A number of parameters which have been reported to influence genetic transformation via Agrobacterium-mediated transformation method were evaluated to increase the frequency of transformation of cabbage (Brassica oleracea subsp. capitata) cv. KY Cross with AtHSP101 gene. The binary vector pCAMHSP was designed and mobilized into two Agrobacterium tumefaciens strains C58 and GV2260. The study was carried out on hypocotyl and shoot tip explants of cabbage cv. KY Cross. Transformation parameters optimized were pre-culture medium, acetosyringone application, bacterial density and inoculation time. The polymerase chain reaction (PCR) assay and production of mRNA of AtHSP101 gene were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The expression of LacZ gene in the transgenic plants also showed that it could be applied as a plant transformation reporter gene in genetic transformation studies. Multiple shoot regeneration of hypocotyl and shoot tip explants of cabbage after co-cultivation with Agrobacterium was optimized and medium containing 2 mg/L BAP was observed to be the best for shoot regeneration after cocultivation. In this study, 45% and 32.5% transformation efficiencies were achieved for hypocotyl and shoot tip explants, respectively using the optimized procedure.
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
OPEN ACCESSMolecules 2015, 20 3648 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.
In the last few years, gold nanoparticle biosensors have been developed for rapid, precise, easy and inexpensive with high specificity and sensitivity detection of human, plant and animal pathogens. Klebsiella pneumoniae serotype K2 is one of the common gram-negative pathogens with high prevalence. Therefore, it is essential to provide the effective and exclusive method to detect the bacteria. Klebsiella pneumoniae serotype K2 strain ATCC9997 genomic DNA was applied to establish the detection protocol either with thiol-capped oligonucleotide probes and gold nanoparticles or polymerase chain reaction based on K2A gene sequence. In the presence of the genomic DNA and oligonucleotide probes, a change in the color of gold nanoparticles and maximum changes in wavelength at 550-650 nm was achieved. In addition, the result showed specificity of 15 × 10 CFU/mL and 9 pg/μL by gold nanoparticles probes. The lower limit of detection obtained by PCR method was 1 pg/μL. Moreover, results demonstrated a great specificity of the designed primers and probes for colorimetric detection assay and PCR. Colorimetric detection using gold nanoparticle probe with advantages such as the lower time required for detection and no need for expensive detection instrumentation compared to the biochemical and molecular methods could be introduced for rapid, accurate detection of the bacteria.
An efficient micropropagation system for strawberry cv. Camarosa was developed. Sterilized runner tips were cultured on hormone-free Murashige and Skoog (MS) medium with 3% sucrose, 1 mL·L−1 Plant Preservative Mixture, and solidified using 0.25% phytagel to produce in vitro stock plants. Shoot tips derived from the in vitro stock plants were cultured on MS media containing 0, 2, 4, and 8 μM thidiazuron (TDZ) and 0, 4, 9, 18, and 27 μM N6-benzylamino-purine (BAP) for shoot induction. Shoots produced on the best shoot induction medium were rooted on MS media containing 1, 2, 3, and 5 μM of either indole-3-butyric acid (IBA) or naphthaleneacetic acid (NAA). Results showed that MS medium with 2 μM TDZ and 4 μM BAP was optimum for shoot multiplication from the shoot tips. The most suitable medium for inducing the highest number of roots per explant, the highest percentage of explant with roots, and the highest mean root length were 1 μM NAA, 1 μM IBA, and hormone-free MS medium, respectively. Plantlets were transplanted into substrate consisting of perlite + vermiculite + cocopeat (2:1:2 v/v/v) resulting in 90% survival. After 1 month, plants were irrigated using Hoagland's solution and runners were produced after 3 months.
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 μm)] and 6-benzylaminopurine [BAP (0, 4, and 9 μm)] added into medium containing Murashige and Skoog (MS) basal salts were compared. The most shoots regenerated per leaf explant were observed with 4-μm TDZ. Regeneration from shoot tips was evaluated with 0-, 2-, 4-, 8-, and 16-μm zeatin, kinetin, or 6-α,α-dimethylallylamino purine (2ip) tested individually. Optimum shoot proliferation was achieved from shoot-tip explants on medium containing 4-μm zeatin. Rooting was best without cytokinins in the medium; however, adequate rooting was obtained on the 4-μm zeatin treatment as well.
Background
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.
Results
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.
Conclusion
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.
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