Research Journal of Biotechnology

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Vol. 10(12) December 2015

Effect of K+ Nutrition on Plasma Membrane H+-ATPase of Suaeda salsa under Salt Stress

Zhou Feng and Hua Chun

Suaeda salsa seedlings grown in Hoagland nutrient solution were treated with different concentrations of NaCl combined with two levels of K+ (0 and 6 mmol/L) to study the effect of K+ nutrition on the growth and activity of leaf plasma membrane proton-translocating adenosine triphosphatase (PM H+-ATPase). The leaf PM H+-ATPase activity was significantly increased with increasing NaCl concentration under high K+ application (6 mmol/L) but little changed under K+ starvation (0 mmol/L). Western blot analysis showed that the subunit of leaf PM H+-ATPase and its protein amount were not affected by NaCl treatments under K+ starvation (0 mmol/L) but increased under high K+ application (6 mmol/L). There was a positive correlation between activity of PM H+-ATPase and the protein amount of PM H+-ATPase. The results suggest that K+ nutrition played an important role in the salt tolerance of S. salsa and that enhancement of PM H+-ATPase activity under salt stress was K+-dependent.

Ecological vs. Conventional Broiler Chickens System rearing in Romania

Simeanu D., Pop I.M., Grădinaru A.C. and Simeanu Cristina

The present study aimed to evaluate the efficiency of the ecological rearing system for broiler chickens in comparison with the conventional one. Our research was carried out in an avian factory from the NE of Romania, using 24,000 subjects belonging to commercial hybrid “Ross-308”, starting with their first day of life. All of the subjects were divided in two batches (L1 and L2), each of them with 12,000 individuals. The individuals from L1 batch were accommodated in a common shelter with permanent layer (the “conventional” system) and the ones from L2 batch into a shelter for meat ducklings adapted for broiler chickens, with free access in paddock (the “ecological” system). A complete mixed fodder was administrated according to their days of life and a pre-biotic treatment in order to determine a good state of health and a good capitalization of feed were used. The microclimate conditions such as the ambient temperature and the relative air moisture were analyzed for the entire period of the study. The individual body weight was determined at each seven days, starting with the first one. At the end of those 42 growing days, the conversion index of feed, losses from the groups and the European efficiency factor were calculated. Our results showed a poor performance of the subjects included in L2 batch compared to the one of the L1 batch and both of them were compared to one of the standard “Ross-308” rearing guide. Only at the seven days’ weighting, the cumulated gain was higher for the individuals included in L2 batch and the conversion index of feed was 12.72% higher for the L2 group compared to the L1 group for the entire period of study. During the entire experimental period, 2.54% of individuals from L1 batch and 8.39% from L2 batch died due to accidental causes, helmints gastroenteritis, lung diseases, fractures etc. The European efficiency factor was 23.92% higher for L2 group than for L1’s.

Apoptotic Potential of Ethyl Acetate Fraction of Cochliobolus Spicifer (Pleosporales: Pleosporaceae) isolated from Phoenix Dactylifera L. on Human Cancer Cell Lines

Nael Abutaha

Endophytes are microorganisms that survive inside the plant tissue and produce compounds of great pharmaceutical benefit. Solvent extracts were tested for antiproliferative activity against the different cancer cell lines employing the 3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Apoptotic potential of the ethyl acetate fraction was determined using acridine orange, ethidium bromide staining and caspase3/7 activity. Cell migration was evaluated by scratch wound healing assay. The ethyl acetate fraction (F1A) showed the most potent cytotoxic activity in dose dependent manner for all the cell lines examined. The IC50 values on MCF7, HepG2 and Jurkat cells were 171.2, 121.34 and 117.90 respectively. The percentage of apoptotic nuclei after treatment with 170μg/ml of extract was significant when compared to control 49±4.9% at the concentration of 60 μg/ml. F1A fraction treatment significantly decreased the migration rate of HepG2 cells. Results indicated the potential for production of bioactive metabolites from endophytes cochliobolus spicifer. Considering the apoptotic potential of the examined F1A, they might have the potential to serve as a template for future anticancer drug development. Further investigation to identify the mode of action is required.

Isolation and characterization of acid- and pepsin- soluble collagens from Lagocephalus gloveri skin

Kirti and Khora Samanta S.

Fish skin is a major by-product of the fish processing industry, causing wastage and pollution, and could provide a valuable source of collagen which has wide biomedical and pharmaceutical applications. Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of Lagocephalus gloveri showed the yield of 8 and 17% respectively and were characterized to type I collagen, containing two different α chains (α1 and α2) and one β chain. Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and were found to be similar. The denaturation temperature (Td) of ASC and PSC was found to be 29.3 °C and 28.4°C respectively. There was no major difference in solubility observed for both ASC and PSC at various pH and for different NaCl concentrations. Hence, there is possibility to use collagen from Lagocephalus gloveri skin as an alternative source of collagen for use in various applications.

Characterization and evaluation of seed extracted proteins by using spectrophotometric and two dimensional electrophoresis (2-DE) method in Sunflower (Helianthus annuus L.)‏

Alireza Shabani

Total sunflower proteins, storage proteins and helianthinin (11S) and 2S albumin fractions in seeds of six sunflower hybrids, Lotto3620, Lotto7R09, LottoR101, Lotto7R0, LottoR28, LottoR02 and Cms8, Mt8, TR3 were analyzed by using optical density approach. Protein contents were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coupled with densitometry. The SDS-PAGE profiles of the seed proteins in the crude extracts for all genotypes showed a very similar number of protein bands in the electrophoretograms. Three polypeptide groups of helianthinin fraction were detected. Two of these were acidic (αMw= 36.8-42.9KDa and α', Mw =31-35.3KDa) while one was basic (β, Mw=21-29KDa). The molecular weight of the 2S albuminproteins ranged from 11.5KDa-20.1KDa. According to our results, there were significant differences among the seed protein concentration in different hybrids since the highest optical absorption ratio and protein concentration observed in LottoR02. In contrast, the lowest optical absorption and consequently protein concentration was observed in Lotto3620.

Research on Gene Introgression from Common Wheat into Aegilops L

Ximin Cao and Aizhi Lv

We carried out a group of experiments to verify possibility of gene introgression from common wheat into Aegilops. The artificial indoor crossbreed was conducted using 7 genotypes from 4 wheat relative species as female and common wheat as male. The experiment result shows that different species have variable cross ability. Among the 4 Aegilops species, the highest cross rate is from the combination of Ae. tauschii T. aestivum (46.49% for genotype Ae42, 22.58% for Y92), the second is from Ae. ovata  T. astivum (14.76% for Y100, 12.11% for Ae23), the third is from Ae. cylindrica T. astivum (2.23% for Ae7, 8.50% for Y145), the lowest is from Ae. speltoides  T. astivum (0.19%). Hybrid embryos from different combinations have different abilities of callus initiation and germination. The hybrid embryos from A. ovata / T. eastivum and Ae. tauschii / T. eastivum have higher level of callus initiation and germination, Ae. cylindrica / T. eastivum has middle level, while the Ae. speltoides has lower level. The interspecific hybrids between Aegilops and common wheat have so low fertility. In back-crosses, the seed-set rate of hybrids of Ae. ovata / T. aestivum is 3.71% and 4.36% respectively back-crossed with male and female parents while for hybrids of A. cylindrica / T. aestivum, it is 0 and 0.33% respectively and for A. tauschii / T. aestivum, 0.33% and 0 respectively. On selfing of the hybrids, the seed-set rate is 0 (no seed set from 9750 florets) for the combination of Ae. cylindrica / T. aestivum, 0.044% (3 selfed seeds out of 6870 florets) for A. ovata / T. aestivum and 0 (no seed set from 7253 florets) for A. tauschii / T. aestivum. The research suggested that the probability of gene introgression from T. aestivum into Aegilops species is very low in nature.

Cloning, sequence characterization and expression analyses of PK gene from pepper cytoplasmic male sterility (CMS)

Jun-heng Lv, Jin-long Huo, Kai Zhao and Ming-hua Deng

Pyruvate kinase (PK) is an important regulatory enzyme of the pathways of both anaerobic glycolysis and gluconeogenesis, it plays an important function in energy metabolism. The complete coding sequence (CDS) of the PK gene was amplified using a reverse transcriptase PCR based on the conserved sequence information of the tomato and other Solanaceae plants and known highly homologous pepper ESTs. 510 amino acids were encoded by the 1533 long cDNA of the pepper PK gene. Nucleotide sequence analysis showed that the encoded amino acids were highly homologous with the seven species: Solanum lycopersicum (97%), Solanum tuberosum (95%), Jatropha curcas (93%), Phaseolus vulgaris (93%), Vitis vinifera (92%), Prunus persica (92%) and Glycine max (92%). The result of the tissue expression showed that the pepper PK gene is over expressed in placenta, seed and flower, moderately in pericarp and stem, weakly in leaf. During the abortion stages, expression levels of PK in anthers of a sterile line were higher than that in the maintainer line.

The quality of meat in two species of fish from Cyprinids family

Valerica Gilca and Ioan Gilca

The biochemical analyses of the meat were determined in two species of cyprinids respectively Cyprinus carpio and Hypophtalmichthys molitrix. We worked on four groups of 10 fishes each (two groups for C. carpio and two H. molitrix). One group from both species was fed with combined feed and the other two groups received natural food (plankton). Measurements were performed in the laboratory of chemical analysis of the Faculty of Animal Sciences, University of Agricultural Sciences and Veterinary Medicine Iasi and we determined the content of meat in proteins, fat, dry substance and minerals. The chemical characteristics of meat were determined on the extracted sample from the lateral musculature of the body. Biochemical analyzes of meat show that protein content is higher when groups fed with special fodder compared to the groups fed on plankton. Therefore, in case of feeding fish with combined feed the protein content of meat increases which proves a good recovery of protein from combined feed in muscle, compared to assimilate protein from plankton. Fat and minerals contents in meat ciprinids studied are both higher in the individuals fed with combined feed than individuals fed natural food. When fat content in meat is higher, the dry matter content in meat is higher. When the fat content is higher, dry matter also increases.

Soil Characteristics of Some Endemic Geophytes growing around Lake District, Turkey

Fakir Hüseyin, Karatepe Yasin and Gürlevik Nevzat

A total of 816 geophyte plant taxa belonging to 73 genera naturally grow in Turkey flora. Considering that they hold to and take root in topsoil, geophyte plant species are potentially the most negatively affected species from global warming as evaporation most intensively occurs in topsoil. Furthermore, since especially bulbous, root, rhizome, tuber and flowers of geophyte plants are used as ornamental, drugs and spices, they are intensively and constantly collected from the nature. This study investigated soil characteristics of some endemic geophytes that are naturally distributed in Lake District of Turkey to facilitate future cultivation and conservation of these species. Nine endemic geophyte plant taxa belonging to 3 families were identified in the study carried out in 2011-2013 period. Of the identified species, 6 were Mediterranean element, while 3 were Irano-Turanian element. Common soil texture in the natural distribution area of plants was found to be clay and loamy clay, with a pH value of 5.5-8.18; lime ratio of 1.1-37.8 % and organic matter content of 0.6-26.7 percent. According to ecological spectrum of plants, 2 species were distributed in macquis areas, 2 species were distributed in rocky areas and 5 species were distributed in forests.

In Vitro Anti-Tumor Activity of L-Glutaminase from Marine Bacillus Subtilis Strain JK-79

Jambulingam Kiruthika, Nachimuthu Saraswathy, Sudhakar Swathi and Ilangovin Sri Vani Uthra

Lung and oral cancer are one of the most common and serious type of cancers. Over nearly sixty lakh people are diagnosed with this condition over year in all over the world and the leukemic, ovarian and oral and colon cancer had made notable steps forward in the past decade. L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukemia. Accumulating evidences suggest the beneficial effects of amino acid-depleting enzymes in lowering the risk of various cancers. The present study was focused to determine the anti-tumor activity of L-glutaminase. The L-glutaminase enzyme was purified from marine Bacillus subtilis strain JK-79 (KC492745) and tested for their antitumour property by employing MTT assay against the human cell line such as HL60(Leukimic Cell lines), KB(Oral Cancer Cell line), A549 (Lung Cancer Cell line),OVCAR3 (Ovarian Cancer cell line) and HT115 (Colon Cancer Cell line). The results show that the L-glutaminase enzyme proves to be very effective against all the different types of cell lines. The IC50 for leukemic cell line is found to be 500 μg/ml and IC50 for lung cancer and colon cancer is 750μg/ml whereas IC50 value for ovarian cancer and oral cancer cell line is found to be 600μg/ml. Thus, L-glutaminase from marine Bacillus subtilis JK-79 is a promising broad range therapeutic agent.

Callogenesis, rhizogenesis and cell suspension culture of bryonia laciniosa

Supe Ujjwala

The present study was carried out to investigate in vitro callogenesis and direct rhizogenesis of bryonia laciniosa. This medicinal plant grows in various regions of Chattisgrah. Leaf explants were incubated on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of auxins likeNAA, IAA, and IBA (0.5 to 3 mg/L) with cytokinins like Kin and BA (0.1 to 0.5 mg/L). Callus induction from leaf explants was achieved at all MS media containing auxins in combination with cytokinins. Most of these treatments revealed 100% callogenesis response. Results showed that the MS medium supplemented with 2 mg/L IBA was the best medium to induce adventitious roots. In this treatment, percentage of rhizogenesis and the number of roots per explant were recorded as 76.82% and 46.400±14.258 respectively. Callus and adventitious root cultures of bryonia laciniosa were established successfully.

In silico mining and characterisation SSR markers for studying the genetic diversity of Anthurium

Sudarshini Venkat K., Lakshaman Reddy D. C. and Aswath C.

Co-dominant genetic markers are required for various genetic studies in Anthurium since the species is highly heterozygous in nature. The recently explored next generation sequencing technology of anthurium has generated large amount of transcribed sequences which are available for identifying simple sequence repeats (SSR) marker. A total of 411416 contig sequences were analysed and 16389 SSRs were identified. Di-nucleotide repeats were the most abundant motif class. A total of thirty random primers were used for studying diversity analysis of 19 anthurium varieties. Seventeen polymorphic SSR primer pairs showed variation in the banding pattern showing 56.6% polymorphism. The mean heterozygosity and polymorphic information content were 0.2 and 0.41 respectively. These SSR markers not only enrich the current resources of molecular markers in anthurium but also would facilitate in various mapping and marker assisted selection.

Spider diversity in transgenic and non-transgenic cotton in rainfed agro ecosystem of central India

Nagrare V. S. Deshmukh Vrushali, Naikwadi Bhausaheb, Bhoyar Paresh and Khadakkar Suvarna

Spider diversity in transgenic and non-transgenic cotton in rainfed agro ecosystem of central India was studied during crop seasons 2013-14 and 2014-15. Fifteen species of spiders belonged to 6 families viz., Araneidae, Oxyopidae, Thomisidae, Salticidae, Tetragnathidae and Theridiidae. Family Araneidae contributed one third spider population (34.56%) followed by Oxyopidae (27%) and Thomisidae (24.53%). Frequent occurrence of Neoscona theisi followed by Oxyopes pankaji and Thomisus spectabilis was recorded. Maximum population of spiders was observed during second fortnight of October to first fortnight of November that coincides with boll development stage. Spider population varied in the cotton crop season in the cotton field, maximum population was observed in the month of October and November. The diversity and evenness indices were calculated for the collected spiders. The Shannon index value was 1.64, Simpson index value was 0.84, while Evenness index was 0.92. Positive correlation of spiders with prey density of cotton sucking pests was observed in case of whitefly and mirid while it was negatively correlated with aphid, leafhopper and thrips. Spider population was negatively correlated with all the weather parameters in transgenic cotton while positively correlated in non-transgenic cotton. No noticeable effect of either genotype variation (transgenic or non-transgenic) or limited pesticide intervention has been observed on spider population.

Evaluation of physiological and morphological responses associated with cotton subjected to drought stress conditions

Bonde Shilpa, Chandrasekhar C. N. and Pusadkar Pratik

Drought is an abiotic stress that affects performance and growth of cotton genotypes. Therefore, the identification of tolerant genotypes to drought is of utmost importance in cotton for germplasm selection and further improvement. The experiment was conducted in three genotypes of cotton (GossypiumhirusutumL.)cvs. Pratima, Anjali and Bunny Bt under pot culture technique by using gravimetric method for study of drought tolerance in cotton. A factorial completely randomized design with three replications was used. The research trials were carried out during the period from August 2012 to April 2013 with pot culture experiment in the glasshouse, Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore. The stress imposed with four watering treatments viz., 100, 75, 50 and 25% of field capacity at squaring stage to understand the effects of water deficit in terms of physiological and morphological parameters studied. The total chlorophyll content (SPAD) values were highest in control as that of with drought stress condition. Gas exchange attributes such as photosynthetic rate, stomatal conductance and transpiration rate were found to be remarkably higher in cotton variety Bunny Bt compared to Pratima and Anjali during water deficit. The morphological characters plant height, number of leaves and angle of branching were reported to be reduced in cotton genotypes with different watering treatments. The study concludes that both physiological and morphological parameters were found to be reduced under drought stress conditions.The drought stress imposed conditions revealed that Bunny Bt was considered as more tolerant and stable genotype.

Development of Inter Simple Sequence Repeat based SCAR marker for sex determination in Carica papaya

Tomar Rukam S., Parakhia M. V., Rathod V. M., Kheni J. V., Thakkar J. R., Thummar V. D., Padhiyar S. M. and Golakiya B. A.

Carica papaya belonging to Caricaceae family is a herbaceous nutritive fruit tree, which has medical applications like anti-helminthes, anti-inflammatory, antiparasitic, antiseptic and antidiabetic. From commercial agricultural point of view planting of female papaya plant is more beneficial, since only female plants are grown for fruits. Sexuality of plant cannot be distinguished before floral initiation. Recently, many PCR based techniques were used for sex determination in plants at earlier stage like ISSR molecular marker and many more. However, they have the limitation of poor reproducibility and stability which can be overcome by conversion of ISSR markers into specific and reproducible marker like sequence-characterized amplified regions (SCAR). In the present study, 100 ISSR DNA markers were screened for sex determination of papaya. Finally four primers with amplicon size UBC-880 (600 bp), UBC-812 (600 bp), UBC-817 (600 bp) and UBC-857 (1200 bp) were selected for gene cloning. Clones were sequenced and new primers derived from these sequences were used to obtain Sequence Characterized Amplified Region (SCAR) fragments. Four SCAR markers with product size of 410 bp form cps-1, 782 bp from cps-2, 427 bp from cps-3 and 487 bp from cps-4 were obtained. These primers were validated in the individual Male and Female plants of “Madhubindu” variety of Carica papaya and were able to detect sexuality of papaya at seedling stage before flowering.

Virulence analysis and molecular characterization of Puccinia triticina pathotypes causing wheat leaf rust in India

Manjunatha C., Aggarwal Rashmi, Bhardwaj S. C. and Sharma Sapna

Leaf rust of wheat caused by Puccinia triticina is one of the most wide spread and important diseases occurring in India. New pathotypes keep on evolving by overcoming the resistance genes present in the wheat varieties. Some of the predominant leaf rust pathotypes in India are 10, 12, 12-1, 12-2, 12-3, 12-4, 12-6, 77-1, 77-2, 77-3, 77-4, 77-5, 77-6, 77-8, 77-9, 104, 104-1, 104-2, 104-3, 104-4, 106, 162, 162-1 and 162-2. In the present study twenty four pathotypes of P. triticina were genetically characterized using URP primers. Out of the 12 URP primers taken for the study, 9 primers gave reproducible bands. Our results showed 40 to 90% polymorphism with amplified bands ranging from 250 to 4000 bp. There was positive correlation between URP based clustering and virulence/avirulence behaviour, geographical origin, evolution time of different leaf rust pathotypes. Some of the URP primers viz., URP 1F, URP 4R, URP 13R and URP 17R grouped all the pathotypes of race 104 group (viz., 104, 104-1, 104-2, 104-3 and 104-4) into a single cluster. In the present study 24 monomorphic bands were produced, these can be further utilised in development of species specific diagnostic marker for early detection of P. triticina. Our results indicate that URP’s are sensitive markers which give reproducible results to study the genetic variability in P. triticina.