Nanomaterial-mediated gene delivery in wheat (Triticum aestivum L.)
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Date
2021
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Punjab Agricultural University, Ludhiana
Abstract
The present study aimed for the synthesis and functionalization of different NMs and conjugation of
plasmid DNA (pGFPGUSplus) onto the functionalized NMs. These NM-plasmid DNA conjugates
have been evaluated as nano-delivery vehicles to deliver the desired plasmid DNA in wheat (Triticum
aestivum L.). The UV-Vis absorption spectroscopy analysis showed a shift in the peak of the NMplasmid
DNA conjugates in comparison to the plasmid DNA absorption peak and the pristine NMs
absorption peak, which showed the intercalation of the plasmid DNA with the respective NMs. The
FT-IR spectroscopy of the respective NMs and NM-plasmid DNA complexes showed the
presence/weakening of functional groups before and after conjugation of plasmid DNA onto the NMs.
Transmission electron microscopy (TEM) analysis revealed the formation of nano-scale (>100 nm)
pristine NMs and NM-plasmid DNA complexes that formed aggregates after conjugation. DNase
treatment and gel electrophoresis of the respective NMs and NM-plasmid DNA complexes confirmed
the binding and accurate loading of plasmid DNA onto the NMs. The NM-plasmid DNA conjugates
were used to transform two different strains of untransformed gram negative rods i.e. Escherichia coli
(DH5alpha) and Agrobacterium tumifaciens (EHA105). The Fourier Transform-Infra Red spectroscopy
studies depicted the shift in bending/stretching vibrations of amino or phosphate groups present in
bacterial polysaccharides after tethering of NM-plasmid DNA conjugates onto the bacterial cells. The
TEM study of the transformed bacterial cultures showed the binding of NPs around and inside the
bacterial cell. The transformed bacterial cultures under fluorescence microscopy showed green
fluorescence due to the delivery of plasmid DNA inside the cells with the aid of NMs. The total soluble
protein content analysis revealed production of different amounts of total intracellular and extracellular
soluble proteins which were increased in a time dependent manner. LDH NPs showed lesser cytotoxicity
towards bacterial cells along-with better viability rate than other NPs. The bacterial cell
viability assay revealed the impact of different NMs on the viability count of the bacterial cells.
Further, the NM-plasmid DNA conjugates were used for transformation of the wheat callus cell
suspensions and entire callus tissues. All the callus cells transformed with different NM-plasmid DNA
complexes exhibited green fluorescence on fluorescence microscopy. Agrobacterium-mediated
infiltration was also performed in entire wheat callus tissues to compare the efficacy of Agroinfiltrations
and nano-delivery vectors. The Layered Double Hydroxides (LDH) and Graphene oxide
(GO) nanosheets showed maximum fluorescence as compared to other NM-plasmid DNA conjugates
as these particles were able to penetrate deep inside the callus tissues while others also showed the
ROS effect. Upon bath-sonicating the wheat callus tissues with the LDH-plasmid DNA and GOplasmid
DNA complexes, the LDH nanosheets showed an increase in the intensity of the green
fluorescence with an increase in sonication incubation period. Therefore, LDH nanosheets delivered the
desired plasmid DNA inside the callus tissue cells more effectively without damaging to callus
cells/hampering the cellular systems.
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Citation
Harkamal Kaur (2021). Nanomaterial-mediated gene delivery in wheat (Triticum aestivum L.) (Unpublished M.Sc. thesis). Punjab Agricultural University, Ludhiana, Punjab, India.