &lt;p&gt;Olympus&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Minufugue centrifugue 5415R&lt;/p&gt; </td><td> &lt;p&gt;Eppendorf&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Murashige and Skoog media&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;M0254.0050&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt;</td><td> &lt;p&gt;Panreac&lt;/p&gt; </td><td> &lt;p&gt;131679&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;NAA&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;N0903&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;NaCl&lt;/p&gt; </td><td> &lt;p&gt;Panreac&lt;/p&gt; </td><td> &lt;p&gt;131659&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/p&gt;</td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;58282&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;NightSea Stereo&lt;/p&gt; </td><td> &lt;p&gt;SFA Moonting Adapter&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Parafilm&lt;/p&gt; </td><td> &lt;p&gt;Anorsa&lt;/p&gt; </td><td> &lt;p&gt;PRFL-001-001&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Peptone&lt;/p&gt; </td><td> &lt;p&gt;Lab Conda&lt;/p&gt; </td><td> &lt;p&gt;1616&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Petri dishes (90 x 14)&lt;/p&gt; </td><td> &lt;p&gt;Anorsa&lt;/p&gt; </td><td> &lt;p&gt;200200&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;pHmetre&lt;/p&gt; </td><td> &lt;p&gt;Crison&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Phytotron&lt;/p&gt; </td><td> &lt;p&gt;Inkoa&lt;/p&gt; </td><td> &lt;p&gt;RFTI-R5485&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Plant Agar&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;P1001&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Refrigeratot&lt;/p&gt; </td><td> &lt;p&gt;Liebherr Medline&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Rifampicin&lt;/p&gt; </td><td> &lt;p&gt;Duchefa&lt;/p&gt; </td><td> &lt;p&gt;R0146&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Spectinomycin&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;59007&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Spectrophotometer&lt;/p&gt; </td><td> &lt;p&gt;Shimadzu&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Square plates (120 x 120)&lt;/p&gt; </td><td> &lt;p&gt;Deltalab&lt;/p&gt; </td><td> &lt;p&gt;200204&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Streptomycin&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;S6501&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sucrose&lt;/p&gt; </td><td> &lt;p&gt;Panreac&lt;/p&gt; </td><td> &lt;p&gt;131621&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Surgical blades&lt;/p&gt; </td><td> &lt;p&gt;Swann-Morton&lt;/p&gt; </td><td> &lt;p&gt;201&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Surgical needle&lt;/p&gt; </td><td> &lt;p&gt;NIPRO&lt;/p&gt; </td><td> &lt;p&gt;015/0204&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Triptone&lt;/p&gt; </td><td> &lt;p&gt;Lab Conda&lt;/p&gt; </td><td> &lt;p&gt;1612&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Triton&lt;/p&gt; </td><td> &lt;p&gt;Serva&lt;/p&gt; </td><td> &lt;p&gt;37240&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Unimax 1010 shaker&lt;/p&gt; </td><td> &lt;p&gt;Heidolph&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Vacuum&lt;/p&gt; </td><td> &lt;p&gt;Dinko&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;x-GlcA (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, sodium salt anhydrous)&lt;/p&gt; </td><td> &lt;p&gt;Biosynth&lt;/p&gt; </td><td> &lt;p&gt;B-7398&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Yeast extract&lt;/p&gt; </td><td> &lt;p&gt;Lab Conda&lt;/p&gt; </td><td> &lt;p&gt;1702.00&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Zeatin riboside&lt;/p&gt; </td><td> &lt;p&gt;Sigma&lt;/p&gt; </td><td> &lt;p&gt;1001042850&lt;/p&gt; </td><td></td></tr></tbody></table>

agrobacterium tumefaciens and agrobacterium rhizogenes-mediated transformation of potato and the promoter activity of a suberin gene by gus staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium rhizogenes-mediated potato transformation 
In this manuscript, the step-by-step procedure set up to obtain transformed root with A. rhizogenes is presented. Figure 1 presents an overview of the procedure, which altogether takes around 5-6 weeks (from injection of A. rhizogenes to obtaining fully developed hairy roots). Then, ...

Watch this Scientific Journal Video about Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining at JoVE.c

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

agrobacterium-tumefaciens-agrobacterium-rhizogenes-mediated

Universidad de Cartagena UDEC

Research

JoVE Journal

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29.4K Views.  Universitat de Girona. Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Video: Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Genetics

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 ...

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.

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.

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

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Biology

High-Efficiency Production of Transgenic Soybean Hairy Roots

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Author Spotlight: Soybean Hairy Root Transformation for the Analysis of Gene Function  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

Bioengineering

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Plant Promoter Analysis: Identification and Characterization of Root Nodule Specific Promoter in the Common Bean

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in understanding the gene regulation and spatiotemporal expression patterns of target genes. On the other hand, it is also critical to establish promoter evaluation tools and genetic transformation techniques that are fast, efficient, and reproducible. In this study, we investigated the spatiotemporal expression pattern of the rhizobial symbiosis-specific nodule inception (NIN) promoter of Phaseolus vulgaris in the transgenic hairy roots. Using plant genome databases and analysis tools we identified, isolated, and cloned the P. vulgaris NIN promoter in a transcriptional fusion to the chimeric reporter &#946;-glucuronidase (GUS) GUS-enhanced::GFP. Further, this protocol describes a rapid and versatile system of genetic transformation in the P. vulgaris using Agrobacterium rhizogenes induced hairy roots. This system generates &#8805;2 cm hairy roots at 10 to 12 days after transformation. Next, we assessed the spatiotemporal expression of NIN promoter in Rhizobium inoculated hairy roots at periodic intervals of post-inoculation. Our results depicted by GUS activity show that the NIN promoter was active during the process of nodulation. Together, the present protocol demonstrates how to identify, isolate, clone, and characterize a plant promoter in the common bean hairy roots. Moreover, this protocol is easy to use in non-specialized laboratories.

Promoter expression analyses are crucial to improving the understanding of gene regulation and the spatiotemporal expression of target genes. Herein we present a protocol to identify, isolate, and clone a plant promoter. Further, we describe the characterization of the nodule-specific promoter in the common bean hairy roots.

The upstream sequences of gene coding sequences are termed as promoter sequences. Studying the expression patterns of promoters are very significant in ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

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Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain