Indonesian Journal of Biotechnology
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Elimination of shallot bulb viruses through heat treatment
No full textShallot (Allium cepa L. Aggregatum group) is usually cultivated vegetatively. As a result, viruses tend to accumulate within the host plants and spread to healthy plants every crop cycle, reducing yield and bulb quality. There are a very limited number of studies about the elimination of shallot viruses through heat treatment. The objective of this research was to eliminate shallot viruses through heat treatment to produce virus-free plantlets. The leaves of Biru Lancor with specifc visual virus symptoms were detected by Reverse Transcription–Polymerase Chain Reaction (RT-PCR). Then bulbs of Biru Lancor that were positively infected by viruses were used as materials for heat treatment. The treatments were a control (without treatment), electric treatment at 15 mA for 10 minutes, heat treatment in an incubator at 37°C for 4 weeks, heat treatment in a waterbath at 45°C for 60 minutes, and combination of heat treatment in an incubator at 37°C for 4 weeks and heat treatment in a waterbath at 45°C for 60 minutes. After being subjected to heat treatment, the pseudo stem were cultivated in the MS Medium + 1 mg/L BAP + 1 mg/L IBA.Virus detection by RT-PCR was conducted 28 days after planting using samples of leaves from each plantlet. The results of this research showed that the treatments of electric treatment at 15 mA for 10 minutes and combination of heat treatment in the incubator at 37°C for 4 weeks and heat treatment in the waterbath at 45°C for 60 minutes could suppress the incidence of Shallot latent virus (SLV) until 100%. Heat treatment might have an important role in the degradation of virus particles by boosting Virus-Induced Gene Silencing (VIGS) as plant responses to virus infection
Sequences Analysis of a Gene Encoding Extracellular Xylanase in Streptomyces costaricanus 45I-3
No full textStreptomyces costaricanus 45I-3 is a bacterial strain belongs to actinomycetes group isolated from peat soil. Thebacterium is known to produce extracellular xylanase. The aims of this study were to analyze DNA sequence andsub-clone gene involved in the synthesis of extracellular xylanase. Complete DNA sequence predicted to encodexylanase genes was isolated from bacterial genome using Inverse Polymerase Chain Reaction (I-PCR). Total DNAsequence of 1664 bp in size obtained from I-PCR consisted of two open reading frames (ORF) in opposite direction.ORF1 was 1029 bp and ORF2 (partial sequence) was 309 bp. Analysis sequence using BlastX indicated that ORF1was homologous with xylanase bacterium enrichment culture clone Xyl8B8 (GenBank accession No. AFH35005.1),i.e. 95% in identity and 99% in similarity. In addition, ORF2 was homologous with glyoxalase bacterium enrichmentculture clone Xyl8B8 (GenBank accession No. AFH35007.1), i.e. 95% in identity and 98% in similarity. Analysis ofamino acid sequence revealed that ORF1 consisted of 2 domains, i.e. glyco-hydrolase 11 (GH11) and CarbohydrateBinding Type 2 (CBM2). Active site was found at 130th amino acid on GH11 domain. Visualization of 3-dimensionstructure showed that 1029 bp fragment is of 19 areas
Black Rice Bran Extracts and Fractions Containing Cyanidin 3-glucoside and Peonidin 3-glucoside Induce Apoptosis in Human Cervical Cancer Cells
No full textAnthocyanin of pigmented rice inhibits the growth of cancer cells. The cytotoxicity and apoptosisinducing properties of local black rice (cv Cempo Ireng) extracts and fractions, which contain anthocyaninincluding cyanidin 3-glucoside and peonidin 3-glucoside, on human cervical cancer cell line (HeLa cells) hasbeen evaluated. The pigmented rice bran was extracted and fractionated using methanol-HCl. The MTT testwas performed on HeLa cell cultures to observe the IC50 value. Preparative TLC was performed to obtain thefractions of black rice bran. Cyanidin 3-glucoside and peonidin 3-glucoside were identified in the pigmentedrice bran extract and fractions using UHPLC. Flowcytometry analysis was performed to measure the percentageof apoptotic cells. Our results suggest that the fractions are more toxic than the methanolic crude extract withIC50 values of 85.95 ± 5.56 μg/mL (the lowest one) and 408.13 ± 51.9 μg/mL, respectively. The concentration ofcyanidin 3-glucoside and peonidin 3-glucoside in the methanolic extract were 1.89 and 0.84 μg/mg, respectively.The apoptosis induction by fractions F2 and F4 (52 and 55%) were significantly higher compared to fractionF3 and F5 (30 and 33%) and doxorubicin (21%). Cyanidin 3-glucoside was detected in F4 (0.14 μg/ml) whilepeonidin 3-glucoside in F2 (0.012 μg/ml), however both were not detected in F3 and F5
An Active of Extracellular Cellulose Degrading Enzyme from Termite Bacterial Endosimbiont
No full textCellulase is an ezyme that specifically cleaves the 1,4-β-glycosidic bond of cellulose to produce thesmall fragments of simple carbohydrate. This work was aimed to characterize the extracellular cellulase fromPaenibacillus spp., which was previously isolated from macro termites, Odontotermes bhagwatii in our laboratory.Two Paenibacillus isolates were used in this experiment, namely Paenibacillus cellulositrophicus SBT1 andPaenibacillus, sp. SBT8. Analysis of the total proteins in the supernatants showed that P. cellulositrophicus SBT1and Paenibacillus sp. SBT8 roughly produced as much as 18.6 mg/l and 24.8 mg/l of extracellular cellulases,respectively. Enzymatic assay showed that SBT1 and SBT8 cellulase exhibited enzymatic acitivity of 0.17 U/mg and 0.12 U/mg, respectively. Temperature dependencies analysis indicated that both cellulases exhibitedmaximum activity at 35oC. At the temperature higher than 55oC, the enzymatic activities of both cellulases wereroughly 20% reduced compared to the maximum activity. SBT1 and SBT8 cellulases were both active at acidicpH. At basic pH (pH 8) the enzymatic activities of both cellulases were reduced roughly 30% compared to thatof acidic pH. Supplementing of Mg2+, Zn2+, and Ca2+ in range of 1-10 mM increased the enzymatic activity ofboth cellulases roughly 33 to 50%
Identifcation of antibiotic producing endophytic microbe isolates from a national park in Java island
No full textEndophytic microbes are potential sources of antibiotics. Some numbers of endophytic bacteria were isolated from plants in Ujung Kulon, Kaliurang, Meru Betiri and Baluran National Park, Bogor Botanical Garden, and Nusakambangan forest, Indonesia. Previous studies have been conducted to examine and obtain endophytic bacteria isolates from the selected plants, which resulted in three selected isolates, namely OOH-1, STG-1, and CMB-2. This research was conducted to determine the molecular identity of OOH-1 and STG-1 isolates, as well as to identify antibiotic compounds produced by STG-1 isolate. Molecular identifcation of selected isolates was based on 16S rRNA gene analysis and amplifed using primers 27F and 1492R. A phylogeny tree was then constructed by comparing the resulting sequences with data from Gene Bank using the BLAST-N program. The identifcation showed that STG-1 isolate had a 99% similarity with Pseudomonas brenneri strain SFML 97-391, and OOH-1 isolate had a 99% similarity with Enterobacter xiangfangensis. Identifcation of antibiotic compounds was done by purifcation and separation of the compounds. Antibiotic activity was also examined based on Lethal Concentration (LC50) on Fusarium oxysporum with a LC50 of 0.01–0.02% against Fusarium oxysporum
High Resolution Microsatellite Marker Analysis of Some Rice Landraces Using Metaphor Agarose Gel Electrophoresis
No full textMicrosatellite markers or simple sequences repeats are DNA - based molecular techniques that areused to see the different among accessions and inbred lines. There are three methods to analysis the results ofthe polymerase chain reaction of microsatellite markers namely polyacrylamide gel electrophoresis (PAGE),capillary electroforesis, and Metaphor Agarose Gel Electroforesis (MAGE), and the Use of MAGE assessedmore easily and economically the polymorphic pattern of DNA markers. This study aimed to obtain fast,effective and efficient in term of easy and cheap technique to identify microsatellite markers of some blackrice cultivars and F2 populations from crosses between black with white rice. The results showed that MAGEsuccessfully separated clearly SSRs alleles with different sizes of less than 25 bp
Formulation of Medium Viscosity Chitosan-Pectin –MJ Protein Nanoparticles Conjugated with Anti-Ep-CAM and Its Cytotoxicity Against T47D Breast Cancer Cell Lines
No full textChitosan nanoparticle could become potential formula to protect protein degradation during therapy,since chitosan nanoparticles have “proton sponge hypothesis” mechanism on its protection. Chitosan and pectinis used as basic formula of drug delivery because of its biodegradable and biocompatible properties. Chitosanpectin nanoparticles can be formulated by polyelectrolit complex. EpCAM showed excessive expression inepithelial cancer cells thus can be used as a therapeutic biomarker. MJ protein, a Ribosome-Inactivating Proteins(RIPs) isolated from Mirabilis jalapa L had a higher cytotoxicity on malignant cells than normal cells. MJ proteinneed to be formulated to protect from proteosome degradation in endosome. The aim of this research was todevelop MJ protein-chitosan-pectin nanoparticles and conjugated with anti EpCAM for breast cancer therapy.Mj protein was extracted from M.jalapa leaves. RIPs activity was assayed by supercoiled DNA cleavage. MJprotein were loaded into chitosan nanoparticles using medium viscous chitosan and pectin as cross-linker withpolyelectrolit complex method. Anti EpCAM was conjugated to MJ protein-chitosan-pectin nanoparticles bycarbodiimide reaction and characterized for its entrapment efficiency, morphology by transmission electronmicroscope, particles size, and zeta potential. MJ protein nanoparticles conjugated anti EpCAM and withoutanti EpCAM were cytotoxicity assayed toward T47D and Vero cell lines. MJ protein was able to cleave thesupercoiled DNA into linear and nicked-circular ones. The nanoparticles optimal concentration of mediumviscous chitosan: MJ protein: pectin was 0.01%: 0.01%: 1% (m/v). A high entrapment efficiency of MJ proteinnanoparticles was 98.97 ± 0.07%. Morphology nanoparticles showed an amorphic structure with 200.00 nmparticles size. The nanoparticles conjugated anti EpCAM showed average particles size 367.67nm, polydispersityindex 0.332, and zeta potential +39.97mV. MJ protein-chitosan-pectin nanoparticles conjugated anti EpCAMand unconjugated both had higher cytotoxicity with the IC50 57.64 μg/mL and 46.84 μg/mL respectivelyagainst T47D and 99.38 μg/mL and 111.34 μg/mL against Vero cell lines compared to MJ protein with IC50 of3075.61 μg/mL against T47D and 3286.88 μg/mL against Vero cell lines. Both MJ protein-nanoparticles couldincrease the cytotoxicity effects about 50 times compared to the unformulated MJ protein activity, howeverhad less specificity toward T47D and Vero cell lines
The effects of population size on genetic parameters and mating system of sandalwood in Gunung Sewu, Indonesia
No full textWe combined feld observations with isoenzyme analysis to compare population demographic and its effects on genetic diversity and mating systems, among six populations of sandalwood in Gunung Sewu, Indonesia, during March to August 2015. This endangered economic-important species was originated from the southeastern parts of Indonesia, but is recently occured as new landraces in Gunung Sewu, Java island. The observed heterozygosity varied from Ho 0.184 to 0.385 in parents, and from Ho 0.083 to 0.348 in offspring levels, based on the degree of clonality and genetic base. Most of genetic variation is distributed within populations, and only 2.7% were presented among populations, that was indicated by the low DST and FST value (HT 0.30; HS 0.276; DST 2.4%; FST 7.98%). A dendrogram indicated a grouping of populations into three clusters. However, there were seemed to be no association between geographical and genetic distance. Genetic depletion occured due to (i) clonality events as result of heavy-exploitation and/or natural disturbance which induced root suckering, (ii) genetic drifts and bottleneck effects, (iii) the founder effects due to parental low diversity, and (iv) the alteration on mating systems to be more inbreeders. Some of the results confrmed a “reproductive assurance prediction” while some others were contradicting this. It seemed that genetic diversity and mating systems are not much affected by population size, but more by the parental heterozygosity and the degree of clonality. Our results emphasized the importance of populations’ genetic base or parental genetic diversity to naturally maintain the genetic and evolutionary processes under equilibrium conditions
Identification of BSA B1 Bacteria and Its Potency of Purified Cellulase to Hydrolyze Chlorella zofingiensis
No full textCellulase has been widely used as biocatalyst in industries. Production of cellulase from microorganismshas many advantages such as short production time and less expense. Our previous study indicated that oneof cellulolytic bacteria from digestive tract of milkfish (Chanos chanos), namely BSA B1, showed the highestcellulase activity. The objective of this study was to determine the phylogenetic of BSA B1 strain using 16SrRNA gene sequence. Furthermore, this study also determine the specific activity of purified cellulase from BSAB1 strain and its potency to hydrolyze Chlorella zofingiensis cellulose. Cellulase was purified using ammoniumsulphate precipitation, dialysis, and ion exchange chromatography. The purified cellulase was used to hydrolyzecellulose of C. zofingiensis. The result demonstrated that BSA B1 strain was closely related with Bacillus aeriusand Bacillus licheniformis. The specific activity of the crude enzyme was 1.543 U mL-1; after dialysis was 4.384 UmL-1; and after chromatography was 7.543 U mL-1. Purified cellulase exhibited activity in hydrolyzed both CMCand C. zofingiensis. Compared to commercial cellulase, purified cellulase had lower activity in hydrolyzed CMCbut higher activity in hydrolyzed C. zofingiensis. Ethanol dehydration could potentially increase the reducingsugar yield in cellulose hydrolysis when used appropriately. Morphology of C. zofingiensis cell has changedafter incubation with cellulases and ethanol dehydration indicated degradation of cell wall
Application of Molecular Biology for Identification of Virus Resistance Gene in Melon
No full textSource of resistance to an Indonesia isolate of Cucumber mosaic virus (CMV-B2) in melon cultivarYamatouri has been reported. Moreover, Creb-2, a locus that confers resistance to CMV-B2 in Yamatouri hasbeen determined as a single dominant gene. To elucidate the resistance mechanism conferred by Creb-2 inmore detail, it is necessary to clone the Creb-2 gene and determine its molecular structure. One approach isby amplification and cloning of melon resistance gene analogs (MRGAs) based on degenerated PCR primersdesigned from conserved amino acids in the NBS-LRR motifs (P-loop, Kinase-2, and the GLPL) and Toll/Interleukin-1 receptor-like region (TIR). This study was aimed to identify and characterize the resistance geneanalogs from Cucumis melo L. cv. Yamatouri by employing polymerase chain reactions (PCR) as a molecularbiology tools with degenerate primers based on conserved motifs of cloned R genes. The application of molecularbiology such as DNA isolation, degenerate primers and PCR condition, cloning, sequencing, linkage analysisand mapping of resistance gene analogs to Creb-2 gene in melon will be widely discussed in this pape