Agrobacterium rhizogenes—mediated transformation of Pisum sativum L. roots as a tool for studying the mycorrhizal and root nodule symbioses

Plant material and bacterial strains

Surface-sterilized seeds of the Pisum sativum cultivars Finale and Frisson were treated with concentrated sulfuric acid for 5 min, washed five times with a large volume of sterile water, and then germinated on agar plates in the dark at 21–23 °C. After germination for 4 days, the seedlings were transferred to the light and placed into sterile plastic containers filled with Jensen medium and subsequently grown for further 3–4 days in a growth chamber at 21 °C and 60% humidity under 16 h/8 h light/dark cycle. The Agrobacterium rhizogenes strains ARqua1 (Quandt, 1993) and AR1193 (Hansen et al., 1989) and the RCAM1026 (WDCM 966) strain of Rhizobium leguminosarum bv. viciae were grown at 28 °C in Tryptone Yeast medium (Sambrook et al., 1989) containing the appropriate antibiotics.

Transformation procedure

In order to induce the formation of hairy roots, the roots of P. sativum seedlings were excised in the hypocotyl region (3–4 mm below the point of cotyledon attachment). The root hypocotyls were then treated with A. rhizogenes ARqua 1 or AR1193 containing appropriate plasmids. For plant transformation, we used the pB7WGD plasmid carrying the gusA/GFP reporter gene construct driven by the CaMV35S promoter, which was introduced into the A. rhizogenes strains by electroporation. Electroporation of A. rhizogenes was performed using GenePulser Xcell (Bio-Rad Laboratories, Hercules, CA, USA) in 1 mm cuvette with voltage −2,500 V, capacitance 25 µF, resistance 200 ohms. The wounded root tip of the seedling was touched to the A. rhizogenes growing on a plate. Plants were placed on Jensen agar in plastic jars (Duchefa, Haarlem, The Netherlands), and the cut root tips were covered with wet wool and foil. The seedlings were co-cultivated with A. rhizogenes for 10–14 days at 21 °C (16 h/8 h light/dark) until a visible callus had developed. Emerging roots were examined using an epifluorescence stereo microscope, and transformed hairy roots were identified and selected based on GFP expression. Following the removal of primary roots, the seedlings were transferred to Emergence medium containing 150 mg/mL of cefotaxime and incubated for 3–4 days. Thereafter, the plants were transferred into pots of vermiculite saturated with Jensen medium containing 1.5 M M NH₄NO₃, grown for 2 days in a growth chamber at 21 °C and 60% humidity under a 16 h/8 h light/dark cycle, and then inoculated with Rhizobium strains (OD₆₀₀ = 0.5). The pots were then placed in large plastic bags and plants were incubated under high humidity conditions for at least 1 week and then under normal conditions. The plants were watered as needed by adding the Jensen solution with 1.5 M M NH₄NO₃.

RNA extraction and quantitative reverse transcription PCR (qRT-PCR)

RNA extraction was performed as described previously (Leppyanen et al., 2017). RNA (from 1 to 2.5 µg) was used for cDNA synthesis with the RevertAid Reverse Transcriptase (Thermo Scientific, Waltham, MA, USA) for 1 h at 42 °C followed by heating to 95 °C, 5 min. Aliquots of the cDNA were diluted 1:10 for qPCR analysis. qRT-PCR analysis was performed using a CFX-96 real-time PCR detection system incorporating a C1000 thermal cycler (Bio-Rad Laboratories). All reactions were done in triplicate and averaged. Cycle threshold (CT) values were obtained with the accompanying software and data were analyzed with the 2 − ΔΔCt method (Livak & Schmittgen, 2001). The relative expression was normalized against the constitutively expressed Ubiquitin gene. RT-PCR for GFP gene expression analysis was performed using Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific). All primer pairs (Table S1) were designed using Vector NTI program and produced by Evrogen (Moscow, Russia; http://www.evrogen.com).

Mycorrhizal colonization

Rhizophagus irregularis (BEG144, International Bank of Glomeromycota (Dijon, France)) was kindly provided by Prof. Dirk Redecker as a soil–root-based inoculum from onion (Allium cepa L.) pot cultures. To obtain mycorrhizal pea plants, pea seedlings were placed in a nurse pot system with chives (Allium schoenoprasum L.) as nurse plants and grown in a growth chamber under conditions as previously described (Shtark et al., 2016). A mineral substrate, silica-rich marl, was used as the growth substrate. Mycorrhizal colonization was determined according to (Trouvelot, Kough & Gianinazzi-Pearson, 1986). Three parameters were considered: M%—intensity of internal colonization of the root system (reflects the proportion of the root length colonized by the fungus), A%—arbuscule abundance in mycorrhizal root fragments (characterizes the functional state of the fungus), V%—vesicle and/or spore abundance in mycorrhizal root fragments. 150 random root pieces (1 cm) were scored per replicate (not less than 750 cm/genotype).

Histochemical staining and microscopy

The root segments and nodules were collected and stained with X-Gluc (0.2 M Na₂PO₄, pH7.0; 0.5 M EDTA, pH8.0; 20 mM K ferricyanide; 20 mM K ferrocyanide; 20 mM X-Gluc; 0.01% Triton X-100) for GUS staining. Tissues were incubated in staining due overnight at 37 °C. In order to assess roots mycorrhizal colonization, roots were stained with Sheaffer Black Ink at room temperature as described previously (Vierheilig et al., 1998). Photographs were obtained using an Olympus BX51 microscope (Olympus Optical Co. Europa GmbH, Hamburg, Germany) equipped with a ColorView II digital camera and analySIS FIVE analytical software (Olympus Soft Imaging Solution GmbH, Hamburg, Germany).

Generation of pea composite plants and subsequent nodulation

In order to select for transformed hairy roots and quantify the efficiency of transformation, we introduced a plasmid carrying a fusion with the gusA/GFP reporter gene driven by the CaMV35S promoter and subsequently examined hairy roots and nodules for green fluorescent protein (GFP) or β-glucuronidase (GUS) staining.

To obtain the composite plants, young etiolated seedlings of the two cultivars Finale and Frisson were used for transformation with the ARqua1 or AR1193 strain of Agrobacterium rhizogenes carrying the gusA/GFP reporter construct. Using both A. rhizogenes strains, root transformation was induced with satisfactory efficiency, although differences were observed in number of induced roots (Table 1). Consistent with the findings of Clemow et al. (2011), we found that AR1193 was a more effective strain for hairy root transformation. In the procedure used in the present study, the plants were incubated in containers filled with Jensen medium placed within ventilated plastic box (Fig. 1A). Seedlings with one to two internodes are quite susceptible to transformation and were freshly sectioned in the hypocotyl region to remove the remainder of the root. In our transformation protocol, the wounded root tip of each seedling was touched to growing on a plate A. rhizogenes containing a plasmid that harbored the gusA/GFP reporter. Following transformation, the pea seedlings were incubated in jars and the root tips were maintained under high-humidity conditions (Fig. 1B).

After 10–14 days, calli started to develop at the wound sites in seedling of the cultivar Frisson (Fig. 1C), whereas the initiation of callus development was slightly more prolonged (16–18 days) for seedlings of the Finale cultivar. Approximately 70%–80% of the wound sites gave rise to callus formation and the subsequent development of transformed roots. At this stage, a number of primary roots appeared (Fig. 1C). These primary roots were removed prior to antibiotic treatment in order to stimulate development of the transformed roots. We found that incubation on an antibiotic-containing medium prevented excessive growth of A. rhizogenes and the development of root abnormalities (Fig. 1D). After incubation, the plants were subsequently transferred to pots saturated with a low-nitrate nutrient solution to prevent nitrogen starvation (Figs. 2 and 3A). Since the seedlings were cultivated under axenic conditions, they may also be used to obtain the cultivated hairy roots at this stage.

After transferring to pots and cultivating for a few days, the plants were inoculated with R. leguminosarum bv. viciae RIAM1026 and we observed that root nodules generally appeared after 2–3 weeks. In order to maintain high humidity conditions after transfer, we placed the pots into large plastic packages in which they were retained for at least 1 week, because it was easy to ventilate the plants during cultivation (Fig. 3A). After 2–3 weeks, we were able to visualize the emergence of nodules on transformed roots using an epifluorescence stereo microscope (Fig. 3B). We found that the efficiency of nodule formation varied depending on the cultivar used, with cv. Finale showing a high level of nodule formation, whereas comparatively fewer nodules developed on the transformed roots of cv. Frisson plants. GFP staining was used as a simple marker to score for transformed roots 3 weeks after infection with R. leguminosarum. We found that approximately 70% of the inoculated plants had at least one transformed root, and that composite plants had an average of two to three such roots, thereby indicating the high frequency of transformation achieved using this protocol, which will greatly facilitate studies involving gene function analysis.

Studying the possibility of mycorrhization for transformed roots in pea

In the present study, we also developed approaches for inoculation of transgenic hairy roots with the AM fungus R. irregularis using a nurse-plant system. As nurse plants, we used four young onion seedlings grown in pots containing Hoagland medium-saturated substrate for 4 weeks. After cultivation on antibiotic-containing medium, the transformed pea seedlings were transferred to the pots containing these nurse plants.

In order to maintain the high humidity conditions after transfer, we placed the pots into large plastic packages for at least 1 week, similar to the procedure used for rhizobial inoculation (Fig. 4A). After 4 weeks of growth, we assessed the efficiency of symbiosis development with AM fungi and detected highly effective inoculation (Figs. 4B and 4C). Expression levels of marker typically associated with AM fungus symbiosis development, namely, the PT4 gene encoding the mycorrhiza-inducible inorganic phosphate transporter, confirmed induction of the symbiotic association (Fig. 4D). These findings indicate that the protocol we describe here may be useful for the analysis of another type of symbiosis in composite pea plants with transformed roots.