Open Access Original Research Article

Improvement of Regeneration of Pelargonium radula via Somatic Embryogenesis

A. R. Zuraida, M. A. Mohd Shukri, M. N. Erny Sabrina, O. Ayu Nazreena

Biotechnology Journal International, Page 166-173
DOI: 10.9734/BBJ/2015/15337

In vitro stem segments of Pelargonium radula cultured for callusing then differentiated into somatic embryos and subsequently regenerated plantlets. Initiation of callus was observed in culture medium containing low concentrations of the plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and/or α-naphthalene acetic acid (NAA). At 0.2 mg/L 2,4-D and 0.2 mg/L NAA was showing the highest rate (92%) of callus induction. The callus showed no sign of browning after sub-cultured. Sub-culturing the callus onto medium with 0.2 mg/L 2,4-D showed the highest in proliferation rate  resulted 13.45g weight of callus. The presence of agar at 6 g/L and 0.5 mg/L Gibberellic acid (GA3)  improved the regeneration of the somatic embryos, which produced maximum number of plantlets (15 plantlets). Agar with concentration of 9 to12 g/L increased the incidence of browning.  The former medium was more successful in plantlet regeneration when the selected embryos were subsequently transferred to regeneration medium with 3 g/L agar, 0.5 mg/L GA3 and 0.5 mg/L Benzylaminopurine (BAP).

Open Access Original Research Article

Selenium Treatment Alleviated Oxidative Alteration Generated by Cadmium in Sunflower Roots

S. Issam, D. Wahbi, C. Yacine

Biotechnology Journal International, Page 174-181
DOI: 10.9734/BBJ/2015/14802

Selenium Treatment Alleviated Oxidative Alteration Generated by Cadmium in Sunflower Roots

The present study investigated the role of selenium (Se) in regulating cadmium (Cd)-induced oxidative stress in sunflower (Helianthus annuus) roots. Short-term exposure of plants to 20 µM Cd intensively increased hydrogen peroxides (H2O2), protein carbonyl (PCO) content and lipid peroxidation as indicated by malondialdehyde (MDA) accumulation. In contrast, Se (5, 10 and 20 μM) pretreatment alleviated the oxidative damages as evidenced by the lowered H2O2, protein carbonyl (PCO) and MDA content. Concomitantly, Se treatment enhanced the activities of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione peroxidase (GPX, EC 1.11.1.9), but lowered that of superoxide dismutase (SOD, EC 1.15.1.1) in the root exposed Cd. Taken together, our results strongly suggest that exogenous Se may improve the tolerance of the plant to the Cd-induced oxidative stress.

Open Access Original Research Article

Purification and Characterization of α-Glucan Phosphorylase Isoform Pho 2 from Spinach Leaves

Ritu Jain, Anil Kumar

Biotechnology Journal International, Page 182-195
DOI: 10.9734/BBJ/2015/15106

α-Glucan phosphorylase is an important enzyme of carbohydrate metabolism. In spinach leaves, it has been reported in two multiple forms viz. Pho 2 (cytosolic) and Pho 1 (plastidial). Here, we extracted and purified Pho 2 form of α-glucan phosphorylase from spinach using salting out with ammonium sulfate, desalting using Sephadex G-25 chromatography, anion exchange chromatography using DEAE-Sepharose and gel filtration chromatography using Sepharose-4-B. The purified enzyme had a specific activity, 150 units/mg protein. There was 38% recovery and 652 fold purification after final Sepharose-4B chromatography. The purified enzyme showed a single protein band on SDS sodium dodecyl sulfate polyacrylamide gel electrophoresis having molecular weight 94,000±2000 daltons. The native molecular weight is found to be 188,000±3000 daltons as determined using gel filtration chromatography over Sephadex G-200. The Pho2 exhibited optimum pH at pH 6.0 with two half pH optima at pH 5.2 and pH 7.0. The optimum temperature of Pho2 is found to be 37ºC with two half temperature optima at 30ºC and 40ºC. The Km value of the enzyme for starch and glucose-1-phosphate is found to be 116 µg/mL and 0.55 mM, respectively.

Open Access Original Research Article

Production and Partial Characterization of Pectinase from Snail (Archachatina marginata) Gut Isolates

E. C. Egwim, T. Caleb

Biotechnology Journal International, Page 196-205
DOI: 10.9734/BBJ/2015/11449

Aim: To identify isolates for industrial production of pectinase.

Methodology: The isolates were screened for pectinolytic activity using pectin as substrate. Enzyme activity was expressed as mg of glucose equivalent released per ml of crude enzyme solution per second. The kinetic parameters of the enzyme were determined to obtain the optimum pH, temperature, Km and Vmax. Pectinase produced from Bacillus subtillis was immobilized on chitosan and its activity was compared with that of free enzyme.

Results: Pectinase from Bacillus subtilis was screened to have the highest enzymatic activity among bacterial isolates while pectinase from Aspergillus niger had the highest enzymatic activity among fungal isolates. High yield of pectinase enzyme was obtained from B. subtilis after 24hrs with activity of 4.01×10-4 mg/ml/sec while high yield of pectinase was obtained from A. niger on the 5th day with activity of 2.07×10-4 mg/ml/sec. The optimum pH for pectinase produced from B. subtilis and A. niger were 8 and 6, respectively. The optimum temperature for pectinase from B. subtilis and A. niger were at 50ºC and 40ºC, respectively. The Vmax and Km of pectinase from B. subtilis and A. niger were 16.88×10-4 (mg/ml/sec) and 10 (mg/ml); 9.29×10-4 (mg/ml/sec) and 30 (mg/ml), respectively. Optimum temperature and pH of immobilized pectinase were 70ºC and 4.0, respectively. Residual activity of immobilized enzyme was 92% after storage at 4ºC for 14 days.

Conclusion: This study revealed that Bacillus subtilis from snail gut may be considered as a good candidate for industrial production of pectinase.

Open Access Original Research Article

Microsatellite DNA Marker Analysis Revealed Low Levels of Genetic Variability in the Wild and Captive Populations of Cirrhinus cirrhosus (Hamilton) (Cyprinidae: Cypriniformes)

Md. Abul Hasanat, Md. Fazlul Awal Mollah, Md. Samsul Alam

Biotechnology Journal International, Page 206-215
DOI: 10.9734/BBJ/2015/14895

Microsatellite DNA Marker Analysis Revealed Low Levels of Genetic Variability in the Wild and Captive Populations of Cirrhinus cirrhosus (Hamilton) (Cyprinidae: Cypriniformes)

Aims: To reveal the genetic variability of wild and captive populations of the Indian major carp, mrigal (Cirrhinus cirrhosus) based on microsatellite DNA markers analysis.

Study Design: Three rivers namely the Halda, the Padma and the Jamuna were selected under wild population category and three hatcheries such as Brahmaputra Hatchery of Mymensingh, Raipur Government Hatchery, Luxmipur, and Sonali Hatchery, Jessore were selected under the captive population category. 

Place and Duration of the Study: Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh from July 2005 to June 2008.

Methodology: DNA was extracted from fin clips of a total of 180 fish, 30 from each of the six populations. Five microsatellite markers MFW1, MFW2, MFW17, Barb54 and Bgon22 were amplified by polymerase chain reaction for each DNA sample and resolved on denatured polyacrylamide gel and visualized by silver nitrate staining.  

Results: Three of the five loci were found to be polymorphic in all the six populations. The observed (HO) and expected heterozygosity (HE) ranged from 0.233 to 0.633 and 0.406 to 0.664 respectively. The FIS values ranged from 0.032 to 0.635 indicating deficiency in heterozygosity. Except the Raipur Hatchery stock, the other five populations showed nonconformity to Hardy-Weinberg Expectation at least in one locus. Significant population differentiation was observed between the Halda-Jamuna, Jamuna-Brahmaputra Hatchery, Jamuna-Raipur Hatchery and the Padma-Raipur Hatchery population pairs. The UPGMA dendrogram based on genetic distances resulted in two major clusters: the Halda river and the Raipur Hatchery population were in one cluster and the remaining four populations were in the other cluster.

Conclusion: The study, as a whole, revealed low levels of genetic variation in terms of allelic richness and heterozygosity in the three major rivers and three selected hatchery stocks of C. cirrhosus in Bangladesh.