Indonesian Journal of Biotechnology
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Early detection of the orchid flowering gene PaFT1 in tobacco cells using a GFP reporter
No full textHere we describe a novel method of using green fluorescence protein (GFP) as a reporter gene for early detection of an integrated TDNA containing the orchid flowering gene, PaFT1 (Phalaenopsis aphrodite Flowering locus T1) in the tobacco genome. Functional assays that report the presence of exogenous DNA early in development are especially useful in plants where the desired phenotype is only apparent after long periods of vegetative growth. The objective of this study is to establish a method for detecting an inserted Phalaenopsis orchid flowering gene and examining its function in tobacco. The p35S::PaFT1 35S::GFP construct was introduced into Agrobacterium tumefaciens strain EHA101. Transformed tobacco leaves were cultured on MS medium with addition of 1 mgL-1 NAA+3 mgL-1 BAP+50 mgL-1 Kanamycin+300 mgL-1 timentin for selection. Results showed bright green GFP fluorescent signals in 11 out of 15 (73%) tobacco leaf cells at a 2month time point after transformation. GFP and PaFT1 fragments were amplified in genomic PCR using GFP and PaFT1 specific primers. The accumulated PaFT1 transcripts were observed in 3 monthold transgenic tobacco plants containing p35S::PaFT135S::GFP. Green florescence was observed only in the transgenic plants at the 5 monthold stage but not in the wild type controls
Secondary metabolite profiling of four host plants leaves of wild silk moth Attacus atlas L.
No full textSecondary metabolites may affect insect herbivores’ host plant preferences. Attacus atlas L. larvae are known have a wider variety of host plants compared with other members of the Attacus genus. This research compared the metabolic profiles of four A. atlas host plants: keben (Barringtonia asiatica (L.) Kurz), dadap (Erythrina lithosperma Miq.), gempol (Nauclea orientalis L.), and soursop (Annona muricata L.). Leaves were collected from Sawit Sari Research Station, Yogyakarta. Terpenoid was extracted by macerating the leaves in ethyl acetate and subjecting them to GC-MS analysis, while alkaloid, tannin, and flavonoid were extracted through percolation. Total alkaloids, tannins, and flavonoids were measured using spectrophotometric analysis. Multivariate data analysis using PAST ver. 3.0 was performed on the GC-MS data. Based on the PCA scatter plot of the GC-MS data, keben leaves were clustered separately from the other three leaves by PC1. Dadap and gempol leaves were clustered together due to the phytol content while caryophyllene was detected only in soursop leaves. Neophytadiene was detected in all of the leaves, suggesting that this terpenoid may serve as a signal to locate the host plants. Keben leaves contained the lowest alkaloids and highest tannins and flavonoids compared with the other leaves. These secondary metabolites may determine the host plant suitability for culturing the A. atlas
Evaluation of antimicrobial activity and identification of yellow pigmented marine sponge-associated fungi from Teluk Awur, Jepara, Central Java
No full textMarine sponge associated fungi are known as potential source of metabolites with various biological activities. Natural pigment is one of metabolite which produced by microorgisms. Several researches reported the antimicrobial activity from natural pigment. Unfortunatelly there are lack of information about marine fungi natural pigment and its producer. The aims of this research were to identify yellow pigmented Indonesian marine sponge-associated fungi, to extract the pigment, and to study the antimicrobial activity of the pigment against clinical MDR bacteria and clinical pathogenic fungi. Sponge associated-fungus isolate MT23 was successfully identified as Trichoderma parareesei. The fungal pigment could be extracted only in methanol with yield 6,22±0,29%. The pigment could inhibitted S. typhi and E. coli MDR strains. The biggest antibacterial activity was shown by concentration 1000µg/mL against S. typhi with inhibition zone was 4.03±0.06 mm
Transient transformation of artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) gene into Artemisia annua L.
No full textGlobal demand of antimalarial drug artemisinin has a gap with production capacity from existing sources since the low content of this compound from Artemia annua L. Genetic engineering-based strategy for A. annua plant on key enzymes in artemisinin biosynthetic pathway is needed. Artemisinic aldehyde ∆ 11 (13) double bond reductase (dbr2) is one of the key enzyme on artemisinin biosynthesis which was studied in this research. Agrobacterium tumefaciens-mediated transformation of A. annua using dbr2 was carried out. Synthetic dbr2 was ligated into pCAMBIA1303 and transformed into Escherichia coli DH5α. pCAMBIA1303-dbr2 plasmid was transformed to A. tumefaciens AGL1. Leaves of A. annua were infected by positive transformant of recombinant A. tumefaciens (OD600 ≈ 1) supplemented with acetosyringone 50 ppm, and Silwet S-408 0.02%. Samples were incubated in desiccators connected with vacuum pump, this method is called infiltration vacuum. Leaves were covered in dark for 45 min, and co-cultivated on MS co-cultivation media for 3 days. All leaves were washed in 300 ppm cefotaxime and divided into 2 parts; 3 leaves for GUS histochemical assay and 300 mg of leaves for HPLC analysis. Transient transformation was done in triplicate. In GUS histochemical assay, pCAMBIA1303 and pCAMBIA-dbr2 showed positive blue spot where coefficient of variance was less than 5%. PCR analysis for genomic DNA of transformed A. annua showed a positive result of inserted dbr2 recombinant indicated by migration profile and direct sequencing analysis. It could be concluded that pCAMBIA-dbr2 construct and transformation into A. annua have been successfully performed
The effect of ethanolic extract of black and white rice bran (Oryza sativa L.) on cancer cells
No full textIndonesia has a wide range of rice cultivars and pigments. This rice can be used as a source of phytochemical compounds for cancer prevention. This research aims to analyze the cytotoxic activities of the ethanolic extract of black rice bran of 4 local cultivars i.e. ‘Cempo Ireng’, ‘Woja Laka’, ‘Toraja’ and ‘IR64’ (white rice) on cancer cells and to determine the compounds groups of those extracts. First step, rice bran was extracted with ethanol. This extract was applied to Raji (a human Burkitt Lymphoma cancer), HepG2 (a human liver cancer), and Vero (a nonhuman cell line) cells in order to measure the cytotoxic activities by using MTT assay. To determine descriptively the compounds groups of phenolics, flavonoids, terpenoids, steroids, and alkaloids the thin layer chromatography method was performed. The IC50 value was analyzed quantitatively by using probit analysis. Results showed that the IC50 values of ethanolic extract of rice bran ‘Woja Laka’, ‘Toraja’, ‘Cempo Ireng’ and ‘IR 64’ on HepG2 cells were 857.23±99.19; 1,896.55±83,8; 1,494.47±87.81 and 727.89±145,97 µg/ml respectively. The IC50 on Raji cells were 816.61±85.31; 1,079.93±28.31; 1,627.82; ±119.82, and 769.33±61.43 µg/ml respectively. The IC50 on Vero cells were 1,295.2±37; 1,232.07±165.51; 1,874.14±169.56, and 724.4±122.79 µg/ml respectively. The ethanolic extracts of rice bran from four cultivars contain phenolics, flavonoids, terpenoids, and steroids. However, alkaloids could not be detected. The variety of rice cultivars indicates the variation of cytotoxic activities on cancer cells. The ethanolic extracts of rice bran from those four rice cultivars contain similar kinds of organic compounds groups but vary in the Rf values
The first evaluation of glucose-6-phospate dehydrogenase defciency (G6PD) gene mutation in malaria endemic region at South Central Timor (SCT) district, Eastern Indonesia 2014–2015
No full textPrimaquine (PQ) is a key drug in the malaria pre-elimination stage. However, PQ can trigger acutehemolysis for people with G6PD defciency (G6PDd). In 2013, 15–25 million Indonesian people were infected with malaria, with 30,000–38,000 deaths each year mostly in eastern Indonesia with API= 15.6 %. Recently, the Ministry of Health of the Republic of Indonesia announced a plan to reach the pre-elimination stage based on WHO guidelines. This study assesses whether eastern Indonesia should proceed with the activities of malaria pre-elimination. A total 555 healthy people in fve subdistricts in eastern Indonesia were selected by systematic random samping. All data were collected using a standard questionnaire, physical examination, and laboratory tests. PCR and DNA sequencing protocols followed respective manufacture’s instructions. Statistical analysis by bivariate with α= 0.05 and 95% CI were performed using the SPSS software package. Based on the nested PCR, the result showed a malaria prevalence of 32.6% with being the dominant species (52.5%). Malaria cases were found in all study sites and not using a bed net was the moost signifcant risk factors with Exp B= 1.54 with 95% CI= 0.99–2.38. G6PDd prevalence was 16.6%, the highest G6PDd ever found in Indonesia with variant molecular dominant 10.883 T>C and one sample with a heterozygous female. Malaria pre-elimination in eastern Indonesia should be delayed. High risk patients should be tested for enzyme G6PD activities before antimalarial administration
Marker Assisted Selection for Bacterial Leaf Blight Rice Mutant Lines Resistant
No full textInduction of mutation using gamma rays for improving of Mira-1 rice variety has been conducted.Rice mutant lines M2 generation have been obtained from mutation by the doses of 25, 50, 75, 100, 150 and200 Gy of gamma rays. Selection of mutant lines tolerant to the disease was only observed in the field neithergenetically. Marker assisted selection is a tool to obtain a new rice variety tolerant to the disease of bacterialleaf blight (BLB) genetically. Xanthomonas oryzae pv.oryzae (Xa) was the pathogen of BLB, and the identificationof rice mutant lines which were containing of Xa5, Xa13 and Xa21 genes have been done using PolymeraseChain Reaction ( PCR ) method. The result showed that one mutant line, and four mutant lines from mutationby the doses of 25 Gy and 150 Gy were containing Xa5, Xa13 and Xa21 genes the same as that of Code ricevariety as positive control, and none in Kencana Bali rice variety as negative control. Mira-1 rice variety as theparent plant was only contains Xa5 and Xa21 genes. The doses of 50 Gy and 100 Gy were very affective onremoving of all bands for identification of those genes. The purpose of this research was to obtain the mutantlines which were contain of those Xa genes as indicator for resistant to BLB disease genetically
Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien”
No full textPhalaenopsis “Sogo Vivien “is a mini orchid hybrid with beautiful flowers and numerous inflorescences.Mass propagation of this orchid is needed to meet the market demand. Objective of this research was toinduce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNAcontaining 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated byAgrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid inductionsystem, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developedinto protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocormsdeveloped from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specificprimers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantletswere positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryoswere also emerged from leaf explants of transformant on hormone-free NP medium and became normalplantlets. It is probably due to the high activity of AtRKD4 genes in orchid
Characterization of Aspergillus Niger 65i6 lipase from solid-state fermentation using Jatropha seed cake medium
No full textJatropha curcas seed cake contains a high amount of protein, and consequently has very high potentialas a medium for lipase production. The objective of this research was to characterize lipase from Aspergillusniger 6516, which was produced by solid-state fermentation on Jatropha curcas seed cake as the medium. The effects of pH and temperature on enzyme activity were evaluated, along with substrate specifcity and enzyme stability. Fermentation was performed at a water concentration of 63% and temperature of 30 °C for 7 days. The results showed that the optimum pH and temperature for Aspergillus niger 6516 lipase activities were 8.0 and 40 °C, respectively. The lipase had the substrate specifcity to hydrolyze long-chain fatty acids and was stable in polar organic solvents. The lipase had a molecular weight, Km and vmax about 19 kDa, 0.27 µmol/ml, and 52.63 µmol/ml/min, respectively. The results also suggested that the produced lipase from Aspergillus niger 6516 was an alkaline lipase. Based on these results, we conclude that Jatropha seed cake is a suitable medium for lipase production
The effect of methanol extract of soybean seeds (Glycine max L.Merr.) on the histology and immunohistochemical distribution of Cyp19 aromatase in rat testis (Rattus norvegicus L.)
No full textSoybean (Glycine max L. Merr.) contains phytoestrogens that have a chemical structure resembling estrogen in the body. They function like estrogen and antiestrogen, affecting the metabolism of sex steroid hormones. This research aimed to determine the effect of the methanol extract of soybean on the histological structure and distribution of immunohistochemical Cyp19 aromatase in rat testis . Twenty males of Wistar rats were divided into 4 groups of 5. The frst group was the control and the second to fourth groups were given soybean extract (250 mg/kg of body weight, 500 mg/kg of body weight) and genistein (0.3 mg/kg of body weight), respectively, for 52 days. The results of this study indicate that the effect of methanol extract from soybean caused weight gain, and the weight of the testis and epididymis decreased. In addition, the histological results showed that seminiferous tubules were reduced in size, became irregular, were separated by a wide interstitium, and spermatogenic cells were decreased. The immunohistochemical results showed that the expression of Cyp19 aromatase in the rats decreased both in spermatocyte cells and Leydig cells. It could be concluded that the methanol extract of soybean induced testicular damage and reduced Cyp19 aromatase expression in rat testis