Exogenous glycine betaine alleviates water-deficit stress in Indian pennywort (Centella asiatica) under greenhouse conditions.

Centella asiatica (Indian pennywort) is a green leafy vegetable containing centelloside' (triterpenoid), a key phytochemical component in traditional medicine. Being a glycophytic species, they exhibit decline in growth performance and yield traits when subjected to water-deficit (WD) conditions. Glycine betaine (GB) is a low molecular-weight organic metabolite that plays a crucial role in abiotic stress conditions in higher plants. The objective of this study was to investigate the potential of GB in alleviating water-deficit stress (in terms of morphological and physiological responses) in two different genotypes of Indian pennywort, "Nakhon Pathom" (NP; high centelloside-yielding genotype) and "Pathum Thani" (PT; low centelloside-yielding genotype). The genotypes of Indian pennywort were propagated by stolon cutting and transplanted into plastic bags containing 2 kg of garden soil. At the flower-initiation stage (30 days after transplantation), uniform plant material was treated exogenously with 0 (control), 25, and 50 mM GB at 100 mL per plant (one-time foliar spray) and then divided into two groups, 1) well watered (WW; irrigated daily with 400 mL fresh water; 98% field capacity) and 2) water deficit (WD; withheld water for 14 days; 72% field capacity). Foliar application of GB (25 mM) significantly improved leaf osmotic potential in NP under WD conditions via osmotic adjustment by free proline and fructose. Differences in leaf temperature (Tleaf) between WD and WW in NP were maximized (+ 1.93 °C) and the gap of Tleaf was reduced in the case of 25-50 mM GB application. Similarly, crop water stress index (CWSI) in NP and PT plants under WD condition was significantly increased by 1.95- and 1.86-fold over the control, respectively; however, it was significantly decreased by exogenous GB application. Increasing Tleaf and CWSI in drought-stressed plants was closely related to stomatal closure, leading to reduced gas exchange parameters, i.e., stomatal conductance (gs), transpiration rate (E), net photosynthetic rate (Pn), and intercellular CO2 concentration (Ci), and consequently decreased plant biomass and total centelloside yield. Overall physiological, morphological, and secondary metabolite traits were enhanced in NP under WD conditions using 25 mM GB exogenous application compared with the control. The study highlights the significance of GB in Indian pennywort production under limited water irrigation (water deficit) with higher vegetable yield and phytochemical stabilization.

Effects of methyl jasmonate on the growth and triterpenoid production of diploid and tetraploid Centella asiatica (L.) Urb. hairy root cultures.

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

Chlorogenic Acids Biosynthesis in Centella asiatica Cells Is not Stimulated by Salicylic Acid Manipulation.

Exogenous application of synthetic and natural elicitors of plant defence has been shown to result in mass production of secondary metabolites with nutraceuticals properties in cultured cells. In particular, salicylic acid (SA) treatment has been reported to induce the production of phenylpropanoids, including cinnamic acid derivatives bound to quinic acid (chlorogenic acids). Centella asiatica is an important medicinal plant with several therapeutic properties owing to its wide spectrum of secondary metabolites. We investigated the effect of SA on C. asiatica cells by monitoring perturbation of chlorogenic acids in particular. Different concentrations of SA were used to treat C. asiatica cells, and extracts from both treated and untreated cells were analysed using an optimised UHPLC-QTOF-MS/MS method. Semi-targeted multivariate data analyses with the aid of principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA) revealed a concentration-dependent metabolic response. Surprisingly, a range of chlorogenic acid derivatives were found to be downregulated as a consequence of SA treatment. Moreover, irbic acid (3,5-O-dicaffeoyl-4-O-malonilquinic acid) was found to be a dominant CGA in C. asiatica cells, although the SA treatment also had a negative effect on its concentration. Overall SA treatment was found to be an ineffective elicitor of CGA production in cultured C. asiatica cells.

Growth and asiaticoside production in multiple shoot cultures of a medicinal herb, Centella asiatica (L.) Urban, under the influence of nutrient manipulations.

Growth and in vitro asiaticoside accumulation in multiple shoot cultures of Centella asiatica (L.) Urban was studied as a function of nutrient manipulations in the culture media. Shoot cultures raised in liquid Murashige and Skoog medium supplemented with 2.5 mg/l kinetin attained a growth index (GI) of 6.06 along with the highest asiaticoside content of 3.8 mg/g dry weight on the 35th day of the culture cycle. The shoot growth and asiaticoside accumulation were found to be influenced by the relative proportions of NH(4)(+)-N:NO(3)(-)-N or Cu(2+) concentration in the medium. Asiaticoside content in shoots increased from 5.3 to 8.9 and 8.7 mg/g dry weight when total nitrogen concentration of 60 mM in the control medium was reduced to 50 and 40 mM with a corresponding change in NH(4) (+):NO(3)(-) ratio from 20:40 to 20:30 or 20:20, respectively. Total nitrogen level higher than 60 mM drastically reduced the asiaticoside concentration in these in vitro shoot cultures. Medium devoid of Cu(2+) significantly favored higher asiaticoside accumulation in the cultured tissue (7.05 mg/g dry weight) along with an improved biomass production (GI = 7.7) when compared with shoots reared on the control medium with 0.10 µM Cu(2+) (GI = 5.8; asiaticoside content = 4.4 mg/g dry weight). Carbohydrate enrichment of the medium by increasing the sucrose concentration from 3.0 to 5.0 or 7.0% was also beneficial for biomass and asiaticoside production with GI = 17.1 and 16.9 and asiaticoside content = 7.2 and 5.2 mg/g dry weight, respectively, in comparison to control cultures maintained on medium containing 3.0% sucrose. The procedure described here provides a viable production platform for generating clean and quality material from Centella with high bioactive content.

Flavonoid and leaf gas exchange responses of Centella asiatica to acute gamma irradiation and carbon dioxide enrichment under controlled environment conditions.

The study was couducted to investigate the effects of gamma irradiation and CO2 on flavonoid content and leaf gas exchange in C.asiatica. For flavonoid determination, the design was a split split plot based on Randomized Complete Block Design (RCBD). For other parameters, the designs were split plots. Statistical tests revealed significant differences in flavonoid contents of Centella asiatica leaves between different growth stages and various CO2 treatments. CO2 400, G20 (400 = ambient CO2; G20 = Plants exposed to 20 Gy) showed 82.90% higher total flavonoid content (TFC) in the 5th week than CO2 400 as control at its best harvest time (4th week). Increasing the concentration of CO2 from 400 to 800 µmol/mol had significant effects on TFC and harvesting time. In fact, 800 µmol/mol resulted in 171.1% and 66.62% increases in TFC for control and irradiated plants, respectively. Moreover, increasing CO2 concentration reduced the harvesting time to three and four weeks for control and irradiated plants, respectively. Enhancing CO2 to 800 µmol/mol resulted in a 193.30% (CO2 800) increase in leaf biomass compared to 400 µmol/mol and 226.34% enhancement in irradiated plants (CO2 800, G20) [800 = Ambient CO2; G20 = Plants exposed to 20 Gy] than CO2 400, G20. In addition, the CO2 800, G20 had the highest amount of flavonoid*biomass in the 4th week. The results of this study indicated that all elevated CO2 treatments had higher PN than the ambient ones. The findings showed that when CO2 level increased from 400 to 800 µmol/mol, stomatal conductance, leaf intercellular CO2 and transpiration rate had the tendency to decrease. However, water use efficiency increased in response to elevated CO2 concentration. Returning to the findings of this study, it is now possible to state that the proposed method (combined CO2 and gamma irradiation) has the potential to increase the product value by reducing the time to harvest, increasing the yield per unit area via boosting photosynthesis capacity, as well as increasing biochemicals (flavonoids) per gram DM.

Mutagenic potential of water from Pelotas Creek in Rio Grande do Sul, Brazil.

Water resource degradation is one of mankind's greatest worries, as it causes direct and indirect damage to the associated biota. We initiated a water monitoring study in Pelotas Creek in 2003 in order to assess the mutagenic effect of the creek's waters. Allium cepa cells exposed to water samples and a chronically exposed macrophyte were analyzed, through evaluation of the mitotic index, mitotic anomalies, interphase anomalies, and total anomalies. Five points were chosen along the lower course of Pelotas Creek, from which water samples and floating pennywort (Hydrocotyle ranunculoides, Apiaceae) were collected in 2006 and 2007. The enteric bacterium Escherichia coli was found at all sampling points; in the physical-chemical analysis, a few variables exceeded permitted limits, pH (from 6 to 9), chloride (250 mg/L), hardness (from 10 to 200 mg CaCO(3)/L), and conductivity (100 microOmega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.Omega/cm). There was an increased number of cytogenetic anomalies in exposed A. cepa cells and in the pennywort in 2006 relative to 2007, which may be explained by the increased rainfall, which was three times greater in 2007 at some stations than in 2006.

Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate.

Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

Comparative analysis of active constituents in Centella asiatica samples from Madagascar: application for ex situ conservation and clonal propagation.

A comparative quantitative analysis of the active triterpenoids in Centella asiatica samples collected in different locations in Madagascar was carried out to evaluate the natural variability in triterpenoid content and to select elite samples for further ex situ germplasm conservation and clonal propagation. The highest asiaticoside content (6.42%) was measured in samples collected in Mangoro region. In vitro propagation of C. asiatica was successfully achieved in hormone-free medium. Although lower asiaticoside content was detected in 8-week-old vitro plants, the Mangoro sample still showed the highest content in this triterpenoid constituent (1.78%). Acetoxycentellynol, a C(15)-polyacetylene, was found to be accumulated up to 18 times more in in vitro plants as compared to plant material collected in situ.

The effect of methyl jasmonate on triterpene and sterol metabolisms of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants.

Considering that exogenously applied methyl jasmonate can enhance secondary metabolite production in a variety of plant species and that 2,3-oxidosqualene is a common precursor of triterpenes and sterols in plants, we have studied Centella asiatica and Galphimia glauca (both synthesizing triterpenoid secondary compounds) and Ruscus aculeatus (which synthesizes steroidal secondary compounds) for their growth rate and content of free sterols and respective secondary compounds, after culturing with or without 100 microM methyl jasmonate. Our results show that elicited plantlets of G. glauca and to a higher degree C. asiatica (up to 152-times more) increased their content of triterpenoids directly synthesized from 2,3-oxidosqualene (ursane saponins and nor-seco-friedelane galphimines, respectively) at the same time as growth decreased. In contrast, the free sterol content of C. asiatica decreased notably, and remained practically unaltered in G. glauca. However, in the case of R. aculeatus, which synthesizes steroidal saponins (mainly spirostane type) indirectly from 2,3-oxidosqualene after the latter is converted to the plant phytosterol-precursor cycloartenol, while the growth rate and free sterol content clearly decreased, the spirostane saponine content was virtually unchanged (aerial part) or somewhat lower (roots) in presence of the same elicitor concentration. Our results suggest that while methyl jasmonate may be used as an inducer of enzymes involved in the triterpenoid synthesis downstream from 2,3-oxidosqualene in both C. asiatica and G. glauca plantlets, in those of C. asiatica and R. aculeatus it inhibited the enzymes involved in sterol synthesis downstream from cycloartenol.

Toxicity and accumulation of lead and chromium in Hydrocotyle umbellata.

The aquatic plant, Hydrocotyle umbellata, was studied for its toxicity and accumulation of lead (Pb) and chromium (Cr) in a synthetic solution. Plants were cultured in a modified Hoagland's nutrient solutions supplemented with 20, 40, 60, 80, and 100 mg Pb/l as lead nitrate [Pb(NO3)2] and 2, 4, 6, 8, and 10 mg Cr/l as potassium dichromate (K2Cr2O7). They were separately harvested after 3, 6, 9, and 12 days. Plants exposed to Pb and Cr showed significant decreases in the biomass productivity and total chlorophyll content when the exposure time and metal concentration were increased. The accumulation of Pb and Cr in the plants was significantly increased, but it was not linear with the exposure time and metal concentration. Both metals were accumulated higher in the roots than in the shoots. The bioconcentration factor of Pb was higher than that of Cr at the same exposure time, indicating a higher accumulation potential of Pb than Cr in H. umbellata. Toxicity symptoms of both metals showed a reduction in the production of new plantlets, withering of petioles, and change in color of roots from light green to dark brown. Pb caused leaf chlorosis, whereas Cr caused leaf necrosis. The toxicity symptoms increased when the exposure time and metal concentration were increased.

Stimulation of asiaticoside accumulation in the whole plant cultures of Centella asiatica (L.) urban by elicitors.

The effects of a number of different elicitors on asiaticoside production in whole plant cultures of Centella asiatica were studied, including yeast extract, CdCl(2), CuCl(2) and methyl jasmonate (MJ). Only MJ and yeast extract stimulated asiaticoside production--1.53 and 1.41-fold, respectively. Maximum asiaticoside production was achieved following treatment with 0.1 mM MJ (116.8 mg/l). The highest asiaticoside production (342.72 mg/l) was obtained after 36 days of elicitation in cultures treated with 0.1 mM MJ and 0.025 mg/l 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea (TDZ). Interestingly, MJ not only stimulated the production of asiaticoside but also had an important role in the senescence of C. asiatica. Although asiaticoside content did not change when TDZ was added to medium containing an elicitor, TDZ did increase shoot growth of C. asiatica. We discuss the interactive roles of MJ and TDZ in secondary metabolic production and biomass in whole plants of C. asiatica.