Research Journal of Biotechnology

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Research Journal of Biotechnology

Phylogenetic relationship among Bipolaris species based on morphological and molecular variability in internal transcribed spacer region of the nuclear ribosomal DNA

Aggarwal Rashmi, Sharma Sapna, Gupta Sangeeta, Jahani Mehdi, Banerjee Sagar, Singh Veer Bahadur, Bashyal Bishnu Maya and Srinivas Petikam

Phytopathogenic fungi belonging to Bipolaris species are responsible for severe economic losses to many cereal crops globally. Molecular variability among thirty eight isolates belonging to five Bipolaris species viz., B. sorokiniana (Bs), B. oryzae (Bo), B. maydis (Bm), B. spicifera (Bsp) and B. tetramera (Bt) was studied by sequence analysis of internal transcribed spacer (ITS) region of nuclear ribosomal DNA by PCR amplification, restriction analysis and direct sequencing. The PCR amplification with ITS1 and ITS 4 universal primers showed a single DNA band of 600bp (approx.) in all the isolates. The length of ITS 1 varied from 164 to 202bp and that of ITS 2 varied from 132 to 233bp. The variation in % G+C of ITS 1 among the isolates was lower than that of ITS 2. In silico analysis of full length ITS region sequences of all Bipolaris spp. resulted in formation of two clusters with more than 90% sequence homology. Cluster I consisted of isolates belonging to B. sorokiniana, B. maydis and B. oryzae while B. tetramera and B. spicifera formed cluster II. Clustering based on restriction enzyme site analysis also grouped the isolates into two major clusters with B. tetramera and B. spicifera isolates grouped together in a separate cluster. Restriction enzymes Hha-1, Hae-III and Rsa-1 produced distinct restriction pattern in different Bipolaris spp. On the basis of sequence analysis it was observed that Indian pathogenic isolates of Bipolaris spp. have evolved distinctly in tandem with their host preference and their variability can conveniently be elucidated by ITS region rDNA sequence. B. tetramera and B. spicifera were highly similar based on morphology and ITS sequences.

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Vegetative yield and photosynthetic rate of kenaf (Hibiscus cannabinus L.) in reclaimed lands

Cheng-Wu Jin, Yan-Lin Sun, Hak-Tae Lim and Dong-Ha Cho

As the land reclamation is widely practiced to satisfy increasing demands of land for human activities, how to utilize reclaimed lands has become a concerned theme. Considering environment protection and wildlife habitat conservation, the draining of wetlands has not been allowed in the latest reclamation projects. To exploit and select salt-tolerant plant species with intrinsic potential for adaptation to reclaimed lands and suitability to grow in such lands becomes an urgently required work. In this study, we investigated the responses of four kenaf (Hibiscus cannabinus L.) cultivars to reclaimed lands. During the experimental period in reclaimed lands, there was no significant decrease found in photosynthesis and relative water content among the four cultivars. Although Dowling was found to show relatively high adaptability expressed by the high growth rate, it showed the lowest vegetative yield. Most of the cultivars showed a large decrease in relative water content in root than in leaf, but Fuhong-952 persistently maintained relatively high water content in root, suggesting that the plant growth of Fuhong-952 was affected little by reclaimed lands. In view of the results of photosynthesis, plant growth and vegetative yield, Tainung-2 was finally considered to be the ideal cultivar for cultivation in reclaimed lands.

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First report on Torque Teno Virus in patients with gastroenteritis in Taiwan

Chien-Hsien Chen and Chia-Peng Yu

Until now, data on the epidemiological (including ages and seasonality) features of Torque teno virus (TTV) infection in acute gastroenteritis (AGE) in Taiwan are limited. The aim of this study investigated TTV Infections in children and adults with gastroenteritis in Taiwan. One hundred fifty five AGE stool samples were collected from August 2011 to July 2012 and detected TTV using polymerase chain reaction method. TTV was isolated in 80 (51.6%) of 155 stool specimens. TTV was isolated from all age groups. Statistical analysis showed that the TTV infectious rate of AGE patients was statistically significant (P < 0.05) in summer, fall and winter season respectively. Data from this study showing the existence of TTV in fecal samples indicated that the high rates of prevalence of TTV among AGE patients might be due to a possible fecal-oral route of transmission of TTV.

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Enhancement of Cognitive Function of Codonopsis lanceolata from Stepwise Steaming Process associated with Antioxidation Activity

Kim Nam Young and Lee Hyeon Yong

It was first found that cognitive function of Codonopsis lanceolata can be improved through the stepwise steaming process and its cognitive function was strongly correlated with high antioxidant activity, possibly due to great increase of phenolic acids during the steaming process. DPPH free radical scavenging ability of the extracts from this process was increased up to 50.44% in treating 1.0 mg ml-1 whose increase was threefold higher than only 8.94% of the fresh Codonopsis lanceolata (FC) extract. As a result of measuring in vivo cognitive functions, for the Morris water maze test, an improved memory ability was 53 sec in admistering the extract from steaming process (SC), compared to only 36 sec of a memory ability in the FC extract. The passive avoidance test also showed that the memory recovery of the mice in treating the SC extract was 50 sec higher than that of the mice in feeding the FC extract. It was also first reported that the gallic acid that was not found in the FC extract, existed through the fifth step steaming process and played an important role in improving cognitive function but also had a synergistic effect with other substances in the SC extracts. These results strongly indicate that the complex extract form, not a single compnent from C. lanceolata would be beneficial for the application to the drugs.

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Wheat × Maize Crosses for Haploid Embryo Production and Comparison of haploid-diploid Embryo Structure

Özkara Arzu and Savaşkan Çiğdem

The aim of this study was to compare haploid and diploid embryo structure derived from two different wheat varieties, Triticum aestivum L. (Gün 91) and Triticum durum Desf. (Mirzabey). For diploid embryos, plants were grown until earing time, dicamba and 2,4-D were also applied to spikes in flowering period and embryos were collected after 18th-21st days of following pollination. For haploid plants, embryo rescue technique was used to derive haploid embryos by using intergenic crosses between both wheat varieties and corn (Zea mays L.). Synthetic auxins were also used to support of haploid embryo production. Two different microscopic measurement, width and length, were taken in each of embryo obtained. The decreasing in width of embryo in dicamba treatment group and also in length of embryo in 2,4-D treatment group were found to be different significantly (p<0.05 and p<0.01) in Gün 91 wheat variety. On the other hand, in Mirzabey, the decreasing of embryo length was also found to be shorter significantly (p<0.05) in 2,4-D treatment group than that of other groups studied. A total of 576 florets were crossed with maize and 0.52% of the florets produced haploid embryos across the two varieties. It was found that the measured sizes of diploid embryos were bigger than that of the haploid embryos as follows: width (60.68%) and length (47.52%) .There was no study about the structure of haploid embryo anatomically.

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Selection of Explants for Organogenic Callus Induction and Direct Shoot Regeneration in Grasspea (Lathyrus sativus L.)

Tripathy Swapan K.

Different explants including root, cotyledon (both whole and sectional), hypocotyl, epicotyls, internode, node, axillary buds, petiole, leaf and shoot tip meristem were tested for their callusing response in B5 medium supplemented with 2,4-D (2mg/l) + BAP (0.5mg/l) and B5 with NAA (2mg/l) + BAP (0.5mg/l) for callus induction; colour, texture and relative growth of calli. The callus induction medium B5 + 2,4-D(2mg/l) + BAP (0.5mg/l) was better for callus induction (%) than B5 + NAA(2mg/l) + BAP (0.5mg/l) for all explants. Leaf pieces followed by internode explant induced very high callus induction frequency and both produced excellent callus growth. Though leaf explants induced very high callus induction frequency(CIF %), but whitish yellow and soft texture of calli in the former and hard, compact, greenish white calli induced in the latter media; were not supposed to be ideal for regeneration. In this regard, internode explants may be opted for organogenic regeneration due to induction of hard, loose, nodular and dark green calli in B5 + NAA(2mg/l) + BAP (0.5mg/l). Nodal and leaf segments yielded high frequency of direct shoot regeneration(15-20%) in B5 + NAA (2mg/l) + BAP (0.5 mg/l). Induction of highly regenerative calli and the direct regeneration system obtained in this investigation could be amenable for genetic transformation in Lathyrus sativus.

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Fermentative production of γ(gamma)-aminobutyric acid by Lactobacillus brevis L2 using wheat bran and millet bran supplemented with glutamate

Can Chen, Donghua Jiang and Aitong Gao

A Lactobacillus strain L2 that produced a higher amount of γ(gamma)-aminobutyric acid (GABA) was isolated from traditional pickles. Phylogenetic analysis based on the 16S rDNA sequence and morphological and biochemical studies indicated that it may belong to Lactobacillus brevis. Under un-optimized conditions in MRS broth with 2% monosodium glutamate (L-MSG), it produced GABA at a concentration of 9.822 g/L after 48 h. On the principle of maximum economic benefit, the next work describes the optimization of fermentation variables for the production of GABA by Lactobacillus brevis L2 using wheat bran (WB) and millet bran (MB) supplemented with L-MSG. Based on a single- factor-at-a-time optimization strategy, we determined the optimal cultivation conditions of the GABA production using WB and MB as described below: initial pH 4.00, reaction temperature 300C and the reaction time 72 h. Subsequently, the D-optimal mixture design was applied to optimize the ratio of the three kinds of components above-mentioned for GABA production. The results showed that when the mixing ratio of WB, MB and L-MSG reached 22.454:20.000:57.546 (g/L), the maximum GABA yield of Lactobacillus brevis L2 reached 27.2069 g/L. Analysis of variance for the regression model suggested that the GABA yield showed close agreement with the model prediction.

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Biomass equations and aboveground biomass carbon stocks of Kermes Oak (Quercus coccifera L.) in Southwestern Turkey

Karatepe Yasin and Lim A. Bahtiyar

The aim of this study was to investigate the aboveground biomass and carbon stocks stored in the biomass on the shrublands of Kermes oak (Quercus coccifera L.), which is a very common species within the maquis vegetation of the Mediterranean Region in Turkey. To this end, the aboveground biomass components (branches, leaves and stems) were cut and collected from 1 m2 plots in 10 study sites with 3 replications and they were weighed separately. Furthermore, carbon concentration in the biomass components was measured. Then, biomass and carbon stocks in the study sites were calculated taking account of the canopy cover. The carbon concentration was found as 51.14% for leaves, 49.15% for branches and 48.56% for stems. The weighted average carbon concentration of the aboveground mass was found to be 49.27%. On the other hand, the carbon density varied from 4.35 to 40.59 Mg ha-1 (mean 16.65 Mg ha-1), which indicated a considerable amount of carbon stock in the kermes oak shrubs. Moreover, biomass equations were also formulated under this study. The basal area at ground level (G, cm2 m-2) and mean shoot length (H, m) were used separately or as G2H in all equations as independent variables. The significance level of all biomass equations is P < 0.001.

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Molecular authentication of Hippophae species and subspecies and their phylogenetic analysis based on nuclear ITS sequences from north western Himalayan region of India

Jadhav M. S. and Sharma T.R.

Sea buckthorn is one of the important medicinal plants, it is also known as gold mine of Himalayas. Traditionally taxonomic classification of genus Hippophae is based on different morphological and biochemical features. These methods alone can be affected by many factors, leading to subtle and ambiguous results. In the present study internal transcribed spacer regions (ITS) markers were used for authenticating and establishing phylogenetic relationship between Hippophae species and sub-species. ITS marker authenticated ecotypes from Lahual region as H. rhamnoides subspecies turkestanica with 98% identity while ecotypes from Spiti, Kinnour and Leh region as H. salicifolia with 97-98% identity. Phylogenetic studies on the ecotypes from four different regions of north western Himalayas revealed three major clades namely H. rhamnoides subspecies turkestanica and H. salicifolia and followed both species specificity and geographic affiliation; an out-group clade of Elaegenus spp. was also obtained. The molecular phylogenetic trees indicated that most of Hippophae species and subspecies are closely related and share common clade while all the out group have separate clade. Present investigation shows the efficiency of ITS markers as molecular tools for species authentication and establishing phylogenetic relationship in Hippophae.

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Nucleotide sequence polymorphism within exon 3 region of leptin and prolactin genes in Herri sheep

Mahmoud A. H., Saleh A. A., Abou-tarboush F. M., Shafey T. M. and Abouheif M. A.

The objective of the present study was to determine the polymorphism in the leptin (LEP) and prolactin (PRL) genes in Herri (n=35) sheep breed of Saudi Arabia. No single nucleotide polymorphism (SNP) was detected in the 156-bp PCR amplicon of PRL exon 3 region. The aligned DNA sequences of the 471-bp fragment of LEP exon 3 showed four non-synonymous SNPs and two synonymous SNPs. The non-synonymous SNPs were found at positions 170 (GGG/AGG-transition), 248 (CTG/CCG-transition), 286 (GTG/TTG-transversion) and 332 (CGG/CAG-transition). The two synonymous SNPs were detected at positions 213 (ACC/GCC transition) and 216 (CCA/CCG transition). The SNP (C) at position 248 was specific to Herri sheep. These sequences were deposited into the GenBank with accession nos. KJ924981-KJ924986. Five genotypes were recognized as homozygotes (01-05) and two were heterozygotes (06 and 07). The most frequent LEP genotype 05 (37%) was also found to be the most common genotype in GenBank database. The present study provided basic information to understand the genetic diversity of Herri sheep breed of Saudi Arabia.

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Expression study of high affinity nitrate transporter gene from Brassica juncea in Arabidopsis T-DNA “knockout”

Verma R., Singh A. K. and Jain V.

Plants have developed sophisticated mechanism to acquire nitrogen (N) efficiently for proper growth and development which is mediated by transporters. Nitrate transporter 2.1 (NRT 2.1) is one of the seven NRT2 family genes that is induced under N starvation. Present study was conducted to investigate the effect of overexpression of BjNRT2.1 (gene Bank accession no JQ305139) transgene from Brassica juncea on nitrate uptake using Arabidopsis mutants. Lacking Atnrt2.1 gene has no IHATS capacity and shows poor growth on low nitrate. Transgenic lines expressing functional 35S:: NRT2.1 transgene exhibit normal growth and restore nitrate transport in the mutant on low nitrate indicating its role in HANT. Genomic southern analysis showed the presence of single copy of T-DNA insertion. Time course analysis of transcript showed strongest degree of induction on the low nitarte concentration as the amount of transcript increased up to 5.2 fold by 30 mins and a 3.4 to 3.8 fold increase was maintained until 2 h. No significant differences were observed at high nitrate provision to induce Nrt2.1 mRNA accumulation. This experiment provides a foundation of future experiments that will help to develop crop plants with better N acquisition efficiency to minimize N fertilizers in agriculture.

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Production of Lignin degrading enzymes namely Laccase, Manganese Peroxidase (MnP) and Lignin Peroxidase (LiP) of selected white-rot fungus in Solid state fermentation (solid media)

Parikh Jatin, Nadagouda Vijaya and Shukla M. D.

The production of the lignin degrading enzymes namely Laccase, Manganese Peroxidase (MnP) and Lignin Peroxidase (LiP) of selected white-rot fungus was examined in this study. Two fungal cultures were studied, both belonging to Ganoderma sp. After screening the cultures for the presence of the enzymes Laccase by Plate Assay techniques, quantitative analysis of all the three enzymes was done by Spectrophotometric method. The effect of different carbon sources namely ligninocellulose containing Rice bran and Wheat bran was examined on the production of these enzymes in solid media. Both the cultures showed diverse enzyme production of all the three enzymes in Rice bran and Wheat bran media. This study demonstrated that media manipulation can change the enzyme production in the fungal cultures. By optimizing the media condition we can maximize the desired enzyme production.

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Molecular Typing of Thermophilic Bacilli isolated from Different Hot Springs of Turkey

Yanmis Derya and Adiguzel Ahmet

In this study, the molecular characterization of 10 thermophilic bacilli isolated from different geothermal areas in Turkey was done by using the fatty acid methyl ester, 16S rRNA sequencing and rep-PCR profiling methods. Depending upon the data obtained from fatty acid analysis, it was determined that there were 8 different fatty acids in the examined 10 bacterial strains and 16:0 and 17:0 anteiso were determined to be the fundamental fatty acid. In consequence of the comparison of 16SrRNA sequence analysis results with the data in GenBank, all the isolates were identified to resemble Aeribacillus pallidus at a rate of ≥98 %. Later on, rep-PCR was performed for the purpose of determining the differences between these isolates. After several primer sets, which were targeting the repetitive DNA elements of REP, ERIC and BOX, had been assessed, the BOXA1R primer was determined to be the most reliable technique for identification and molecular characterization of A. pallidus strains. Therefore it was reasonable to state that, rep-PCR finger printing using the BOXA1R primer may be well considered as a promising genotypic tool for the identification and taxonomic characterization of thermophilic bacteria from species to strain level.

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A Comparative analysis of ISSR and RAPD markers for studying genetic diversity in Trigonella foenum-graecum genotypes

Tomar R. S., Parakhia M. V., Rathod V. M., Thakkar J. R. and Golakiya B. A.

Genetic diversity of thirty genotypes of fenugreek was evaluated using RAPD (Random amplified polymorphic DNA) and ISSR (Inter-simple sequence repeat) markers. Fenugreek popularly known as “Methi” belongs to family Fabaceae and subfamily Papilionaceae, which have many medicinal values. RAPD and ISSR are helpful for accessing genetic diversity of rare and endemic species and also for resolving genetic relationship among population. A total of 30 RAPD primers and 20 ISSR primers were used to detect genetic variation. The amplified products of the RAPD and ISSR profiles ranged from 250 to 1300 bp. A higher proportion of polymorphic bands were observed using RAPD (76.78 %) than ISSR (68.08%). The dendrogram developed by RAPD and ISSR analyses revealed that genotypes are clustered into two main groups. According to RAPD analysis the highest similarity (94%) was between AM-329 and AFG-5. However, minimum similarity was found between AFG-6 and other varieties (46%) and in case of ISSR analysis the highest genetic similarity (89%) was between AM-326 and CO-2 as well as between AM-202 and HISSARSUVARNA. Minimum similarity was found between AM-288 and rest of the genotypes. The result of the present study can be used to identify phylogenetic relationship between genotypes and could be used in breeding programs and conservation of medicinal plant.

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