Introduction

Aerial parts and root bark extracts of Bonellia macrocarpa possess antimicrobial, cytotoxic and antiproliferative activities against various cancer cell lines (Vila-Luna et al. 2017). Bonediol cannot be commercially produced because B. macrocarpa is a wild plant and there is no a protocol for organ nor tissue culture. Hairy root cultures have been investigated due to its potential to produce the valuable metabolites that are present in wild type roots (Guillon et al. 2006). The aim of this protocol is to describe the establishment of hairy root of B. macrocarpa induced by infection with Agrobacterium rhizogenes ATCC15834/pTDT and bonediol production in transformed root cultures.

Methods

Seedling growth (6–10 weeks)

  1. 1.

    Wash 50 seeds in a laminar air flow cabinet with liquid soap for 15 min.

  2. 2.

    Rinse the seeds three times in sterile water.

  3. 3.

    Add 200 mL of 70% ethanol for 3 min, discard the ethanol and repeat step 2.

  4. 4.

    Add 200 ml of 30% sodium hypochlorite for 20 min, discard supernatant and repeat step 2. CRITICAL STEP (CS): make the solution immediately before the experiment.

  5. 5.

    Incubate seeds on MS with 30 g/L sucrose, 5 mg/L gibberellic acid and 2.5 g/L Phytagel ®, pH 5.6.

  6. 6.

    Place seeds in a dark growth chamber at 25 ± 2 °C for 10 weeks, then transfer to continuous light. CS: darkness stimulates B. macrocarpa germination.

Agrobacterium rhizogenes growth (2 days)

  1. 1.

    Inoculate A. rhizogenes ATCC15834/pTDT in 20 mL of YMB medium 28 °C, 180 rpm. CS: always use an inoculum directly from a glycerol stock.

  2. 2.

    Incubate 24 h in darkness at 28 °C, 180 rpm. CS: the light affects motility and infectivity of Agrobacterium.

  3. 3.

    Subculture 1 mL of bacterial suspension (step 2) in 20 mL YMB and add 200 µM acetosyringone, incubate in darkness 8 h at 28 °C 180 rpm for Agrobacterium-mediated transformation. Culture until it reaches OD600 0.4. CS: 100 mM acetosyringone solution prepared with DMSO and sterilized by filtration. Acetosyringone is termolabile.

Agrobacterium rhizogenes maintenance by criococonservacion (1 day)

  1. 1.

    Place 7 mL of the grown culture [see step 1 in “Agrobacterium rhizogenes growth (2 days)”] with 3 mL of glycerol in 300 mL sterile glass jar. Mix and aliquot 500 µL in Eppendorf tubes. CS: place them in liquid nitrogen and store immediately at − 80 °C.

Induction of hairy roots by Agrobacterium rhizogenes-mediated transformation (7–11 weeks)

  1. 1.

    Hypocotyls, roots and cotyledons from 10 week-old seedlings are harvested in order to provide explants for Agrobacterium transformation. CS: explants superficially and longitudinally wounded with a scalpel promote infection of Agrobacterium.

  2. 2.

    Place explants in a 300 mL sterile glass jar and add 20 mL of bacterial culture [see step 1 in “Agrobacterium rhizogenes growth (2 days)”], immediately place in a glass jar in desiccator and apply vacuum for 10 min. CS: shorter or longer time could affect the frequency of transformation.

  3. 3.

    Transfer the infected explants on MS with 30 g/L sucrose, 200 mg/L glutamine, 200 µM acetosyringone and 2.5 g/L Phytagel, pH 5.6. CS: make sure of accurate sterilization conditions because the glutamine and acetosyringone could be loss.

  4. 4.

    Culture explants in a dark growth chamber at 25 ± 2 °C for 3 days.

  5. 5.

    After 3-days of cultivation, wash the explants with liquid MS with 250 mg/L cefotaxime. CS: be sure to decrease the bacterial load to avoid hypersensitive response in explants.

  6. 6.

    Transfer the disinfected explants on MS with 30 g/L sucrose, 200 mg/L glutamine, 200 mg/L cefotaxime and 2.5 g/L Phytagel, pH 5.6. CS: transfer the explants to fresh medium every 20 days.

  7. 7.

    Culture transformed explants in a growth chamber under darkness at 25 ± 2 °C. CS: keep explants under darkness to avoid oxidative stress.

Hairy root culture (10 weeks)

  1. 1.

    Place emerged roots of 3 cm long from infected explants in 25 mL liquid MS with 250 mg/L cefotaxime, pH 5.6. CS: transfer the roots to fresh medium every 10 days to induce hairy root growth and eliminate the residual bacteria.

  2. 2.

    Culture roots at 25 ± 2 °C and 100 rpm under continuous light.

Verification of the transgenic nature of hairy roots (2–3 h)

  1. 1.

    Finely cut one sample of cultivated root (1–2 mm thick). CS: use a microtome.

  2. 2.

    Place sample on a slide, add one drop of glycerol.

  3. 3.

    Slide and excite with a single beam excitation at 488 nm and emission at 520 nm with a confocal laser scanning microscope. Use a negative tissue specimen as control of autofluorescence.

Gene integration analysis (6–8 h)

  1. 1.

    Isolate DNA of A. rhizogenes, uninfected and infected roots according Kit Termo Scientific Phire Plant Direct ®. CS: strictly follow the manufacture’s indications.

  2. 2.

    For PCR reactions, use 1 µL of DNA and 10 µL 2X Phire Plant Direct PCR Master Mix, 0.5 µM of each primer (Primers for rolC gene: F5′-TGTGACAAGCAGCGATGAGC-3′ and R5′-GATTGCAAACTTGCACTCGC-3′; and for VirD 2 gene: F5′ATGCCCGATCGAGCTCAAGT-3′ and R5′-CCTGACCCAAACATCTCGGCTGCCCA-3′), and sterile water to a final volume of 20 µL.

  3. 3.

    Run PCR for rolC, initial denaturation at 98 °C for 5 min, 40 cycles of amplification (98 °C 5 s, 54.8 °C 5 s and 72 °C 20 s) and 72 °C for 1 min; for virD 2 , follow the same program but annealing temperature at 56 °C.

  4. 4.

    Visualize by gel electrophoresis stained with SYBR Green. CAUTION: SYBR Green is carcinogenic.

Identification and quantification of bonediol (3 days)

  1. 1.

    Dry hairy roots of 10 weeks old in a freezer dryer.

  2. 2.

    Macerate dried hairy roots (3 g) at room temperature with 450 mL methanol for 36 h (Caamal-Fuentes et al. 2011). CS: macerate every 12 h until reaching 450 mL methanol.

  3. 3.

    Evaporate the methanol with a rotary evaporator at 45 ± 1 °C and with vacuum; and resuspend dry extract in 10 mL methanol grade HPLC. CS: keep the extract in refrigeration.

  4. 4.

    For the standard curve, prepare stock standard solution of 1 mg of bonediol/mL in methanol; and prepare a series of standard solutions of 0.05, 0.1, 0.3, 0.5, 0.7 and 0.9 mg/mL.

  5. 5.

    Quantify bonediol with a Model Flexar HPLC and inject samples into a Zorbax ODS column (5 µm x 250 mm x 4.6 mm).

  6. 6.

    Use deionized water added with 0.05% of formic acid (A) and acetonitrile with 0.05% formic acid 60/40 (v/v) as the mobile phases (B). Start with 30% A, change from 30 to 5% A in 5 min and constant 5% A for 15 min more at a flow rate of 0.8 mL/min. Detection is carried out at 260 nm. 20 µl of the sample is injected and the column is maintained at room temperature (23 °C). CS: a post-run of 1 min of 100% B for cleaner the column between analyses.

Results and discussion

Different explants such as cotyledons, hypocotyls and roots of plantlets were used for hairy root induction in B. macrocarpa. The emerged roots were visible 4 weeks after inoculation. No roots emerged in uninfected control explants. The transformation frequency depends of the type of explant, 7.14% cotyledons, 24.99% hypocotyls and 10.71% roots. Low transformation frequency could be due to hypersensitive response because is the first defence responses of plants, and generally characterized by a rapid and localized cell death around the site of infection, due to the accumulation of antimicrobial agents or the production of reactive oxygen species (Pitzschke 2013).

The transgenic nature of the roots emerging was confirmed trough tomato protein (TDT). Infected explants with A. rhizogenes pTDT showed red fluorescence, while that red fluorescence was not detected in control (Fig. 1a).

Fig. 1
figure 1

Hairy roots of B. macrocarpa induced by A. rhizogenes ATCC15834 pTDT. Induction of the hairy roots from cotyledons of B. macrocarpa. a, 4 weeks old hairy roots; b, 10 weeks old hairy roots without plant growth. Quantification of bonediol in the methanol extracts of hairy roots of B. macrocarpa by HPLC. c, external standard of bonediol used for the identification; d, methanolic extract of culture of hairy roots after 10 weeks of culture. Light and fluorescence microscopy of hairy roots of B. macrocarpa. e and g, root explant from a hairy root; f and h, root explant form plantlet as negative control. PCR analysis of hairy roots of B. macrocarpa. i Lane 1, molecular weight marker; lanes 2 and 5, amplification of A. rhizogenes; lanes 3 and 6, transformed root; lanes 4 and 7, untransformed root; lanes 2–4 amplification with rolC primers; lanes 5–7, amplification with virD 2 primers

Full size image

Bonediol was produced in the cultures of transformed roots. In the extract obtained with 10-week culture samples, 2.78 mg bonediol/g dried weight of transformed root were quantified. Presence of bonediol was identified after 7 weeks (Fig. 1c, d). These results suggest the possibility of using massive cultivation of hairy roots of B. macrocarpa for the biosynthesis of this metabolite, because the bonediol production is higher in hairy roots that in plants. For troubleshooting advice see Table 1.

Table 1 Troubleshooting for bonediol production in Bonellia macrocarpa hairy root culture
Full size table

Both transformed root from B. macrocarpa and A. rhizogenes contain rolC gene but it was not detected in the untransformed root. Amplification of virD 2 gen was only detected in A. rhizogenes (Fig. 1b). Light and fluorescence microscopy of hairy roots of B. macrocarpa were presented in Fig. 1e, g. Root explant from a hairy root (Fig. 1f) and root explant form plantlet as negative control (Fig. 1h). PCR analysis of hairy roots of B. macrocarpa (Fig. 1i).