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 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.
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.
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.
The authors have nothing to disclose.
We acknowledge Jiří Macas (Biology Centre CAS, České Budějovice, 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 "SINGING PLANT" (no. CZ.02.1.01/0.0/0.0/16_026/0008446) and the INTER-COST project LTC20004.
1.50 mL tubes | Eppendorf | 125.215 | |
10% solution of commercial bleach | SAVO | ||
1-naphthaleneacetic acid (NAA) | Duchefa | N0903 | Callus regeneration medium |
2.0 mL tubes | Eppendorf | 108.132/108.078 | |
3M micropore tape | Micropore | ||
6-Benzylaminopurine (BAP) | Duchefa | B0904 | Callus regeneration medium, Shoot elongation medium |
70% ethanol | |||
bacteriological agar | HiMedia | RM201 | LB medium |
Bacteriological peptone | Oxoid | LP0037 | LB and YEB media |
Beef extract | Roth | X975.1 | YEB medium |
Bottles | DURAN | L300025 | |
Cefotaxime sodium | Duchefa | C0111 | Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium |
chloroform | Serva | 3955301 | |
CTAB Hexadecyltrimethylammonium bromide | Sigma | 52365 | |
dNTP mix | Thermo Fisher Scientific | R0193 | |
EDTA – Titriplex III, (Ethylenendinitrilo)tetraacetic Acid, Disodium Salt, Dihydrate | Sigma | ES134-250G | |
elctroporation cuvette | |||
electrophesis agar, peqGOLD universal | VWR | 732-2789 | |
electrophoresis chamber | BIO-RAD | ||
electrophoresis gel reader | BIO-RAD | ||
electroporator GenePulser Xcell | BIO-RAD | ||
ethidium bromide | AppliChem | ||
Gene Pulser/MicroPulser electroporation cuvettes, 0.2 cm gap | BIO-RAD | 1652082 | |
Gene Ruler DNA ladder mix | Thermo Fisher Scientific | SM0331 | |
Gibberellic acid (GA3) | Duchefa | G0907 | Shoot elongation medium |
glycerol | Sigma | G5516-1L | |
HEPES (2-(4-(2-hydroxyethyl)-1-pirerazinyl)-ethansulfonique | Merck | 1101100250 | |
indole-3-butyric acid (IBA) | Duchefa | I0902 | Root induction medium |
kanamycin monosulfate | Duchefa | K0126 | |
Magenta GA-7 Plant Culture Box w/ Lid | Plant Media | V8505-100 | |
Measuring cylinder | |||
MES monohydrate | Duchefa | M1503 | Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium, Medium for germination, Plant growing medium |
Murashige and Skoog medium (MS) | Duchefa | M0237 | Medium for germination, Plant growing medium |
Murashige and Skoog medium (MS) + B5 vitamins | Duchefa | M0231 | Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium |
needle Agani 26G x 1/2 – 0.45 x 13mm | Terumo | ||
pH meter | |||
Phytagel | Sigma | P8169 | Callus regeneration medium, Root induction medium, Medium for germination |
PVP 40 (polyvinylpyrolidone Mr 40000) | Sigma | 9003-39-8 | |
Redtaq DNA Polymerase,Taq for routine PCR with inert dye, 10X buffer included | Sigma | D4309-250UN | |
Retsh mill | Qiagen | ||
sodium chloride | Lachner | 30093-APO | LB medium |
square Petri Dishes | Corning | GOSSBP124-05 | |
sucrose | Penta | 24970-31000 | Hairy root growing medium, Callus regeneration medium, Shoot elongation medium, Root induction medium, Medium for germination, Plant growing medium |
Syringe filter | Carl Roth | P666.1 | Rotylabo syringe filters 0.22 µm pore size |
thermomixer | Eppendorf | ||
Ticarcillin disodium | Duchefa | T0180 | Hairy root growing medium |
Tris(hydroxymethyl)aminomethan | Serva | 3719003 | |
ultrapure water | Millipore Milli-Q purified water | ||
Yeast extract | Duchefa | Y1333 | LB medium |