Kee-Won Yu

Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O2−), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 μM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O2− accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O2− stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.

Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng (Eleuterococcus senticosus)

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation during somatic embryogenesis in Eleuterococcus senticosus, using two methods to evaluate DNA methylation rates: (1) direct determination of 5-methyl-deoxycytidine (5mdC) amounts in genomic DNA by HPLC separation and quantification of nucleosides and (2) methylation-sensitive amplification polymorphism (MSAP) technique. The tissue assayed include embryogenic and non-embryogenic callus. Leaf segments were cultured on modified Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Opaque and friable embryogenic callus formed on modified MS medium with 2.26 μM 2,4-D. Initiation and development of somatic embryos occurred in the same medium containing 2.26 μM 2,4-D. Non-embryogenic callus failed to regenerate even after prolonged maintenance and subculture on to the same media. Dark treatment significantly increased the number of mature somatic embryos. HPLC analyses on genomic DNA from embryogenic and non-embryogenic callus showed that global DNA methylation rates were significantly lowered in embryogenic calli. Similarly, 16.99% of 5′-CCGG-3 sites in the genome of non-embryogenic callus were cytosine methylated, where as 11.20% were methylated in case of embryogenic callus tissue, as detected by MSAP technique. Hypermethylation of DNA in non-embryogenic callus compared with embryogenic callus reflects the marked expression of this molecular feature, which may well contribute to the developmental gene expression.

Improvement of ginsenoside production by jasmonic acid and some other elicitors in hairy root culture of ginseng (Panax ginseng C. A. Meyer).

SummaryHairy root cultures of Panax ginseng, established after the infection of root sections with Agrobacterium rhizogenes KCTC 2703, were cultured in phytohormone-free Murashige and Skoog (MS) liquid medium containing different concentrations of jasmonic acid and some other elicitors, in order to promote ginsenoside accumulation. Jasmonic acid in the range 1.0−5.0 mg l−1 (4.8–23.8 μM) strongly improved total ginsenoside production in ginseng hairy roots. Peptone (300 mg l−1) also showed some effect on ginsenoside improvement; however its effect was much weaker than that of jasmonic acid. Ginsenoside content and productivity were 58.65 and 504.39 mg g−1, respectively. The Rb group of ginsenoside content was increased remarkably by jasmonic acid, while Rg group ginsenoside content changed only slightly compared to controls. However, jasmonic acid also strongly inhibited ginseng hairy root growth.

Effects of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng (Panax ginseng C. A. Meyer)

We investigated the effects on ginseng adventitious root growth and ginsenoside production when macro-element concentrations and nitrogen source were manipulated in the culture media. Biomass growth was greatest in the medium supplemented with 0.5-strength NH4PO3, whereas ginsenoside accumulation was highest (9.90 mg g-1 DW) in the absence of NH4PO3. At levels of 1.0-strength KNO3, root growth was maximum, but a 2.0 strength of KNO3 led to the greatest ginsenoside content (9.85 mg g-l). High concentrations of MgSO4 were most favorable for both root growth and ginsenoside accumulation (up to 8.89 mg g-1 DW). Root growth and ginsenoside content also increased in proportion to the concentration of CaCI2 in the medium, with the greatest accumulation of ginsenoside (8.91 mg g-1 DW) occurring at a 2.0 strength. The NH4/NO3-- ratio also influenced adventitious root growth and ginsenoside production; both parameters were greater when the NO3- concentration was higher than that of NH4+. Maximum root growth was achieved at an NH4+/NO3- ratio of 7.19/18.50, while ginsenoside production was greatest (83.37 mg L-1) when NO3- was used as the sole N source.

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm3 of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm−3 indolebutyric acid, 0.5 mg dm−3 kinetin and 30 g dm−3 sucrose. A 1 % CO2 supply was found beneficial for the production of cell mass; however, increasing CO2 concentration to 2.5 and 5 % decreased the biomass accumulation. CO2 enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO2 supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.

Effects of different irradiances on the photosynthetic process during ex-vitro acclimation of Anoectochilus plantlets

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 µmol(photon) m−2 s−1], intermediate irradiance, II [180 µmol(photon) m−2 s−1], and high irradiance, HI [300 µmol(photon) m−2 s−1] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (PN) and transpiration (E), stomatal conductance (gs), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (ΦPS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, PN, E, gs, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (Fv/Fm), probability of an absorbed photon reaching an open PS2 reaction centre (Fv′/Fm′), ETR, ΦPS2, and energy efficiency of CO2 fixation (ΦCO2/ΦPS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.

Plant Regeneration from Adventitious Roots of Rehmannia glutinosa Liboschitz and Bioreactor Culture

This experiment was carried out to develop rapid mass propagation via shoot organogenesis system from adventitious roots of Rehmannia glutinosa. The induction of adventitious roots from leaf explants was most favorable to MS solid medium supplemented with 2mg/L IBA. However, the growth of adventitious roots was highest when they were cultured on 1/3 strength MS liquid medium supplemented with 2mg/L IBA. When the adventitious roots were grown in 10L bioreactor, 10g roots as initial inoculum was increased to 225g after 6 weeks of culture. The harvested roots were cultured onto solid medium to induce plant regeneration. The optimal adventitious shoot formation was observed on MS medium supplemented with 2mg/L BA. Rooting of individual shoots was induced after transfer to half strength MS medium without growth regulators. Plantlets after acclimatization were successfully transplanted in the field and no phenotypic variation was observed among them.