| Plant latin name | Glycyrrhiza uralensis Fisher |
| Literature code | Glycyrrhiza_uralensis-Ref-3 |
| Reference | Kojoma M et al., Plant Biotechnology 27: 59–66 (2010) |
| Summary | Dried roots and stolons of licorice plants (Glycyrrhiza uralensis) are among the most important drugs
(Glycyrrhizae radix) in traditional oriental medicine; they are also important commercial products used worldwide in sweetening and flavoring. Here, we describe the establishment of an in vitro stolon culture system for G. uralensis. Stolon formation was induced in single node stems (with axillary buds) grown in Murashige and Skoog (MS) liquid culture medium, supplemented with 0.01μM α -naphthaleneacetic acid (NAA). Stolons were cultured at 26°C in the dark on a rotary shaker, gyrating at 100 rpm. The same NAA concentration produced the highest rates of stolon proliferation (6.58-fold in 4 weeks). A 6% sucrose concentration also enhanced stolon proliferation (6.34-fold in 4 weeks). GC/MS analysis confirmed the accumulation of small amounts of glycyrrhizin (14 μgg-1 dry weight) in cultured stolons. Interestingly, betulinic acid and oleanoic acid production in vitro were higher than in field-grown stolons. Adventitious root and shoot
regeneration from cultured stolons were readily achieved under illuminated conditions in MS medium containing 0.01mM of NAA and 0.2% gerlite. Regenerated plants produced glycyrrhizin (7,600 μgg-1 dry weight) in their roots. Our in vitro stolon culture system is suitable for studying glycyrrhizin biosynthesis and for rapid propagation of elite licorice clones. |
| Objectives | Establishment of in vitro stolon system for the propagation of G. uralensis and study of
glycyrrhizin and other secondary metabolite production |
| Materials | Seeds of G. uralensis (Accession No. 13905) were obtained from the agricultural research field of the Division of Hokkaido Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation (NIBIO), Japan |
| Explant | Seeds |
| Initial culture | Seeds were washed with tap water for 3 h and surface-sterilized in 70% (v/v) EtOH for 1 min and then in a 2% (v/v) sodium hypochlorite solution (10% commercial bleach), containing 0.02% (v/v) Tween 20 for 15 min, followed by three rinses in sterile water. Seeds were placed on Murashige and Skoog (MS) medium, containing 3% (w/v)
sucrose and solidified with 0.2% (w/v) gelrite (San-Ei Gen F.F.I., Inc. Osaka, Japan) in plastic tubes (ø 30×115 mm) containing 10 ml of medium. The cultures were incubated at 23℃ with 40μmol photons m-2s-1 of fluorescent light (16 h d-1). After one month of seed culture, one germinated plantlet was
selected for the propagation of clonal explants for all subsequent experiments, to exclude genetic differences among the seeds. Single-node cuttings from stem segments with axillary buds were cultured, essentially as described by Kohjyouma et al. (1995)(Glycyrrhyza _glabra-Rep-1). The medium consisted of MS containing 3% sucrose and 0.2% gelrite, supplemented with 0.1 μM α -naphthaleneacetic acid (NAA). |
| Shoot multiplication | To examine the effects of gibbelleric acid (GA3) and NAA on in vitro stolon induction, stem segments (1 cm) with axillary buds were cultured in 100 ml MS liquid medium containing 3% sucrose and GA3 (0, 0.01, 0.1, 1, and 10 μM) or NAA (0, 0.01, 0.1, 1, and 10 μM) (15 segments per polycarbonate culture bottle, ø80×102 mm) at 26℃ in the dark on a rotary shaker gyrating at 100 rpm. Segments were transferred to fresh medium (same composition) after two weeks in culture. After two further weeks (total four weeks) in culture, analyses were conducted. The highest frequency of stolon induction occurred using 0.01 μM NAA (40.0%), which also produced the longest stolon lengths after four weeks in culture (10.31±4.07 cm). Few cases of
induction or elongation of stolons using GA3 treatments were observed as well as abnormal stolon morphologies and
enlarged stolon buds (0.01μM GA3) and thin
stolons (1 and 10 μM).
To examine the effects of NAA on the proliferation of cultured stolon, the stolons (2–3 cm long, 10 segments/bottle, total 2 g fresh weight) were inoculated (as before) into MS liquid medium containing 3% sucrose and NAA (0, 0.01, 0.1, 1, and 10μM) in polycarbonate culture bottles and cultured as mentioned above. The segments were transferred to fresh medium (same composition) after two weeks in culture. After two further weeks (total four weeks) in culture, analyses were conducted. The largest effect response was obtained in medium supplemented with 0.01 μM NAA (12.94±0.40 g fresh weight/bottle). There was a 6.58-fold increase in proliferation per bottle after four weeks in culture. Under these conditions, stolons grew vigorously.
To examine the effects of sucrose on the proliferation of the cultured stolon, the stolons (2–3 cm long, ten segments/bottle, total 2 g fresh weight) were inoculated (as before) into MS liquid medium supplemented with sucrose (1, 3, 6, and 9%, without phytohormones) in polycarbonate culture bottles and cultured as mentioned above. The segments were transferred to fresh medium (same composition) after two
weeks in culture. After two further weeks (total four weeks) in culture, analyses were conducted. The largest effect was obtained in medium supplemented with 6% sucrose
(12.82±0.17 g fresh weight/bottle). Stolons proliferated 6.34-fold in four weeks of culture. Higher concentrations of sucrose reduced stolon growth. The stimulatory effect of sucrose on stolons was unaffected by the presence of 0.01μM NAA. |
| Rooting | To examine the effects of auxin on rooting and shoot regeneration from stolons, stolon segments (about 5 cm long), each with one bud primordium, were transferred to MS medium with 3% sucrose, 0.2% gelrite and 0.01 or 0.1μM of 2,4-D, IAA, IBA, or NAA in a polycarbonate culture vessel (80.5×80.5×100 mm, Technopot® Sumitomo Bakelite, Co. Ltd. Tokyo, Japan) containing 80 ml medium and cultured at 23℃, under 40μ mol photons m-2s-1 fluorescent lights (16 h d-1). After four weeks of culture, there was a high frequency of rooting in medium with NAA (0.01 and 0.1μM), IBA (0.01 and 0.1μM),
and without phytohormones ( 87%). Elongation of roots and shoots was also vigorous under these conditions. Generally shoot and root development was most vigorous in 0.01μM NAA. The continuing culture of plantlets in phytohormone-free medium resulted in a gradual loss of vigor in growth and shoot
development. Hence, medium supplemented with 0.01μM NAA was the most suitable
for the adventitious root formation and shoot
development required to achieve whole plant
regeneration. Cultured stolons from which
whole plants regenerated on solid medium retained a pale whitish or light-brown color similar to that of intact field-grown stolons. |
| Acclimation | After acclimation, regenerated plants were cultivated at 23℃,
under 40 μmol photons m-2s-1 fluorescent lights (16 h d-1). Regenerated plants were easily acclimated to pot cultivation after in vitro culture. |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | The regenerated plants grew vigorously and formed thickened roots similar to those of field plants. Glycyrrhizain in the roots of six-month-old regenerated plants was quantitatively analyzed by HPLC according to the procedures of The Japanese Pharmacopoeia. Regenerated plants contained glycyrrhizin in the
roots, (7,600 μgg-1 dry weight), despite the short cultivation period of 6 months. |
| Ingredients analyzed | Glycyrrhizin, triterpenoids
(oleanolic acid and betulinic acid), triterpenols (β-amyrin, α-amyrin, and lupeol), and sterols |
| Extraction | For glycyrrhizin in roots of regenerated plants: Roots were dried (50℃, 12 h) and ground to a fine powder. The dried root samples (ca. 200 mg) were extracted with 50% EtOH (5 ml) for 30 min at room temperature. These were then treated with ultrasonication for 10 min at room temperature. This operation was repeated twice, and the extracts were made up to a final volume of 10 ml. The extract (20μl) was subjected to HPLC analysis.
For triterpenoid and sterol in cultured stolons：Freeze-dried plant materials
were extracted twice with CHCl3-MeOH (1 : 1) and twice with MeOH-H2O (1: 1) at 80℃. Internal standards [25,26,26,26,27,27,27-2H7]cholesterol (98% D, Cambridge Isotope Laboratories, Inc., Andover, MA, USA), [3,28,28,28-2H4] β-amyrin, [28,28,28-2H3]α-amyrin, and [28,28,28-2H3] lupeol (Ohyama et al. 2007) were added to the extracts. The
extract was injected into a polyamide cartridge column (Discavery® DPA-6S, 500 mg, SUPELCO) and eluted successively with MeOH, 50% MeOH, H2O, and 1% NH3. The
MeOH eluent was dried in vacuo, and the residue was separated on silica gel preparative TLC plates and developed
twice using hexane–ethyl acetate (3 : 1), to yield triterpenol, sterol, oleanolic acid, and betulinic acid fractions. Each fraction
was trimethylsilylated with N-methyl-N-trimethylsilyltrifluoroacetamide at 80℃ for 30 min and analyzed using GC/MS. |
| Analitical methods | For glycyrrhizin in roots of regenerated plants: The HPLC system used consisted of an LC-2000 Plus system (JASCO, Tokyo, Japan) and a TSKgel ODS-80TS QA column (150×4.6
mm, TOSOH). The column temperature was maintained at 30℃. A solvent system consisting of 2.1% (v/v) acetic acid-
CH3CN (3 : 2) was used at a flow rate of 0.6 ml min-1. The eluent was monitored by absorption at 254 nm. A glycyrrhizin
standard was purchased from Tokyo Chemical Industry (Tokyo, Japan). Three regenerated plants were analyzed.
For triterpenoid and sterol in cultured stolons：Sterol and triterpenol quantifications were carried out using previously described methods (Ohyama et al. 2007).
Oleanolic acid and betulinic acid were quantified using standard calibration curves with coefficients of determination,
r2>0.999. The curves were constructed using the peak area ratio (m/z 482) of TMS derivatives. After drying, water and 1% NH3 eluents were analyzed using LC/MS; a mass spectrometer (QSTAR Pulsar, API)
connected to an HPLC (1100 series, Agilent Technologies) for identifications. For glycyrrhizin identification, LC/MS analyses
were performed with two solvent systems and with both negative and positive ion scans. For HPLC, SenshuPak ODS-II 25 cm×0.2 mm column with a flow rate of
0.2 ml min-1 were used. System 1: Eluent A was 0.1% (w/v) acetic acid in
acetonitrile; eluent B was 0.1% (w/v) acetic acid in water. After an 80% B hold for 2 min, the gradient was linear from 80% to 5% B in 10 min; this was followed by a 5% B hold for a further 8 min and a re-equilibration period of 5 min. The column temperature was 35℃. System 2 operated under the same
conditions as System 1, but with Eluent A being 0.1% (w/v) acetic acid in MeOH. Mass spectrometer settings were optimized using authentic glycyrrhizin in TOF/MS scan mode,
with electrospray ionization; the resulting ion, [M H] ([M H] ), was readily detected. Thus, product ion scan (MS/MS) analyses were performed by scanning at m/z 821.3 (823.3). Glycyrrhizin quantifications were calculated from the ratio of the peak area at m/z 821.3 from product ion scans using a calibration curve of authentic compound (with coefficient of determination: r2>0.999). |
| Notes | Ohyama K et al., Chem Pharm Bull, 55:1518–1521 (2007). |