Open Access Original Research Article

Effect of Cytokinins on In vitro Propagation of Boucerosia umbellata (Haw.) Wight & Arn. (Syn.: Caralluma umbellata Haw.) from Nodal Explants of Field Grown Plants

B. Susheela, S. Sandhya Rani, T. Pullaiah

Biotechnology Journal International, Page 1-11
DOI: 10.9734/BBJ/2016/23676

An efficient protocol was described for the rapid in vitro multiplication of medicinal plant Boucerosia umbellata via enhanced axillary bud proliferation from nodal explants collected from young shoots of three-month-old field grown plants. Effects of cytokinins 6-benzylaminopurine (BAP), Thiadiazuron (TDZ), Kinetin (Kin) and 2-isopentenyl adenine (2iP) individually and in combination have been investigated. MS medium supplemented with BAP (2 mg/l) and TDZ (1 mg/l) gave maximum response for multiple shoot induction with a shoot sprouting frequency of 86% and mean shoot number of 3.9±0.16 per node and 2.04±0.05 cm shoot length. Rooting was best achieved on ½ strength MS (Murashige and Skoog, 1962) medium supplemented with Naphthalene Acetic Acid (NAA) (0.1 mg/l). The regenerated plantlets were successfully established in the garden with 73% survival rate.

Open Access Original Research Article

Contribution of GFP Expressing Dermal Papillae Cells to the Formation of Chimeric Embryos and their Survival in Uterine Environment

Alla Madich, Gavin D. Richardson, Colin A. B. Jahoda

Biotechnology Journal International, Page 1-12
DOI: 10.9734/BBJ/2016/23393

Aims: This is an innovative cell-based research project focused on the hair follicle dermal papillae (DP) as a source of key-element cells for regeneration of hair growth. DP is major dermal compartment which has a role in hair formation at embryogenesis and is able to differentiate down an endothelial lineage for in vitro functional assays. In the present study, we have tested the potential of DP cells from the lower end bulb region of hair follicles for multilineage differentiation              in vivo. We have postulated that DP cells’ epigenetics can be changed under the influence of embryonic microenvironment when they are injected into early embryos which would demonstrate plasticity, regenerative and inductive properties of hair follicle dermal cells.

Study Design: Pilot research.

Place and Duration of Study: School of Biological and Biomedical Sciences, University of Durham, the UK, between February 2007 and December 2009.

Methodology: To identify the capacity of functional contribution to organogenesis we microinjected various numbers of Green Fluorescence Protein (GFP)-expressing Dermal Papillae cells (GFP-DP cells) into mice blastocysts and transferred embryos to the foster mothers for the generation of chimeric fetuses.

Results: GFP-expressing cells were detected in 3% of the epiblast egg cylinders formed from microinjected blastocysts cultured in absence or presence of mouse embryonic fibroblasts (MEFs). Surgical transfer of microinjected blastocysts resulted in 4.5% of fetuses developing fully. GFP- DP cell lineages were detected in tissue samples under fluorescence and confirmed immunohistochemically. Our finding displayed subcultural localization of GFP as a proof of chimaeric tissue from unborn pups carrying DP cell lineages which had multiplied in bone marrow, parenchyma and connective tissue, brain and hair bulbs.

Conclusion: The results confirm the capability of a small population of DP cells to convert into embryonic stem-like cells, multiply and contribute to organs and tissue. This makes them a reasonable source for regenerative studies and replacement therapy.

Open Access Original Research Article

Isolation and Identification of Actinomycetes from Mangrove Soil and Extraction of Secondary Metabolites for Antibacterial Activity

Anasuya Priyadarshini, Sameer Kumar Singdevsachan, Subodh Kumar Tripathy, Yugal Kishore Mohanta, Jayanta Kumar Patra, Bijay Kumar Sethi

Biotechnology Journal International, Page 12(2)
DOI: 10.9734/BBJ/2016/24102

The mangrove ecosystem of India is an extensively unexplored source for actinomycetes with the potential to produce secondary metabolites of biological importance. In this study, twenty two actinomycetes were isolated from different soil samples collected from the Bhitarkanika mangrove forest along Odisha coast, India. These isolates were identified as Streptomyces sp. based on their morphological, physiological and biochemical characteristics as described in the International Streptomyces Project. Out of twenty two actinomycetes (designated as BSA-1 to BSA-22) isolates, only four isolates (BSA-5, BSA-10, BSA-11 and BSA-15) displayed significant antimicrobial properties in term of antagonistic activity against six human pathogenic bacterial strains (Staphylococcus aureus, Shigella flexneri, Bacillus licheniformis, Bacillus brevis, Pseudomonas aeruginosa and Escherichia coli). All these isolates exhibited excellent antimicrobial activity in a range of 14.0-22.0 mm as inhibition zone against the above studied human pathogens with highest activity displayed by the isolate BSA-11. The isolate, Streptomyces sp. BSA-11 was further identified up to the species level by 16S rRNA gene sequence analysis. The BLAST analysis confirmed that Streptomyces sp. BSA-11 was homologous to Streptomyces himastatinicus of order Actinomycetles and class Actinobacteria. The novel actinomycete, Streptomyces himastatinicus BSA-11 from Bhitarkanika has the ability to produce extracellular potent bioactive compounds which can be a potential source of many antimicrobials.

Open Access Original Research Article

Ochratoxin A Production by Cocoa Infested Aspergillus Species in Ondo State, Nigeria

Olukayode Olugbenga Orole

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BBJ/2016/24139

Aim: To determine ochratoxin A production by cocoa bean seeds infested Aspergillus species in Ondo State, Nigeria.

Place and Duration of Study: Cocoa bean seeds were randomly collected from six local government areas namely Idanre, Akure, Owo, Akure, Oba-Akoko, and Ile-Oluji known to be highest cocoa seed producers. Samples were collected for a period of one year duration.

Methodology: Sterilized cocoa seeds were plated on potato Dextrose Agar and Sabourard Dextrose agar after which toxigenic strains were inoculated on Czapek Yeast Extract Agar and the resulting culture homogenized and spotted on TLC plate for viewing. Suspected ochratoxin A producers were inoculated separately on sterile cocoa bean seeds for 21 days after which OTA extraction were done and quantified using HPLC.

Results: Fourteen Aspergilli were isolated and identified. Twelve in Ikpenmen, eleven in Akure while the last seven were isolated from samples collected in Idanre. Aspergillus alutaceus were only isolated from Ikpenmen for the 12 months. Aspergillus ochraceus, A. niger, A. niger aggregate, A. carbonarius, A. aculeatus, A terreus, A vesicolor all produced OTA at varying concentration.                  A. ochraceus isolated from Ikpenmen samples produced the highest amount of OTA (372 µg/kg) while samples infested with A. niger aggregate from Akure alone produced OTA 109 µg/kg.                        A. carbonarius from all collection venues produced ochratoxin A OTA produced in Oba-Akoko by specie A. carbonarius had the highest value of 539 µg/kg culture.

Conclusion: The study showed that Aspergillus ochraceus, A. niger, A. niger aggregate,                           A. carbonarius, A. aculeatus, A terreus, A vesicolor were the major OTA producers contaminating stored cocoa produce in Ondo State, Nigeria.

Open Access Original Research Article

Factors Affecting on Induction of Microrhizomes in Ginger (Zingiber officinale Rosc.), Cultivar Local from Sri Lanka

D. B. R. Swarnathilaka, N. S. Kottearachchi, W. J. S. K. Weerakkody

Biotechnology Journal International, Page 1-7
DOI: 10.9734/BBJ/2016/23903

Aims: In-vitro produced microrhizomes in ginger are favored as planting material over other conventional planting materials as they are free from soil born pathogens. This study was conducted to develop an efficient protocol for production of healthy microrhizomes including the investigation of effect of different growth regulators, sucrose concentration and photoperiod exposure levels.

Place and Duration of Study: The entire study was conducted in plant tissue culture research unit of the Department of Export Agriculture, Walpita, Sri Lanka between November 2013 and August 2015.

Methodology: In-vitro produced shoots established in hormone free medium were cultured in Murashige and Skoog (1962) (MS) medium fortified with eight treatments of 6-benzylaminopurine (BAP) (0.0, 2.0, 0.4 and 6.0 mg L-1 of BAP) and Naphthalene acetic acid (NAA) (0.1 and 0.2 mg L-1 of NAA) structuring in factorial design to study the effect of growth regulators for formation of microrhizomes. In-vitro produced shoots were cultured on MS medium fortified with different concentration of sucrose (30, 60, 90 and 120gL-1) separately. Effect of the solid/liquid nature of the medium and the different photoperiod level on induction of microrhizome was also studied. 

Results: Results revealed that medium containing 4.0 mgL-1 BAP with 0.1 mgL-1 NAA showed the best response followed by 6.0 mgL-1 BAP with 0.1 mgL-1 NAA for induction of microrhizomes within 60 days. Increased level of NAA did not enhance mocrorhizome induction. Results of different concentration of sucrose revealed that MS medium fortified with 90 g L-1sucrose recorded the highest fresh and dry weight of microrhizomes followed by the treatment with 60 g L-1 sucrose. However, plantlets supplemented with more than 90 g L-1 of sucrose exhibited lower weight of microrhizomes, but higher root induction and root fresh weight probably due to accumulation of water. Different photoperiod exposure levels revealed that 16 hrs of light and 4 hrs of dark condition with solid medium produced highest fresh weight (3.72 g) and highest number of microrhizomes (9.6).

Conclusion: Murashige and Skoog (1962) medium supplemented with 4 mgL-1 BAP, 0.1 mgL-1 NAA and 90 gL-1 sucrose in solid form with 16-h photoperiod for 10 weeks of culture duration were the best conditions for induction microrhizomes in ginger, cultivar Local.