The effect of Agrobacterium densities and inoculation times on gene transformation efficiency in rubber tree

Rubber tree belonging to the genus Hevea is an economically important crop of Thailand and South-east Asia. To optimize its agronomical trait for glyphosate-resistant, in vitro gene transformation through Agrobacterium tumefaciens was investigated. The bacteria carrying plasmid pCAMBIA 1304, harboring gus as screenable marker genes and EPSPs gene was used. The shoot tips were immersed in A. tumefaciens suspension at optical densities (OD600) at 0.3, 0.6 and 0.9 for various times (15, 30 and 60 min). The results revealed that shoot explants immersed in A. tumefaciens suspension at OD600 of 0.6 for 30 min gave the higher survival rate after being cultured on glyphosate containing MS medium for one and half months. Assessment of transformed shoots revealed positive results in GUS histochemical assay. The presence of the gus and EPSPs genes in transformed rubber tree were confirmed by polymerase chain reaction (PCR) technique, dot blot hybridization and Southern PCR hybridization. Specific primers for the gus and EPSPs genes were designed to amplify a 919 and 1,600 bps DNA fragment, respectively.Keywords: Transgenesis, glyphosate, inoculation time, Agrobacterium density, HeveaAfrican Journal of Biotechnology, Vol 13(23) 2321-232

Effect of seasonal collection on callus Induction, proliferation and somatic embryogenesis from anther cultures of Heveabrasiliensis MuellArg

Hevea brasiliensis that is grown in Thailand and Southeast Asia is a very valuable source of natural rubber. However, few studies have been conducted to evaluate the effects of seasonal effect on plantlet regenera-tion through somatic embryogenesis from anther culture. Thus, this study discussed the effect of season on callus induction subsequent to plantlet regeneration from culturing of anther. Male flowers were aseptically opened and anthers were excised to culture on callus induction medium (CIM) which was MS supplemented with 5% sucrose, 1 mg.L-1 2,4-D, 1 mg.L-1 KN, 1 mg.L-1 NAA. Somatic embryo (SE) developed on MS medium supplemented with 3% sucrose, 0.2 mg.L-1 NAA, 1 mg.L-1 BA, 3 mg.L-1 KN and 0.05 mg.L-1 GA3. This medium is so called embryo induction medium (EIM).Anther collectedfrom different season gave different result in callus induction, proliferation and somatic embryogenesis, healthy anther collected in February 15, 2013 gave the best result in callus induction (86.25%), proliferation (422 mgFW) and somatic embryogenesis (20%). Complete plantlets (15.63%)were obtained on MS medium supplemented with 5% sucrose, 0.5 mg.L-1 KN, 0.2 mg.L-1 IAA, 1 mg.L-1 BA, 0.3 mg.L-1 GA3 and 0.2% phytagel. Seasonal collection plays influence rolein successful somatic embryogenesis in tissue culture of rubber tree.Keywords: Hevea brasiliensis, rubber, antherculture, callus induction, seasonal collection, somatic embryogenesis.African Journal of Biotechnology, Vol 13(31) 3560-356

Antibiotics Stimulate Formation of Vesicles in Staphylococcus aureus in both Phage-Dependent and -Independent Fashions and via Different Routes

Bacterial membrane vesicles research has so far mainly focussed on Gram-negative bacteria. Only recently Gram-positive bacteria have been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MVs formation, the underlying mechanisms are poorly understood. In this study we show that antibiotics can induce MVs through different routes in the important human pathogen DNA damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of but not in their phage-devoid counterparts. β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is higher than that of MVs formed by β-lactam antibiotics-induced blebbing. The purified MVs derived from protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both and in whole blood. In addition, the MVs protected from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium