An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD₆₀₀ of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

Strawberry ( Fragaria × ananassa cv. Camarosa) was evaluated to determine a high-frequency shoot regeneration response for leaf and shoot-tip explants. For direct organogenesis from strawberry leaves, combinations of moderate concentrations of thidiazuron [TDZ (0, 2, and 4 μ m )] and 6-benzylaminopurine [BAP (0, 4, and 9 μ m )] added into medium containing Murashige and Skoog (MS) basal salts were compared. The most shoots regenerated per leaf explant were observed with 4-μ m TDZ. Regeneration from shoot tips was evaluated with 0-, 2-, 4-, 8-, and 16-μ m zeatin, kinetin, or 6-α,α-dimethylallylamino purine (2ip) tested individually. Optimum shoot proliferation was achieved from shoot-tip explants on medium containing 4-μ m zeatin. Rooting was best without cytokinins in the medium; however, adequate rooting was obtained on the 4-μ m zeatin treatment as well.

Due to the advantages of molecular methods over biochemical methods, the use of molecular methods for diagnosing nosocomial infections such as Pseudomonas can be an appropriate and rapid way to choose the right diagnosis and treatment of infection and prevent further complications caused by the infection. The present article provides a description of the development of a nanoparticle-based detection technique for sensitive and specific deoxyribonucleic acid-based diagnostic of Pseudomonas aeruginosa. Specific thiolated oligonucleotide probes for one of the hypervariable regions of the 16S rDNA gene were designed and applied for colorimetric detection of the bacteria. The results of gold nanoprobe-nucleic sequence amplification indicated the probe attached to gold nanoparticles in the presence of the target deoxyribonucleic acid. It caused aggregation of gold nanoparticles in the form of connected networks resulting in color change and indicating the presence of the target molecule in the sample, which could be observed by the naked eye. In addition, the wavelength of gold nanoparticles changed from 524 to 558 nm. Multiplex polymerase chain reactions were performed using four specific genes of Pseudomonas aeruginosa (oprL, oprI, toxA, and 16S rDNA). The sensitivity and specificity of the two techniques were assessed. According to the observations, the specificity of both techniques was 100%, and the sensitivity was 0.5 ng/μL and 0.01 ng/μL of genomic deoxyribonucleic acid for multiplex polymerase chain reaction and colorimetric assay, respectively. The sensitivity of colorimetric detection was about 50 times higher than the polymerase chain reaction using the 16SrDNA gene. The results of our study proved to be highly specific with potential use for early detection of Pseudomonas aeruginosa.

Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.