Agriculture Reference
In-Depth Information
wall and cell membrane by a suitable agent helps in nanoparticle-mediated DNA
transfer in regenerative calli and soft tissues. The ability of NPs to penetrate plant
cell wall also helps precise manipulation of gene expression at the single-cell level
by delivering DNA and its activators in a controlled fashion (McKnight et al.
2003
).
Honeycomb mesoporous silica nanoparticles (MSN) with 3-nm pores can transport
DNA and chemicals into isolated plant cells and intact leaves. MSNs loaded with
gene and its chemical inducers were capped with gold nanoparticles at the ends to
protect the molecules from leaching out. Removal of gold nanoparticle cap enabled
release of chemicals and triggered gene expression in plants under controlled
conditions. Incubation of protoplast with fluorescently labeled MSNs revealed
that surface modification of MSNs with triethylene glycol was necessary to pene-
trate the cells. The modification of MSNs with triethylene glycol also allowed
adsorption of plasmid DNA on MSN surface. Plasmid DNA was released from the
MSN upon its entry into the protoplasts; the plasmid DNA released allows expres-
sion of marker gene like GFP in the cell, which can be detected by fluorescence
microscopy. This method can detect marker expression of 1,000-fold less than that
required for the conventional delivery method. Efficiency of this delivery method
has pronounced applications in various protoplast-based gene expression studies.
Now-a-days, particle bombardment or gene gun is one of the popular methods to
transfer DNA into intact plant cells (Klein et al.
1989
; Deng et al.
2001
). In particle
bombardment method, the particles used are typically made of gold, due to their
ability to adsorb DNA and nontoxic nature toward cells. MSNs are not suitable for
gene transfer by particle bombardment method, since they are too light to be used
for this method. This problem can be overcome by capping MSNs with gold
nanoparticles, which will increase their momentum after acceleration by the gene
gun. Plasmid DNA transferred by particle bombardment method using gold-capped
MSNs was successfully demonstrated in intact tobacco and maize tissues. In
particle bombardment method, simultaneous delivery of both DNA and effector
molecules to the specific sites results in the expression of genes. Future studies
include pore enlargement and multifunctionalization of MSNs to provide target-
specific delivery of proteins, nucleotides, and chemicals in plant cells.
Some of the approaches, which can be used to improve nanoparticle-based gene
delivery into plant cells, are given below:
1. The surface charge in protoplast and plasma membrane vary from
10 to
30 mV (Reid et al.
2002
). Modifications to this surface charge can be done
to enhance the adhesion of bioconjugate DNA, by introducing a positive charge
over their surface. Wiesman et al. (
2007
) have demonstrated similar concept.
2. The porosity of protoplast is 0.1-5,000 nm (Berestovsky et al.
2001
), and in
plasma membrane, it varies between 1 and 50 nm (Berestovsky et al.
2001
).
3. Stearylamine, triethylamine, and natural biodegradable polymers like chitosan
can impart a positive charge on DNA conjugate nanomaterial surface
(Li et al.
2011
). By using the above approaches, mechanisms of nanoparticles-
mediated gene transfer are presented in a simplified manner in Fig.
10.5
.
