Khảo sát quy trình khử trùng mẫu, ảnh hưởng của cường độ ánh sáng, nồng độ môi trường agar lên sự hình thành mô sẹo rong Kappaphycus alvarezii (Doty) Doty (Rhodophyta) trong điều kiện in vitro

Objective of this study was to ascertain the optimum condition for callus induction of K. alvarezii (Doty) in vitro such as to determine the explants sterilization process, the effect of intensity of light and the concentration of agar. Fresh thalli treated with 0.5% - 1% detergent for 5 mins followed by 0.5% - 1% betadine for 2 – 3 mins and incubated with 0.5% - 1% broad spectrum antibiotic mixture in PES medium for 1 day produced 95 – 98% bacteria free healthy explants.  Two independent experiments with light intensity and agar contentration of the environment were carried out at 5 different levels of 0, 5, 25, 50, 70 µmol photon.m2.s-1 and 9 agar concentrations of 0.5%, 0.75%; 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.5%, 3.0%. The highest callus induction rate was (96 ± 3.5 – 98 ± 2.1%) at 5 - 25 µmol photon.m-2.s-1 and (87 ± 5.8% – 90 ± 5.0%) in 1% - 3% agar concentration after 2 weeks of explants. The highest callus living rate was 98% at the light intensity of 25 µmol photon.m-2.s-1 and (75 ± 5.7 – 84 ± 1.1%) in 0.75 – 1.5% agar concentration after 2 months of explants. The highest callus re-induction rate was 50 – 55% at the light intensity of 5 – 25 µmol photon.m-2.s-1 and 60 – 65% in 1 – 1.5% agar concentration. Callus was not observed in dark condition (0 µmol photon.m-2.s-1). These calluses, that were strong, big and had filamentous type, will be a good material for the next production stage of embryonic callus production and seedling regeneration from micropropagules.Mục đích của nghiên cứu là xác định điều kiện tối ưu lên sự hình thành mô sẹo của rong sụn Kappaphycus alvarezii (Doty) trong điều kiện in vitro như: quy trình khử trùng mẫu, ảnh hưởng của cường độ ánh sáng và nồng độ môi trường agar. Kết quả rong được khử trùng với 0,5% - 1% chất tẩy rửa trong thời gian 5 phút, kết hợp với 0,5% - 1% betadine trong thời gian 2 – 3 phút, cuối cùng xử lí với 0,5% - 1% kháng sinh phổ rộng trong thời gian 1 ngày thu được hơn 95 – 98 % mẫu rong vô khuẩn. Hai thí nghiệm độc lập được bố trí với ánh sáng và hàm lượng agar trong môi trường thạch, ở 5 mức ánh sáng (0, 5, 25, 50, 70 µmol photon/m2/s) và ở 9 mức nồng độ agar (0,5%; 0,75%; 1,0%, 1,25%, 1,5%, 1,75%, 2,0%, 2,5%, 3,0 %). Kết quả tỷ lệ hình thành mô sẹo cao nhất là (96 ± 3,5 – 98 ± 2,1%) ở 5 - 25 µmol photon/m2/s và (87 ± 5,8% – 90 ± 5,0%) ở nồng độ agar 1% - 3% sau 2 tuần cấy mô. Tỷ lệ sống của mô sẹo cao nhất (98%) ở cường độ ánh sáng 25 µmol photon/ m2/s và ở nồng độ agar 0,75 – 1,5% là (75 ± 5,7 – 84 ± 1,1%) sau 2 tháng cấy mô. Tỷ lệ tái sản xuất mô sẹo cao nhất là 50 – 55% ở cường độ ánh sáng 5 – 25 µmol photon.m-2.s-1 và 60 – 65% ở nồng độ agar 1 – 1.5%. Không có mô sẹo hình thành ở điều kiện tối (0 µmol photon/m-2/s). Những mô sẹo phát triển tốt, có dạng sợi, cụm mô to sẽ là vật liệu tốt để làm những thí nghiệm tiếp theo ở công đoạn sản xuất phôi mô sẹo và tái sinh cây con từ phôi mô sẹo

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

The ameliorating effect of Acadian marine plant extract against ionic liquids-induced oxidative stress and DNA damage in marine macroalga Ulva lactuca

Ionic liquids (ILs) are generally considered as the green replacement for conventional volatile organic solvents. Nonetheless, their high solubility in water with proven toxic effects on aquatic biota has questioned their green credentials. In the present study, the detoxification potential of Acadian marine plant extract powder (AMPEP) prepared from the brown alga Ascophyllum nodosum was investigated against the 1-alkyl-3-methylimidazolium bromide [C12mim]Br ionic liquid-induced toxicity and oxidative stress in marine macroalga Ulva lactuca. The IL ([C12mim]Br) at LC50 (70 μM) exposure triggered the generation of reactive oxygen species (ROS) such as O2·-, H2O2 and OH· causing membrane and DNA damage together with inhibition of antioxidant systems in the alga. The supplementation of AMPEP (150 μg mL-1) to the culture medium significantly reduced the accumulation of ROS and lipid peroxidation together with the inhibition of lipoxygenase (LOX) activity specially LOX-2 and LOX-3 isoforms. This is for the first time wherein comet assay was performed to ascertain the protective role of AMPEP against DNA damage in algal tissue grown in medium supplemented with IL and AMPEP. The AMPEP showed protective role against DNA damage (5-45 % tail DNA) when compared to those of grown in IL alone (45-70 % tail DNA). Further, specific isomorphs of different antioxidant enzymes such as superoxide dismutase (Mn-SOD-1, ~150 kDa), ascorbate peroxidase (APX-4, ~55 kDa), glutathione peroxidase (GSH-Px-2, ~55 kDa) and glutathione reductase (GR-1, ~180 kDa) responded specifically to AMPEP supplementation. It is evident from these findings that AMPEP could possibly be used for circumventing the negative effects arising from ILs-induced toxicity in marine ecosystem. © 2012 Springer Science+Business Media B.V

Algal lipids, fatty acids and sterols

Algae are photosynthetic organisms with ubiquitous distribution and contain varied forms of lipids owing to their extreme habitat diversity. This chapter presents detailed information on the structure and distribution of lipids, fatty acids and sterols in algae together with the genes and enzymes involved in their metabolism. The developments in acclimatory roles of lipids, fatty acids and sterols in response to changes in environmental factors such as nutrients, light, temperature and salinity have been discussed. Further, the current status of lipidomics in algae has also been discussed presuming its promising implications in elucidation of novel lipids and understanding of complex metabolic pathways. © 2013 Woodhead Publishing Limited All rights reserved

Nitrate and phosphate regimes induced lipidomic and biochemical changes in the intertidal macroalga ulva lactuca (ulvophyceae, chlorophyta)

This study was carried out in order to understand the lipid and biochemical alterations resulting from different nutritional regimes of nitrate and phosphate in Ulva lactuca. The algal thalli cultured in artificial seawater (ASW) showed higher levels of carbohydrates and non-polar lipids and increased phosphatase activities, accompanied by degradation of polar lipids, proteins and pigments. Further, higher levels of lipid hydroperoxides indicated reative oxygen species (ROS)-mediated non-enzymatic lipid peroxidation due to nutritional limitation-induced oxidative stress. Those thalli cultured in ASW supplemented with nitrate showed responses corresponding to nitrate addition, such as an increase in pigments, monogalactosyldiacylglycerols, polyunsaturated fatty acids and nitrate reductase. In addition, these thalli showed partial induction of phosphatases, low phospholipids, and high sulfolipid and 1,2-diacylglyceryl-3-O-4′-(N,N,N-trimethyl)-homoserine (DGTS) due to phosphate limitation. Similarly, algal thalli cultured in ASW supplemented with phosphate showed down-regulation of phosphatases, an increase in phospholipids due to availability of phosphate as well as a decrease in nitrate reductase, pigment, monogalactosyldiacylglycerols and polyunsaturated fatty acids due to nitrate limitation. On the other hand, algal thalli cultured in ASW supplemented with both nitrate and phosphate showed recovery of lost pigments and proteins, a high monogalactosyldiacylglycerol/digalactosyldiacylglycerol ratio, high unsaturation and high oxylipin levels (both C18 and C20). Further, the accumulation of indole-3-acetic acid in nutrient-limited thalli and of kinetin and kinetin riboside in nutrient-supplemented thalli indicated their antagonistic roles under nutrient stress. Thus, U. lactuca copes with nitrate and phosphate nutritional stress by altering the metabolic pathways involved in lipid biosynthesis including a shift in lipid classes, fatty acids, oxylipins and indole-3-acetic acid/kinetin cross-talk. © 2013 The Author

Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca: Oxidative stress and DNA damage

The green credentials of ionic liquids (ILs) are being increasingly questioned due to the growing evidence of their toxicity to aquatic ecosystems, although the mechanisms of toxicity are unknown. This study provides insights into the mechanism of toxicity and biological effects of 1-alkyl-3- methylimidazolium bromide [C nmim]Br (n = 4 to 16) on the marine macroalga Ulva lactuca. The cell viability of this alga during IL exposure was found to be negatively correlated to the chain length of the alkyl group. The IL ([C 12mim]Br) exposure triggers the generation of reactive oxygen species (ROS viz. O 2•-, H 2O 2, and OH •), damage of the membrane and DNA, and inhibition of antioxidant systems in the alga. The enhanced production of ROS and lipid peroxidation in the alga subjected to LC 50 concentration for 4 days was largely attributed to lipoxygenase (LOX) activity coupled with the induction of two LOX isoforms (∼80 kDa and ∼55 kDa). Pretreatment of the algal thallus with enzyme inhibitors such as diphenylene iodonium, sodium azide, cantharidin, and oxadiazoloquinoxalin-1-one, prior to [C 12mim]Br exposure showed the regulation of ROS by the activation of membrane bound NADPH-oxidase and cytochrome oxidase. The IL exposure resulted in the accumulation of n-3 and n-6 fatty acids at 0.5 LC 50 concentration indicating the induction of desaturase enzymes. Furthermore, antioxidant enzyme activities such as superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) were enhanced by 1.3-2.0-fold, while glutathione peroxidase (GSH-Px) diminished, together with a higher regeneration rate of reduced ascorbate and glutathione. The isoforms of antioxidant enzymes, namely, Mn-SOD (∼85 kDa), APX (∼125 and 45 kDa), and GR (∼135 kDa) regulated differentially to IL exposure. The comet assay performed for the first time for seaweeds revealed the significant induction of DNA damage (>50-70% increase in % tail DNA over control) in alga exposed to ≥ LC 50 concentration. © 2011 American Chemical Society

Purification and characterization of exo-β-agarase from an endophytic marine bacterium and its catalytic potential in bioconversion of red algal cell wall polysaccharides into galactans

An extracellular exo-β-agarase was characterized from an endophytic bacterial strain Pseudomonas sp. isolated from the red alga Gracilaria dura. The enzyme was purified to homogeneity with a recovery of 28.2% and a purity fold of 8.33. The purified enzyme was composed of single polypeptide with a molecular mass of about 66 kDa. The enzyme exhibited a maximum activity of 81.74 U mL-1 and a specific activity of 615.5 U mg-1. The optimal pH and temperature for its maximum activity were 9.0 and 35 °C respectively. The enzyme stabilized its activity in alkaline pH 7-11 and high salt concentration up to 4 mol dm-3. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose while the bacterium when incubated with G. dura biomass yielded galactose 20% on dry wt basis. The agarolytic ability of the former was further confirmed by release of protoplasts from G. dura tissue through digestion of cell wall polysaccharides. The bacterium investigated in this study could possibly be used for bioconversion of marine red algal polysaccharides into energy feedstock and the purified enzyme for preparation of compounds having pharmaceutical importance. © 2013 Elsevier Ltd

Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

Regeneration of protoplasts into de novo plants was reported for a large number of seaweed species. The regeneration of protoplasts into different morphotypes as a result of epigenetic variations was discussed for the first time in this study. The loci assessed for methylation modifications in normal filamentous thalli showed a frequency of 32. 43% as unmethylated DNA, 24. 32% as a hemimethylated, and 20. 27% as a methylation of internal cytosine at both the strands. The corresponding methylation values for disk-type thalli were 27. 02%, 32. 43%, and 14. 86%, respectively. The hypermethylation condition was apparent in the disk-type thalli with methylation ratio of 72. 97% compared to that of normal filamentous thalli with 67. 56%. The frequency of methylation polymorphic sites among the two morphotypes was 53%. The present study reveals the distinct expression of cytosine methylation and is thus correlated to differential morphogenesis of plants regenerated from cultured cells. The number of protoplasts regenerating into filamentous thalli declined with increasing temperature from 15°C, 20°C, 25°C, and 30°C. The disk-type variant had higher thermal stability at 30°C over normal filamentous thalli. Further, this variant could maintain itself for more than a year in the laboratory indicating its suitability for in vitro germplasm maintenance and propagation. © 2012 The Author(s)