We acknowledge Ji&#345;&#237; Macas (Biology Centre CAS, &#268;esk&#233; Bud&#283;jovice, Czech Republic) for providing the agrobacterial strain. The Core Facility Plants Sciences of CEITEC MU is acknowledged for its technical support. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic with the European Regional Development Fund-Project &#34;SINGING PLANT&#34; (no. CZ.02.1.01/0.0/0.0/16_026/0008446) and the INTER-COST project LTC20004.

Agrobacterium-Mediated Hairy Root Induction and Regeneration in Plants

<ol><li>Lee, L. Y., Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-DNA+binary+vectors+and+systems%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">T-DNA binary vectors and systems.</a> Plant Physiology. 146 (2), 325-332 (2008).</li>
<li>Christey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Ri-mediated+transformation+for+production+of+transgenic+plants%5BTitle%5D)+AND+%22In+Vitro+Cellular+%26+Developmental+Biology+-+Plant%22%5BJournal%5D)">Use of Ri-mediated transformation for production of transgenic plants.</a> In Vitro Cellular & Developmental Biology - Plant. 37 (6), 687-700 (2001).</li>
<li>Gelvin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agrobacterium-Mediated+Plant+Transformation%3A+the+Biology+behind+the+%E2%80%9CGene-Jockeying%E2%80%9D+Tool%5BTitle%5D)+AND+%22Microbiology+and+Molecular+Biology+Reviews%22%5BJournal%5D)">Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool.</a> Microbiology and Molecular Biology Reviews. 67 (1), 16-37 (2003).</li>
<li>Georgiev, M. I., Agostini, E., Ludwig-Müller, J., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetically+transformed+roots%3A+from+plant+disease+to+biotechnological+resource%5BTitle%5D)+AND+%22Trends+in+Biotechnology%22%5BJournal%5D)">Genetically transformed roots: from plant disease to biotechnological resource.</a> Trends in Biotechnology. 30 (10), 528-537 (2012).</li>
<li>Gutierrez-Valdes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hairy+root+cultures%E2%80%94a+versatile+tool+with+multiple+applications%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Hairy root cultures—a versatile tool with multiple applications.</a> Frontiers in Plant Science. 11, 33(2020).</li>
<li>Niazian, M., Belzile, F., Torkamaneh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9+in+planta+hairy+root+transformation%3A+a+powerful+platform+for+functional+analysis+of+root+traits+in+Soybean%5BTitle%5D)+AND+%22Plants%22%5BJournal%5D)">CRISPR/Cas9 in planta hairy root transformation: a powerful platform for functional analysis of root traits in Soybean.</a> Plants. 11 (8), 1044(2022).</li>
<li>Petit, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Further+extension+of+the+opine+concept%3A+plasmids+in+Agrobacterium+rhizogenes+cooperate+for+opine+degradation%5BTitle%5D)+AND+%22Molecular+and+General+Genetics+MGG%22%5BJournal%5D)">Further extension of the opine concept: plasmids in Agrobacterium rhizogenes cooperate for opine degradation.</a> Molecular and General Genetics MGG. 190 (2), 204-214 (1983).</li>
<li>Ozyigit, I. I., Dogan, I., Tarhan, E. A. Agrobacterium rhizogenes-mediated transformation and its biotechnological applications in crops. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOzyigit%2C+II+%22Crop+improvement%22">Crop improvement</a>. , Springer. Boston, MA. (2013).</li>
<li>Jedličková, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hairy+root+transformation+system+as+a+tool+for+CRISPR%2FCas9-directed+genome+editing+in+oilseed+rape+%28Brassica+napus%29%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Hairy root transformation system as a tool for CRISPR/Cas9-directed genome editing in oilseed rape (Brassica napus).</a> Frontiers in Plant Science. 13, 919290(2022).</li>
<li>Steinbauerová, V., Neumann, P., Macas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+evidence+for+splicing+of+intron-containing+transcripts+of+plant+LTR+retrotransposon+Ogre%5BTitle%5D)+AND+%22Molecular+Genetics+and+Genomics%22%5BJournal%5D)">Experimental evidence for splicing of intron-containing transcripts of plant LTR retrotransposon Ogre.</a> Molecular Genetics and Genomics. 280 (5), 427-436 (2008).</li>
<li>Neumann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Centromeres+off+the+hook%3A+massive+changes+in+centromere+size+and+structure+following+duplication+of+CenH3+gene+in+Fabeae+species%5BTitle%5D)+AND+%22Molecular+Biology+and+Evolution%22%5BJournal%5D)">Centromeres off the hook: massive changes in centromere size and structure following duplication of CenH3 gene in Fabeae species.</a> Molecular Biology and Evolution. 32 (7), 1862-1879 (2015).</li>
<li>Montazeri, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Comparative+analysis+of+the+hairy+root+induction+methods+in+Hypericum+perforatum%5BTitle%5D)+AND+%22Journal+of+Plant+Molecular+Breeding%22%5BJournal%5D)">A Comparative analysis of the hairy root induction methods in Hypericum perforatum.</a> Journal of Plant Molecular Breeding. 7 (1), 67-76 (2019).</li>
<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peat-based+hairy+root+transformation+using+Rhizobium+rhizogenes+as+a+rapid+and+efficient+tool+for+easily+exploring+potential+genes+related+to+root-knot+nematode+parasitism+and+host+response%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">Peat-based hairy root transformation using Rhizobium rhizogenes as a rapid and efficient tool for easily exploring potential genes related to root-knot nematode parasitism and host response.</a> Plant Methods. 19 (1), 22(2023).</li>
<li>Christey, M. C., Sinclair, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+transgenic+kale+%28Brassica+oleracea+var.+acephala%29%2C+rape+%28B.+napus%29+and+turnip+%28B.+campestris+var.+rapifera%29+plants+via+Agrobacterium+rhizogenes+mediated+transformation%5BTitle%5D)+AND+%22Plant+Science%22%5BJournal%5D)">Regeneration of transgenic kale (Brassica oleracea var. acephala), rape (B. napus) and turnip (B. campestris var. rapifera) plants via Agrobacterium rhizogenes mediated transformation.</a> Plant Science. 87 (2), 161-169 (1992).</li>
<li>Christey, M. C., Sinclair, B. K., Braun, R. H., Wyke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+transgenic+vegetable+brassicas+%28Brassica+oleracea+and+B.+campestris%29+via+Ri-mediated+transformation%5BTitle%5D)+AND+%22Plant+Cell+Reports%22%5BJournal%5D)">Regeneration of transgenic vegetable brassicas (Brassica oleracea and B. campestris) via Ri-mediated transformation.</a> Plant Cell Reports. 16 (9), 587-593 (1997).</li>
</ol>

The authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

We developed a simple protocol for hairy root transformation and subsequent regeneration in B. napus and A. thaliana. This process includes injection-based hairy root induction in the hypocotyl (B. napus) or primary inflorescence stem (A. thaliana). The method of injecting the hypocotyl with agrobacterial strain C58C1 carrying a Ri plasmid was also effective in the Fabaceae family10,11, besides the members of Brassicaceae presented in this study.
An alternative to the injection-based method is the immersion-based transformation consisting of explant immersion in a bacterial suspension, followed by co-cultivation of the explant with agrobacteria. The advantage of the injection-based method over the immersion method is the time saved by the absence of some protocol steps: explant preparation, a test of the co-cultivation time, and culture on a medium containing hormone for induction of hairy roots. Although both approaches are effective for hairy root induction, higher transformation efficiency was observed in some species with the injection-based method compared to the explant immersion one12,13. Moreover, injection-based transformation is also useful for generating composite plants (transgenic hairy roots and wild-type shoots). After cutting off the original roots of the transformed plant, hairy roots support the plant growth, and the transgene can be studied in the context of the whole plant.
The critical step of hairy root induction is injecting the inoculum into the hypocotyl, or primary inflorescence stem. The hypocotyls of B. napus are breakable, and cutting the whole hypocotyl can easily happen. The same can be observed with A. thaliana because of the inflorescence stem thinness. If a comparison of the transformation efficiency of different species/cultivars is required, we recommend that one person perform all experiments to avoid the error caused by manipulation and skill in injecting the plants.
We developed an effective protocol for hairy root regeneration in B. napus DH12075 and A. thaliana Col-0. As regeneration is a highly variable process, some protocol modifications may be applied to a species or cultivar of choice. For instance, the hairy root-derived shoots can be elicited by a different auxin/cytokinin ratio (1:1) in B. oleracea14. Alternatively, cytokinin thidiazuron can be used instead of BAP, such as in the case of B. campestris hairy roots15.
Multiple insertions of the Ri plasmid T-DNA into the plant genome represent a potential limitation of the hairy root transformation and regeneration system. In such cases, no plants free of TL/TR from the Ri plasmid are uncovered after a segregation analysis of T1 seedlings. Thus, we recommend generating several independent hairy root lines for each transgene.
Hairy root cultures are an extremely powerful tool for gene functional studies mainly because of their rapid establishment and cheap maintenance (no hormones needed in cultivation media). This protocol covers the methods for hairy root induction and regeneration in B. napus and A. thaliana, which can be used to study the transgene of interest directly in hairy root cultures, in the context of the whole plant using composite plants, or after the regeneration of the transgenic plants.

Plant transformation is the bottleneck of any genetic study in plant biology. A soil-borne bacterium, Agrobacterium tumefaciens, is widely used as a means for gene delivery by floral dip or tissue culture to generate transformants. A. tumefaciens infects plants at a wounding site and causes tumors due to the transfer and integration of T-DNA from a Tumor-inducing (Ti) plasmid into the host plant genome. Engineered A. tumefaciens strains with modified Ti plasmid without the wild-type T-DNA and a binary vector with artificial T-DNA and cloning sites for inserting a gene of interest are commonly used as an efficient plant transformation system1. However, many model species and crops are recalcitrant to floral dip or in vitro plant regeneration or have long growth cycles, impacting the efficiency of this transformation system.
Agrobacterium rhizogenes induces the formation of adventitious roots, or hairy roots, at the wounding site after infecting a host plant. Similar to A. tumefaciens, A. rhizogenes transfers a T-DNA from a Root-inducing (Ri) plasmid to the host plant genome, causing the development of transgenic hairy roots. This process is controlled mainly by the root oncogenic loci (rol) genes2,3. Using agrobacterial strains carrying both the Ri plasmid and an artificial binary vector encoding a gene of interest, hairy root cultures have been used to produce recombinant proteins, analyze the function of promoters or genes, or edit genomes using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)/CRISPR-associated protein 9 (Cas9)4,5,6.
Our protocol uses the transconjugant strain Ti-less A. tumefaciens C58C1 carrying the Ri plasmid pRiA4b7. The T-DNA of the Ri plasmid consists of two regions, right and left T-DNA (TR-DNA and TL-DNA, respectively), that can independently integrate into the plant genome8. Exploiting this system, the lengthy explant transformation process in Brassica napus DH12075 cultivar was optimized9. The protocol detailed below allows for the regeneration of selected hairy root lines and obtaining T1 plants carrying the transgene of interest and free of rol genes in roughly 1 year. The injection-based hairy root transformation can be used in other Brassicaceae species, as shown by transforming Arabidopsis thaliana Col-0. While hypocotyl is used to transform B. napus, A. thaliana is injected into the primary inflorescence stem.

<table><tbody><tr><td>1.50 mL tubes</td><td>Eppendorf</td><td>125.215</td><td></td></tr><tr><td>10% solution of commercial bleach&amp;nbsp;</td><td>SAVO</td><td></td><td></td></tr><tr><td>1-naphthaleneacetic acid (NAA)&amp;nbsp;</td><td>Duchefa</td><td>N0903</td><td>Callus regeneration medium</td></tr><tr><td>2.0 mL tubes</td><td>Eppendorf</td><td>108.132/108.078</td><td></td></tr><tr><td>3M micropore tape</td><td>Micropore</td><td></td><td></td></tr><tr><td>6-Benzylaminopurine (BAP)&amp;nbsp;</td><td>Duchefa</td><td>B0904</td><td>Callus regeneration medium, Shoot elongation medium&amp;nbsp;</td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>bacteriological agar</td><td>HiMedia</td><td>RM201</td><td>LB medium</td></tr><tr><td>Bacteriological peptone</td><td>Oxoid</td><td>LP0037</td><td>LB and YEB media</td></tr><tr><td>Beef extract</td><td>Roth</td><td>X975.1</td><td>YEB medium</td></tr><tr><td>Bottles</td><td>DURAN</td><td>L300025</td><td></td></tr><tr><td>Cefotaxime sodium</td><td>Duchefa</td><td>C0111</td><td>Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium</td></tr><tr><td>chloroform</td><td>Serva</td><td>3955301</td><td></td></tr><tr><td>CTAB Hexadecyltrimethylammonium bromide</td><td>Sigma</td><td>52365</td><td></td></tr><tr><td>dNTP mix</td><td>Thermo Fisher Scientific</td><td>R0193</td><td></td></tr><tr><td>EDTA - Titriplex III, (Ethylenendinitrilo)tetraacetic Acid, Disodium Salt, Dihydrate</td><td>Sigma</td><td>ES134-250G</td><td></td></tr><tr><td>elctroporation cuvette</td><td></td><td></td><td></td></tr><tr><td>electrophesis agar, peqGOLD universal</td><td>VWR</td><td>732-2789</td><td></td></tr><tr><td>electrophoresis chamber</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>electrophoresis gel reader</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>electroporator GenePulser Xcell</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>ethidium bromide</td><td>AppliChem</td><td></td><td></td></tr><tr><td>Gene Pulser/MicroPulser electroporation cuvettes, 0.2 cm gap</td><td>BIO-RAD</td><td>1652082</td><td></td></tr><tr><td>Gene Ruler DNA ladder mix</td><td>Thermo Fisher Scientific</td><td>SM0331</td><td></td></tr><tr><td>Gibberellic acid (GA3)</td><td>Duchefa</td><td>G0907</td><td>Shoot elongation medium&amp;nbsp;</td></tr><tr><td>glycerol</td><td>Sigma</td><td>G5516-1L</td><td></td></tr><tr><td>HEPES (2-(4-(2-hydroxyethyl)-1-pirerazinyl)-ethansulfonique</td><td>Merck</td><td>1101100250</td><td></td></tr><tr><td>indole-3-butyric acid (IBA)</td><td>Duchefa</td><td>I0902</td><td>Root induction medium</td></tr><tr><td>kanamycin monosulfate</td><td>Duchefa</td><td>K0126</td><td></td></tr><tr><td>Magenta GA-7 Plant Culture Box w/ Lid</td><td>Plant Media</td><td>V8505-100</td><td></td></tr><tr><td>Measuring cylinder</td><td></td><td></td><td></td></tr><tr><td>MES monohydrate</td><td>Duchefa</td><td>M1503</td><td>Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium, Medium for germination, Plant growing medium</td></tr><tr><td>Murashige and Skoog medium (MS)</td><td>Duchefa</td><td>M0237</td><td>Medium for germination, Plant growing medium</td></tr><tr><td>Murashige and Skoog medium (MS) + B5 vitamins&amp;nbsp;</td><td>Duchefa</td><td>M0231</td><td>Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium</td></tr><tr><td>needle Agani 26G x 1/2 - 0.45 x 13mm</td><td>Terumo</td><td></td><td></td></tr><tr><td>pH meter</td><td></td><td></td><td></td></tr><tr><td>Phytagel</td><td>Sigma</td><td>P8169</td><td>Callus regeneration medium, Root induction medium, Medium for germination</td></tr><tr><td>PVP 40 (polyvinylpyrolidone Mr 40000)</td><td>Sigma</td><td>9003-39-8</td><td></td></tr><tr><td>Redtaq DNA Polymerase,Taq for routine PCR with inert dye, 10X buffer included</td><td>Sigma</td><td>D4309-250UN</td><td></td></tr><tr><td>Retsh mill</td><td>Qiagen</td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Lachner</td><td>30093-APO</td><td>LB medium</td></tr><tr><td>square Petri Dishes</td><td>Corning</td><td>GOSSBP124-05</td><td></td></tr><tr><td>sucrose</td><td>Penta</td><td>24970-31000</td><td>Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium, Medium for germination, Plant growing medium</td></tr><tr><td>Syringe filter</td><td>Carl Roth</td><td>P666.1</td><td>Rotylabo syringe filters 0.22 &amp;micro;m pore size</td></tr><tr><td>thermomixer</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Ticarcillin disodium</td><td>Duchefa</td><td>T0180</td><td>Hairy root growing medium</td></tr><tr><td>Tris(hydroxymethyl)aminomethan</td><td>Serva</td><td>3719003</td><td></td></tr><tr><td>ultrapure water</td><td>Millipore Milli-Q purified water&amp;nbsp;</td><td></td><td></td></tr><tr><td>Yeast extract</td><td>Duchefa</td><td>Y1333</td><td>LB medium</td></tr></tbody></table>

hairy root transformation and regeneration in arabidopsis thaliana and brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a Root-inducing (Ri) plasmid induces the formation of hairy roots at the wounding site after the transfer of T-DNA from the Ri plasmid into the plant genome. The protocol describes in detail the procedure of the injection-based hairy root induction in Brassica napus DH12075 and Arabidopsis thaliana Col-0. The hairy roots may be used to analyze a transgene of interest or processed for the generation of transgenic plants. Regeneration medium containing cytokinin 6-benzylaminopurine (5 mg/L) and auxin 1-naphthaleneacetic acid (8 mg/L) successfully elicits shoot formation in both species. The protocol covers the genotyping and selection of regenerants and T1 plants to obtain plants carrying a transgene of interest and free of T-DNA from the Ri plasmid. An alternative process leading to the formation of a composite plant is also depicted. In this case, hairy roots are kept on the shoot (instead of the natural roots), which enables the study of a transgene in hairy root cultures in the context of the whole plant.

We have previously optimized a protocol for injection-based hairy root induction in three cultivars of Brassica napus, namely DH12075, Topas DH4079, and Westar9. To apply this transformation protocol to the model species A. thaliana, the primary inflorescence stems of 1-month-old plantlets were injected with an agrobacterial inoculum. Hairy roots emerged at the site of injection after 2-4 weeks. Hairy roots were excised and cultivated on the solid medium. The comparison of the method in these two species is depicted in Figure 1.
Selected hairy root lines were transferred to the regeneration medium to induce shoot formation. In A. thaliana, yellow calli were induced within 14 days in all 10 tested hairy root lines. First shoot primordia visible as dark green spots emerged within 3 weeks after transfer to the regeneration medium (Figure 2). After 4 weeks of culture, shoots covered the hairy roots in 9 of 10 hairy root lines (90% regeneration efficiency). In some cases, adventitious roots were elicited from the callus (Figure 2H). One line did not regenerate even after 3 months on the Regeneration medium (every 4 weeks, the hairy roots were transferred to a fresh medium). Thus, the regeneration efficiency of A. thaliana hairy roots resembles the efficiency of B. napus DH120759.
Hairy root regenerants of B. napus and A. thaliana display a dwarf phenotype (Figure 3), a typical feature of the hairy root-derived plants2. We also observed dense root systems, wrinkled leaves, and changes in flowering time. This so-called hairy root (or Ri) phenotype is caused by the rol genes from the Ri plasmid inserted into the plant genome. The insertion of the Ri T-DNA and the transgene encoded on a binary vector may be independent or linked. Thus, a segregation analysis of T1 progeny created by self-pollination helps to identify rol-free plants expressing the transgene of interest. Genotyping of the T1 plants is performed by PCR primers specific for the ORFs of TL and TR and the transgene of interest. The absence of agrobacterial contamination is verified by the absence of PCR products of virC primers (Figure 1).
<img alt="Hairy root induction, PCR detection diagram; transgene analysis, composite plant transformation process." class="xfigimg" src="/files/ftp_upload/66223/66223fig01.jpg" data-altupdatedat="1747929244000" data-alt="Figure 1"> 
Figure 1: Summary of the procedure in A. thaliana and B. napus. The injection of the Agrobacterium inoculum in the hypocotyl or primary inflorescence stem induces the development of hairy roots. The hairy roots can replace the native roots to generate a composite plant that will be genotyped and analyzed (blue arrows). Cultured hairy roots can be regenerated into T0 plants, propagated in T1 plants, and genotyped (green arrows). The hairy roots can also be sub-cultured for functional analysis (black arrow). Examples of genotyping results are presented. <a href="https://www.jove.com/files/ftp_upload/66223/66223fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fungal growth progression over four weeks, microscopy images; colony development observation." class="xfigimg" src="/files/ftp_upload/66223/66223fig02.jpg" data-altupdatedat="1747929244000" data-alt="Figure 2"> 
Figure 2: Regeneration of hairy roots in A. thaliana. (A) Hairy root culture 1 day after its transfer to plates. (B, C) Calli developed within 2 weeks of culture on regeneration medium. (D, E) Shoot primordia emerged after 3 weeks of culture. (G, H) Shoots are formed after 4 weeks. (H) Adventitious roots developed from the callus. (C, F, I) Non-regenerating hairy root line. Scale bars represent 1 cm. <a href="https://www.jove.com/files/ftp_upload/66223/66223fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Arabidopsis and Brassica napus regeneration comparison; wild-type vs. hairy root-derived plants." class="xfigimg" src="/files/ftp_upload/66223/66223fig03.jpg" data-altupdatedat="1747929244000" data-alt="Figure 3"> 
Figure 3: Representative photos of B. napus and A. thaliana wild-type plants and hairy-root-derived regenerants (T0 plants). Note the Ri phenotype of the regenerants. Scale bars represent 2 cm. <a href="https://www.jove.com/files/ftp_upload/66223/66223fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus at JoVE.com

Author Spotlight: Optimizing Hairy Root-Based Transformation Protocols for Enhanced Efficiency in Brassicaceae

The protocol describes hairy root induction using Arabidopsis primary inflorescence stems and Brassica napus hypocotyls. The hairy roots can be cultured and used as explants to regenerate transgenic plants.

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Hairy root transformation represents a versatile tool for plant biotechnology in various species. Infection by an Agrobacterium strain carrying a ...

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3.5K Views.  Masaryk University. Ons onderzoek was gericht op het ontwikkelen van een protocol voor een efficiënte transformatie in Brassicaceae. Met behulp van een transformatie op basis van haarwortels hebben we de transformatie-efficiëntie in Brassica napus drastisch verbeterd, wat cruciaal is voor ons onderzoek. We hebben het protocol ook getest in modelsoorten Arabidopsis thaliana.We hebben het protocol geoptimaliseerd voor voorjaarscultivars DH12075 in Brassica napus. De volgende uitdaging is het optimaliseren...

Video: Auteur in de schijnwerpers: Optimalisatie van op harige wortels gebaseerde transformatieprotocollen voor verbeterde efficiëntie in Brassicaceae

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

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 ...

Genetics

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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 ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

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 ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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

One-Step Hairy Root Transformation Method for Advancing Root-Related Biology

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Author Spotlight: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

Hairy Root Transformation and Regeneration in Arabidopsis thaliana and Brassica napus

Samenvatting

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