Shoot Multiplication and Callus Induction of Labisia pumila var. alata as Influenced by Different Plant Growth Regulators Treatments and Its Polyphenolic Activities Compared with the Wild Plant.

This study aims to investigate whether the in vitro-cultured L. pumila var. alata has higher antioxidant activity than its wild plant. An 8-week-old L. pumila var. alata nodal segment and leaf explants were cultured onto Murashige and Skoog (MS) medium supplemented with various cytokinins (zeatin, kinetin, and 6-benzylaminopurine (BAP)) for shoot multiplication and auxins (2,4-dichlorophenoxyacetic acid (2,4-D) and picloram) for callus induction, respectively. The results showed that 2 mg/L zeatin produced the optimal results for shoot and leaf development, and 0.5 mg/L 2,4-D produced the highest callus induction results (60%). After this, 0.5 mg/L 2,4-D was combined with 0.25 mg/L cytokinins and supplemented to the MS medium. The optimal results for callus induction (100%) with yellowish to greenish and compact texture were obtained using 0.5 mg/L 2,4-D combined with 0.25 mg/L zeatin. Leaves obtained from in vitro plantlets and wild plants as well as callus were extracted and analyzed for their antioxidant activities (DPPH and FRAP methods) and polyphenolic properties (total flavonoid and total phenolic content). When compared with leaf extracts of in vitro plantlets and wild plants of L. pumila var. alata, the callus extract displayed significantly higher antioxidant activities and total phenolic and flavonoid content. Hence, callus culture potentially can be adapted for antioxidant and polyphenolic production to satisfy pharmaceutical and nutraceutical needs while conserving wild L. pumila var. alata.

Analysis of anatomical changes and cadmium distribution in Aegiceras corniculatum (L.) Blanco roots under cadmium stress.

Heavy metal stress changes the morphological and anatomical structure of plant organs. In this study, we determined the anatomical changes and Cd distribution in the roots of Aegiceras corniculatum (L.) Blanco (Black mangrove) under Cd stress. The results showed that Cd levels in A. corniculatum root tissues decreased in the following order: endodermis > pith > xylem > epidermis and exodermis > phloem > cortex. The endodermis secondary casparian strip replaces exodermis casparian strip and plays a role in the "retardation mechanism", which sort of compensates for the missing exodermis retardation effect. The xylem and pith both show high affinity for Cd and contain enriched Cd. This creates a low-Cd environment for phloem and protects the nutrient transport function of the vasculature against Cd toxicity. The present study provides new evidences suggesting that Cd regional enrichment and anatomical structure changes are an adaptive strategy of mangrove plants to HM tolerance.

Distribution correlations of cadmium to calcium, phosphorus, sodium and chloridion in mangrove Aegiceras corniculatum root tissues.

Nutriment distributions might influence Cd distribution and Cd tolerance in mangrove plant roots. To demonstrate this, Aegiceras corniculatum was stressed by Cd, and the distributions of Cd, Ca, P, Na and Cl in plant roots were detected with the aid of SEM-EDX. It was found that endodermis, pith and xylem were the predominant tissues for retardation and regional enrichment of Cd. Na and Cl distributions suggest a critical role of salt resistance tissues on Cd tolerance in roots. P participated in Cd retardation and regional enrichment of endodermis and xylem. P, Na, Cl and Ca distribution had a high correlation to that of Cd in roots. The synergetic accumulation between Ca and Cd could be a crucial mechanism for Cd tolerance in A. corniculatum roots. In conclusion, the research of Cd and nutriment distributions in A. corniculatum roots deepens the understanding on Cd tolerance in mangrove plants.

Response of phenolic metabolism to cadmium and phenanthrene and its influence on pollutant translocations in the mangrove plant Aegiceras corniculatum (L.) Blanco (Ac).

Polyphenolic compounds are abundant in mangrove plants, playing a pivotal role in the detoxification of pollutants extruded from surrounding environments into plant tissues. The present study aimed to examine the variations of phenolic compounds, namely total polyphenolics, soluble tannins, condensed tannins and lignin, in the mangrove plant Aegiceras corniculatum (L.) due to the presence of exogenous cadmium and phenanthrene and to explore the influence of phenolic metabolism on biological translocation of these pollutants from roots to leaves. After a 6-week exposure to cadmium and phenanthrene, significant accumulations of both pollutants were observed. All determined phenolic compounds in both leaves and roots at high dosage levels were enhanced compared to the uncontaminated plant. Elevations of polyphenols in both treatments are possibly a result of stimulation in the activity of phenylalanine ammonia-lyase (PAL) and the enrichment of soluble sugar. Additionally, a significantly positive dosage relationship between polyphenolic metabolism intensity and phenanthrene contamination levels was found, while the trend observed in cadmium treatment was weak since cadmium at high levels inhibited phenolic production. The enrichment of polyphenols led to a decline in the biological translocation of these pollutants from roots to leaves. The immobilization of pollutants in the plant roots is possibly linked to the adsorption potential of polyphenols. These results will improve the understanding of the tolerance of mangrove plants to exogenous pollutants and will guide the selection of plants in phytoremediation because of the variability of polyphenol concentrations among species.

Short-term enhancement effect of nitrogen addition on microbial degradation and plant uptake of polybrominated diphenyl ethers (PBDEs) in contaminated mangrove soil.

Effects of nitrogen (N) addition on the microbial degradation and uptake of a mixture of BDE-47 and -209 by Aegiceras corniculatum, a typical mangrove plant species were investigated. At the end of 3-month experiment, a significant dissipation of BDE-47 was observed in the planted soil, and this dissipation, particularly in rhizosphere soil, was significantly accelerated by the frequent addition of N in the form of ammonium chloride. The removal percentage of BDE-47 in the rhizosphere soil without N addition was 47.3% and increased to 58.2% with N. However, the unplanted soil only removed less than 25% BDE-47, irrespective to N supply. The N addition in planted treatments significantly increased soil N content, urease and dehydrogenase activities, and the abundances of total bacteria and dehalogenating bacteria, leading to more microbial degradation of BDE-47. The N addition also enhanced the root uptake and translocation of PBDEs to above-ground tissues of A. corniculatum. These results suggested that N addition could enhance the phytoremediation of BDE-47-contaminated soil within a short period of time. Different from BDE-47, BDE-209 in all contaminated soils was difficult to be removed due to its persistence and low bioavailability.

Cloning of the Aegiceras corniculatum class I chitinase gene (AcCHI I) and the response of AcCHI I mRNA expression to cadmium stress.

Chitinases in terrestrial plants have been reported these are involved in heavy metal tolerance/detoxification. This is the first attempt to reveal chitinase gene (AcCHI I) and its function on metal detoxification in mangroves Aegiceras corniculatum. RT-PCR and RACE techniques were used to clone AcCHI I, while real-time quantitative PCR was employed to assess AcCHI I mRNA expressions in response to Cadmium (Cd). The deduced AcCHI I protein consists of 316 amino acids, including a signal peptide region, a chitin-binding domain (CBD) and a catalytic domain. Protein homology modeling was performed to identify potential features in AcCHI I. The CBD structure of AcCHI I might be critical for metal tolerance/homeostasis of the plant. Clear tissue-specific differences in AcCHI I expression were detected, with higher transcript levels detected in leaves. Results demonstrated that a short duration of Cd exposure (e.g., 3 days) promoted AcCHI I expression in roots. Upregulated expression was also detected in leaves under 10 mg/kg Cd concentration stress. The present study demonstrates that AcCHI I may play an important role in Cd tolerance/homeostasis in the plant. Further studies of the AcCHI I protein, gene overexpression, the promoter and upstream regulation will be necessary for clarifying the functions of AcCHI I.

Allocation of secondary metabolites, photosynthetic capacity, and antioxidant activity of Kacip Fatimah (Labisia pumila Benth) in response to CO2 and light intensity.

A split plot 3 by 4 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites, soluble sugar, phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity, leaf gas exchange, chlorophyll content, antioxidant activity (DPPH), and lipid peroxidation under three levels of CO2 (400, 800, and 1200 µ mol/mol) and four levels of light intensity (225, 500, 625, and 900 µ mol/m(2)/s) over 15 weeks in Labisia pumila. The production of plant secondary metabolites, sugar, chlorophyll content, antioxidant activity, and malondialdehyde content was influenced by the interactions between CO2 and irradiance. The highest accumulation of secondary metabolites, sugar, maliondialdehyde, and DPPH activity was observed under CO2 at 1200 µ mol/mol + light intensity at 225 µ mol/m(2)/s. Meanwhile, at 400 µ mol/mol CO2 + 900 µ mol/m(2)/s light intensity the production of chlorophyll and maliondialdehyde content was the highest. As CO2 levels increased from 400 to 1200 µ mol/mol the photosynthesis, stomatal conductance, f v /f m (maximum efficiency of photosystem II), and PAL activity were enhanced. The production of secondary metabolites displayed a significant negative relationship with maliondialdehyde indicating lowered oxidative stress under high CO2 and low irradiance improved the production of plant secondary metabolites that simultaneously enhanced the antioxidant activity (DPPH), thus improving the medicinal value of Labisia pumila under this condition.

Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata.

OBJECTIVE: Labisia pumila var. alata, commonly known as 'Kacip Fatimah' or 'Selusuh Fatimah' in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. METHODS: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. RESULTS: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34 ± 19.55)% and (70.40 ± 14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00 ± 7.07)% and (77.78 ± 16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5 ± 5.0) and (30.0 ± 8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00 ± 0.00)%) that was obtained in 1 mg/L zeatin after (11.0 ± 2.8) d of culture. CONCLUSIONS: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.

Primary, secondary metabolites, photosynthetic capacity and antioxidant activity of the Malaysian Herb Kacip Fatimah (Labisia Pumila Benth) exposed to potassium fertilization under greenhouse conditions.

A randomized complete block design was used to characterize the relationship between production of total phenolics, flavonoids, ascorbic acid, carbohydrate content, leaf gas exchange, phenylalanine ammonia-lyase (PAL), soluble protein, invertase and antioxidant enzyme activities (ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) in Labisia pumila Benth var. alata under four levels of potassium fertilization experiments (0, 90, 180 and 270 kg K/ha) conducted for 12 weeks. It was found that the production of total phenolics, flavonoids, ascorbic acid and carbohydrate content was affected by the interaction between potassium fertilization and plant parts. As the potassium fertilization levels increased from 0 to 270 kg K/ha, the production of soluble protein and PAL activity increased steadily. At the highest potassium fertilization (270 kg K/ha) L. pumila exhibited significantly higher net photosynthesis (A), stomatal conductance (g(s)), intercellular CO(2) (C(i)), apparent quantum yield (ξ) and lower dark respiration rates (R(d)), compared to the other treatments. It was found that the production of total phenolics, flavonoids and ascorbic acid are also higher under 270 kg K/ha compared to 180, 90 and 0 kg K/ha. Furthermore, from the present study, the invertase activity was also found to be higher in 270 kg K/ha treatment. The antioxidant enzyme activities (APX, CAT and SOD) were lower under high potassium fertilization (270 kg K/ha) and have a significant negative correlation with total phenolics and flavonoid production. From this study, it was observed that the up-regulation of leaf gas exchange and downregulation of APX, CAT and SOD activities under high supplementation of potassium fertilizer enhanced the carbohydrate content that simultaneously increased the production of L. pumila secondary metabolites, thus increasing the health promoting effects of this plant.

Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolata.

UNLABELLED: A method has been developed to genetically transform the medicinal plant Maesa lanceolata. Initially, we tested conditions for transient expression of GFP-bearing constructs in agroinfiltrated leaves. Leaf tissues of M. lanceolata were infiltrated with Agrobacterium tumefaciens carrying a nuclear-targeted GFP construct to allow the quantification of the transformation efficiency. The number of transfected cells was depended on the bacterial density, bacterial strains, the co-cultivation time, and presence of acetosyringone. The transient transformation assay generated the highest ratio of transfected cells over non-transfected cells upon 5 days post-infiltration using A. tumefaciens strain LBA4404 at an OD600 = 1.0 in the presence of 100 µM acetosyringone and in the absence of a viral suppressor construct. In a second series of experiments we set up a stable transformation protocol that resulted in the regeneration of kanamycin-resistant plants expressing nuclear GFP. This transformation protocol will be used to introduce overexpression and RNAi constructs into M. lanceolata plants that may interfere with triterpenoid saponin biosynthesis. KEY MESSAGE: We have developed a transformation protocol for saponin producing Maesa lanceolata. Using the protocol reported here, now we are able to generate the tools for the modification of saponin production.

Increased carbon dioxide concentration improves the antioxidative properties of the Malaysian herb kacip fatimah (Labisia pumila Blume).

A randomized complete randomized design (RCBD) 3 by 3 experiment was designed to investigate and distinguish the relationships among production of secondary metabolites (total phenolics, TP; total flavonoids, TF), gluthatione (GSH), oxidized gluthatione (GSSG), soluble carbohydrate and antioxidant activities of the Malaysian medicinal herb Labisia pumila Blume under three levels of CO2 enrichment (400, 800 and 1,200 µmol mol⁻¹) for 15 weeks. It was found that the treatment effects were solely contributed by interaction of CO2 levels and secondary metabolites distribution in plant parts, GSH, GSHH and antioxidant activities (peroxyl radicals (ROO), superoxide radicals (O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH). The records of secondary metabolites, glutahione, oxidized gluthathione and antioxidant activities in a descending manner came from the leaf enriched with 1,200 µmol/mol CO2 > leaf 800 µmol/mol CO2 > leaf 400 µmol/mol CO2 > stem 1,200 µmol/mol CO2 > stem 800 µmol/mol CO2 > stem 400 µmol/mol CO2 > root 1,200 µmol/mol CO2 > root 800 µmol/mol CO2 > root 400 µmol/mol CO2. Correlation analyses revealed strong significant positive coefficients of antioxidant activities with total phenolics, flavonoids, GSH and GSHH indicating that an increase in antioxidative activity of L. pumila under elevated CO2 might be up-regulated by the increase in production of total phenolics, total flavonoids, GSH, GSHH and soluble sugar. This study implied that the medicinal potential of herbal plant such as L. pumila can be enhanced under elevated CO2, which had simultaneously improved the antioxidative activity that indicated by the high oxygen radical absorbance activity against ROO, O2, H2O2, and OH radicals.

Effects of environmental stresses on the responses of mangrove plants to spent lubricating oil.

The influence of different environmental stresses, including salinity (5-35‰), tidal cycle (6/6, 12/12 and 24/24 h of high/low tidal regimes) and nutrient addition (1-6 times background nitrogen and phosphorus content) on Bruguiera gymnorrhiza and Aegiceras corniculatum grown in sediment contaminated with spent lubricating oil (7.5 L m(-2)) were investigated. The oil-treated 1-year-old mangrove seedlings subject to low (5‰) and high (35‰) salinity had significantly more reduction in growth, more release of superoxide radical (O2·-) and higher activity of superoxide dismutase (SOD) than those subject to moderate salinity (15‰). Extended flooding (24/24 h of high/low tidal regime) enhanced O2·- release and malondialdehyde (MDA) content in both oil-treated species but had little negative effects on biomass production (P>0.05) except the stem of A. corniculatum (P=0.012). The addition of nutrients had no beneficial or even posed harmful effects on the growth and cellular responses of the oil-treated seedlings.

Mixed heavy metals tolerance and radial oxygen loss in mangrove seedlings.

The effects of a mixture of heavy metals (Pb, Zn and Cu) on growth, radial oxygen loss (ROL) and the spatial pattern of ROL were investigated in mangrove seedlings of three species: Aegiceras corniculatum, Avicennia marina and Bruguiera gymnorrhiza. Heavy metals inhibited the growth of seedlings and led to decreased ROL and changes in the "tight" barrier spatial pattern of ROL. There was a significant positive correlation between the amount of ROL from the roots of seedlings and metal tolerance. The species with the highest ROL amount, B. gymnorrhiza, were also the most tolerant to heavy metals. The "tight" barrier spatial ROL pattern was also related to metal tolerance in the seedlings. Therefore, we conclude that both ROL amount and "tight" barrier spatial ROL pattern in the roots of the mangrove seedlings play an important role in resistance to heavy metal toxicity.

Effects of used lubricating oil on two mangroves Aegiceras corniculatum and Avicennia marina.

An outdoor experiment was set up to investigate the effects of used lubricating oil (5 L/m2) on Aegiceras corniculatum Blanco. and Avicennia marina (Forsk) Vierh., two salt-excreting mangroves. A. marina was more sensitive to used lubricating oil than A. corniculatum and canopy-oiling resulted in more direct physical damage and stronger lethal effects than base-oiling. When treated with canopy-oiling, half of A. corniculatum plants survived for the whole treatment time (90 d); but, for A. marina, high mortality (83%) resulted from canopy-oiling within 3 weeks and no plants survived for 80 d. Base-oiling had no lethal effects onA. corniculatum plants even at the termination of this experiment, but 83% of A. marina plants died 80 d after treatment. Forty days after canopy-oiling, 93% of A. corniculatum leaves fell and no live leaves remained on A. marina plants. By the end of the experiment, base-oiling treatment resulted in about 45% of A. corniculatum leaves falling, while all A. marina leaves and buds were burned to die. Lubricating oil resulted in physiological damage to A. corniculatum leaves, including decreases in chlorophyll and carotenoid contents, nitrate reductase, peroxidase and superoxide dismutase activities, and increases in malonaldehyde contents. For both species, oil pollution significantly reduced leaf, root, and total biomass, but did not significantly affect stem biomass. Oil pollution resulted in damage to the xylem vessels of fine roots but not to those of mediate roots.

Effect of NaCl on leaf salt secretion and antioxidative enzyme level in roots of a mangrove, Aegiceras corniculatum.

Short-term salt (NaCl) treatment on Aegiceras corniculatum in roots and leaves showed no change in fresh and dry weight of leaves, roots and leaf area. There was no significant change in total soluble root protein, photosynthetic pigments of leaves and spectral characteristics of thylakoids. However, the activity of antioxidative enzymes (catalase, ascorbate peroxidase and guaiacol peroxidase) in roots decreased by 72, 58 and 80% respectively after 96 hr of treatment (300 mM of NaCl). Secretion of salts from the leaf salt glands and salt accumulation on upper surface of the leaves were quantified that revealed linear increase of salt secretion of leaf with increase in period of salt treatment. It was concluded that loss of activities of antioxidative enzymes at high salt treatment, caused leaf senescence in spite of high rates of salt secretion by Aegiceras corniculatum.