Anti-fungal effects of Essential Oil and Nano-emulsion of some Medicinal Plants on the Human Pathogenic Fungus Candida albicansAntifungal potential of Essential oil Nanoemulsions…: Antifungal potential of Essential oil Nanoemulsions…

Interest in essential oils is continuously increasing due to their biological activities in various fields, from pharmaceuticals to food and agriculture. Essential oil is unstable and hydrophobic under normal storage conditions. Thus, it can quickly lose its anti-fungal and antibacterial activity. The new method of nano-emulsion production has been proposed as an effective solution to increase their stability and activity. In this study, the nano-emulsions of Myrthus communis, Rosmarinus officinalis, and Eryngium campestre essential oils were formulated using different proportions and evaluated on Candida albicans. Nano-emulsions were prepared using an ultrasonic method, and the field emission scanning electron microscopy (FESEM) apparatus determined the size of the prepared nano-emulsion particles. The dilution method determined the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). Also, the bioassay of plant compounds was investigated using the agar well diffusion method. Mean droplet size of M. communis, R. officinalis, and E. campestre nanoparticles were reported in 200-500, 100-200, and 200-300 nm, respectively. The lowest MIC value was obtained for the M. communis (12.5) and R. officinalis (12.5) nano-emulsion. The activity of the essential oil and nano-emulsion of the tested plants against C. albicans was confirmed with an inhibition zone diameter of 3.9-27.5 mm. Examining the time growth curve of C. albicans showed that nano-emulsions were significantly more effective on the fungal pathogen than essential oils. Results show that using nano-emulsions increases the antimicrobial properties of essential oils from medicinal plants

Design, construction and cloning of pCAMBIA-MiAMP1 vector for enhancing disease resistance in plants using Agrobacterium- mediated transformation

ABSTRACT Objective: To design, construct and clone a pCAMBIA-MiAMP1 vector that can be used to enhance disease resistance in plants through Agrobacterium-mediated transformation. Methodology and results: The cDNA sequence encoding MiAMP1 antimicrobial peptide as a defense resistance gene, which had been cloned into the cloning site of pGEM-T T-vector, was obtained from Australia. The pGEM-T-MiAMP1 was transformed into E.coli and the identification of transformed clones among antibiotic resistant bacteria carried out by lacZ expression using blue/white screening system. The presence of MiAMP1 gene was confirmed using PCR analysis with primers M13, restriction enzymes NcoI and BamHI and DNA sequencing. Based on the sequence, the orientation and restriction map of target gene in plasmid was defined. After designing the specific PCR primers flanking the coding region of the MiAMP1 gene and amplifying the complete cDNA with additional restriction sites, either PCR product, or the binary vector pCAMBIA1305.1 were digested with restriction enzymes NcoI and BglII. Subsequently, the insert and the vector were ligated with T4 DNA ligase to produce binary vector pCAMBIA-MiAMP1 for genetic transformation of plants. This construction contains the full coding region of the MiAMP1 antimicrobial peptide and is flanked at its 5' end by the strong constitutive promoter of CaMV35S and at its 3' end by the polyadenylation sequence of NOS polyA. The designed expression vector also contains other elements that are useful for plant transformation such as a kanamycin resistance gene (npt II) and a hygromycin resistance gene (hpt II) that can be used as markers for selecting transformed plants, while the GUSPlus™ reporter gene contains the intron. The presence of MiAMP1 gene in the pCAMBIA-MiAMP1 construction was confirmed among antibiotic resistant colonies by PCR method, restriction enzymes analysis and DNA sequencing. The designed construction was transferred into Agrobacterium tumefaciens strain AGL0 by tri-parental mating method with the helper plasmid pRK2013. Conclusion and application of findings: pCAMBIA-MiAMP1 construction can be used to genetically transform plants to enhance resistance to fungi e.g. Leptosphaeria maculans and Sclerotinia sclerotiorum. Currently, it is being used in Agrobacterium-mediated transformation of canola and sunflower

Optimization of in vitro sterilization protocol for obtaining contamination-free cultures of Tilia platyphyllos

Abstract. Payamnour V, Ghasemi Bezdi K, Mehrdad M, Ahmadi A. 2014. Optimization of in vitro sterilization protocol for obtaining contamination free cultures of Tilia platyphyllos. Nusantara Bioscience 6: 7-12. Tilia platyphyllos is one of threatened species of Caspian forests. Tissue culture techniques are applied for culture, regeneration and genetic resources preservation. Utilizing an accurate sterilization procedure is important to reduce the cost, time and energy. The aim of this present study was to provide optimization of in vitro sterilization protocol to obtain contamination-free cultures of T. platyphyllos. Explants were collected randomly from the best individuals of T. platyphyllos, which were located in Tooskestan forest of Gorgan, Iran. Results revealed that the optimum protocol for sterilization was when explants were exposed in pre-sterilizing solution of 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite (NaOCl) solution (5% Cl activated) for 20 minutes and then explants were exposed in sterilizing solution containing 600 mg L-1 ascorbic acid and 10% sodium hypochlorite.</jats:p

Improved accumulation of betulin and betulinic acid in cell suspension culture of <i>Betula pendula</i> roth by abiotic and biotic elicitors

Betulin (B) and betulinic acid (BA) are two triterpenes with diverse pharmacological and physiological actions. Elicitation of Betula pendula Roth cell cultures by elicitors is an excellent strategy to increase B and BA levels. Six abiotic and biotic elicitors were studied to improve accumulation of B and BA in the cell culture of B. pendula. The B and BA production in treated cells was verified by HPLC. The results showed the maximum growth index (7) on day 3 in cells treated with 0.5 mg L−1 chlorocholine chloride (CCC). The increased accumulation of BA in the cells treated with 200 mg L−1 of chitosan was found to be 5.9 × (6.5 mg g−1 DW) higher over control cells. Treating the cells with 2 mg L−1 of CCC, after 7 days, led to 149.3× enhancement of B content (19.4 mg g−1 DW) over the controls. Production of this triterpenoid at a much shorter time with a much higher growth rate can be economic and lead to producing large amounts of B and BA for anti-cancer and HIV drugs preparation.</p