الفهرس 
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Abstract
SUMMARY
The main aim of the present study is to evaluate chitosan
nanoparticles as a carrier of DNA through transformation E. coli, plant
cells, and plant single nodes.
Two different molecular weights chitosan samples were used as
DNA carrier for transformation. Viscometry method was used to
determine sample molecular weights. Sample (A) was 501 KDa, located
in medium molecular weight chitosan range, while sample (B) was 827.9
KDa located in high molecular weight chitosan range. Titration method
was used to determine the degree of deacetylation (DDA). Sample (A)
DDA was 66.26% and Sample (B) was 76.95%. Chitosan /DNA complex
sizes determined by transmission electron microscopy were 109, 125 n.m.
for chitosan samples A and B, respectively. Chitosan /DNA complex
formation, and protection effect from anionic surfactant was examined by
agarose gel electrophoresis proved that chitosan nanoparticals can protect
DNA molecule in gene delivery.
Chitosan effect was determined in both E. coli and plant cells.
Effect of chitosan on E. coli leads to reduced bacterial population as the
concentration increases. Sample (A) higher lethal effect on competent
cells 55.11% than its effect on normal cells 47.6% in the lowest
concentration, and 99.98%, 97.37% for the highest one, respectively. The
obtained results indicate that sample (A) had higher lethal effect sample
(B) in E.coli normal cells 43.5% in lowest concentration and 94.4% for
the highest one. In case of competent cells lowest concentration was led
to 54.29%, and the highest was 99.4%.
For plant cells, chitosan sample (B) 54.24% had higher lethal
effect than sample (A) 47.37% in their highest concentration, and
42.98%, 37.03% in the lowest concentration, respectively.
The transformation efficiency of bacteria by incubated nanoparticles
including plasmid DNA was examined by colony forming unit
(CFU) on Luria-Bertani (LB) + ampicillin plates. The transformation
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Nour-El-Hoda M. Sorour (2017), Ms.C, Fac. Agric., Ain Shams Univ.
efficiency of chitosan DNA complex in case of normal cells was
(1.53E+07, 1.08E+08, 5.61E+08, 3.50E+09) for type (A) with chitosan
concentration in complexes was more efficient than type (B) (2.83E+07,
1.09E+08, 3.93E+08, 2.14E+09) transformation efficiency gradually
increased chitosan with concentration in complexes. The transformation
efficiency of bacterial competent cells in the same manner of normal cells
(5.35E+07, 3.40E+08, 3.75E+09, 7.20E+11) for chitosan type (A) and
(7.00E+07, 2.29E+08, 1.03E+09 2.53E+10) for type (B) while control
transformation was 4.48E+08.
The effect of different vortex times on frequency of plant cell
transformation was studied. The transformation percentage of plant cell
was determined by detecting green fluorescent protein (GFP) expression
using fluorescent microscope. Without vortex, sample (A) was gave
(4.81%, 16.72%, 22.73%, 32.24%), while sample (B) gave (9.57%,
10.95%, 6.48%, 14.57%) gradually with increased chitosan concentration
in chitosan DNA complex. In case of 30 Sec. vortex, transformation
percentage for sample (A) were (8.16%, 10.34%, 24.09%, 29.30%), and
for sample (B) were (10.52%, 10.32%, 5.49%, 9.36%). The percentage
was achieved by 60 Sec. vortex, sample (A) was gave (9.62%, 13.93%,
17.05%, and 21.11%), sample (B) (7.09%. 6.08%, 4.32%, 8.83%).
Transformation of Plant single nodes that determined by green
fluorescent protein (GFP) expression under U.V. light was obviously
succeed for both types of chitosan