The thionin gene is genetically modelled in the Arabidopsis thaliana plant according to previous study of Abdel-Razik et al. . Onion cultivar was used as a transgenic model (Giza red). They were purchased from Agricultural Research Center and regenerated on MS media .
Obtainig of thionin gene and Cloning
Edward's protocol was used to extract total genomic DNA from Arabidopsis thaliana . For each 25 µl PCR reaction, 50ng of template DNA was used. Also, contained 12.5 µl of 2X master mix (Biolene), 1 µl of each forward and reverse primer (50nmole/base), and sterile d.dH2O to fill the remaining 25 µl. The primers were designed with the snap gene® (2.3.3) and had the following sequences: Thio-60F: 5’ GCTGAATTCATGGAGGACAAAAGA 3´, Thio-60R: 5´ GCTAAGCTTTCATAGACTAAAATCAAT 3´. The PCR reaction was run for 40 cycles: 1 minute at 95oC, 1 minute at 64oC, and 1 minute at 72oC. On a 1.2% (w/v) agarose gel, thionin insert were run. GeneJETTM PCR Purification Kit was used to purify the amplified PCR product (Thermo K0701). pMiniT Vector (NEB® PCR Cloning Kit, #E1202S - 10-beta Competent E. coli) was ligated to the Thio-60 product. Following the manual's instructions.
Confirmation of Bacterial Transformation
Colony PCR was used to differentiate non-recombinant and recombinant colonies, and colony PCR was used to apply colony PCR. On LB agar plates supplemented with 100 µg/L ampicillin, bacteria containing modified plasmid were grown. Single colonies were used as templates for PCR reactions using the same thionin primers, followed by electrophoresis on a 1.2 percent gel.
Chitosan nanoparticle transformation
Degree of Deacetylation
Titration method was used to determine the degree of deacetylation for chitosan nanoparticles according to Czechowska-Biskup et al.  as follow: “Dried chitosan (0.2 g) was dissolved in 20ml of 0.1M hydrochloric acid and 25cm3. After 30min continuous stirring 25ml of dH2O was added with continuous stirring for another 30 minutes. After complete dissolve of chitosan, titration with a 0.1mol·dm-3 sodium hydroxide solution was performed. The degree of deacetylation (DA or DD) of chitosan was calculated as:
Where: m: weight of sample, V1, V2: consumed volumes of 0.1 mol·dm-3 sodium hydroxide solution, 2.03: coefficient of the molecular weight of chitin monomer unit, 0.0042: coefficient of the difference between molecular weights of chitin and chitosan monomer units”.
UV-Visible Spectra Measurements and Zeta Potential and Size
Another characterization method for chitosan nanoparticles is using UV-visible spectroscopy “JASCO V-630 UV-visible spectrophotometer (serial: C285061148) with Spectra Measurement software”. The measured zeta potential and size was estimated using Malvern (7.2) software.
Transmission electron microscopy
The examined CS nanoparticles were prepared as follow: “copper grids mesh coated with carbon 400 and handled on 1 drop of the prepared complex (CS/pDNA) and left for 1.5 min. The grid was stained with 1 drop of filtered solution of 2% uranyl acetate for 1.5 min, with removal of excess uranyl acetate . The grids were dried for 10 min then photographed with transmission electron microscope (TEM) in the Regional Center for Mycology and Biotechnology, Al-Azhar University”.
Preparation of chitosan-DNA nanoparticles complex (CS/pDNA)
Mansouri et al.  designed the protocol of CS/DNA formation as follow: “chitosan nanoparticles (CS) were dissolved in 25mM acetic acid and adjusted to pH 5.5 at a concentration of 0.08%. Both CS and recombinant pMiniT were incubated in water bath at 55oC for 15min and then added equally to each other with stirring on a vortex for 1min”.
Transformation of Chitosan/pDNA into Allium cepa
The method of chitosan nanoparticle transformation into plant tissue was developed by : “Seedlings of A. cepa were inoculated by syringe containing CS/pDNA complex. After that the explants were transferred on to MS medium supplemented with (2mg/L BA and 1mg/l kin) hormones and 100µg/L ampicillin then incubated at 25±1°C for 4 weeks to regenerate plants”.
Molecular analysis of Transgenic Allium cepa Lines
DNA from both leaves of non-transgenic and transgenic Allium cepa lines was isolated. DNA fragments of thio-60 transgenes were amplified by PCR with the same thionin primers and the same PCR program conditions.
A fungal-resistance assay of transgenic plants was used to accomplish this. The thionin genes in transgenic plants were tested for antimicrobial activity against A. niger. Pathogenicity bioassay was performed via three methods: 1) Spore suspension infecting whole Allium cepa shoot; 2) infection of whole in vitro plant and 2) spore suspension infecting plants out-jars (after 2 weeks of growth).
Spore suspension infecting whole Allium cepa shoot
In another experiment, spore suspension was prepared by immersing fungal discs in 5ml of sterile distilled water to release the spores. The spores were collected with a sterile Pasteur pipette, and their concentration was adjusted to 2x105spores/ml using sterile water. This assay was applied for the whole shoot with some modifications.
The whole shoot from mature transgenic and non-transgenic A. cepa, grown in vitro for 4 weeks, were placed in a Petri dish with wet filter paper and inoculated with the spore suspension (100µl each). After inoculation, the shoots were incubated at room temperature under 16 h light/8 h dark conditions and high humidity for a week. Pictures were taken 5 days after inoculation .
Disease Resistance Assay
For fungal infection: this follows  and  with some modification. “Fungi grew on potato dextrose agar till the surface covered with the fungal mycelia. Then, a block of the agar with mycelia was placed near to the base of in vitro transgenic and non-transgenic control plants (3 weeks old) grown on 100ml MS medium and incubated at 25oC with 16h light /8h dark. Photographing for the results was recorded 2 weeks after inoculation”.
Spore Suspension with Infected plant
The spore suspension of A. niger was prepared by submerging fungal discs in 5 ml of sterile dH2O to release the spores (adjust concentration to 2x105 spores/ml using sterile dH2O). This assay was applied for whole of plants from mature transgenic and non-transformed A. cepa, grown in vitro for 4 weeks, put in a Petri plate with wet filter paper, and inoculated with the spore suspension (100μl each). After inoculation, these infected leaves were incubated at room temperature and high humidity for 7 days then photographed to record the infection symptoms .
Inhibitory Protein Bioassay
The antifungal activity of the transgenic Allium's product thionin proteins was tested against A. niger spores. Bradford's method  of protein extraction was used to extract the proteins. Then, with some modifications, the inhibitory effect of protein extracts on spore germination bioassay was used, as described by Maji et al. 2005. “The pathogens' spore suspension was made aseptically from a 7-day-old pure culture. On separate sterile eppendorf tubes, 100 µl of spore suspension and 100 µl of crude protein extracts were taken. One tube was kept as a control, with no extract added. Triplicates of each treatment were kept. The tubes were incubated at 25±2°C for 24 h. After the incubation period, observations were made under microscope to calculate the percentage inhibition” (using 100x magnification power under JENLAB microscope).
The data in this study were analyzed using one-way anova in SPSS 21 software for calculating means and the significance for 10 individuals for each sample. The data analysis of gel was performed using BioRAD Quantity One software (4.6.2).