Agriculture Reference
In-Depth Information
following steps: (a) adhesion of the protoplast to the lysosomal surface, (b) fusion
of liposomes and protoplast after adhesion, (c) formation of endosome within the
cytoplasm, and (d) release of plasmids inside the cell. The liposomal technique has
been successfully used to deliver DNA into the protoplasts of a number of plant
species (e.g., tobacco, petunia, carrot, etc.). Deshayes et al. (
1985
) developed
positively charged liposomes bearing
E. coli
plasmid (pLGV23neo) having a
kanamycin resistance gene, which were used in the development of transgenic
tobacco. Protoplasts isolated from the leaves of transgenic tobacco were resistant
to 100 mg/ml kanamycin.
10.3.10 Gene Gun (Particle Bombardment)
Gene gun is the gene transfer technology by applying mechanical force. The
particle bombardment is the “biolistic particle delivery system” or a gene bom-
bardment system, in which acceleration of the particles is achieved by using a pulse
of helium. With the help of high pressure with helium, DNA can pass through cell
membranes into the cytoplasm or even in the nucleus by evading the enzymatic
degradation (Uchida et al.
2009
). In this technique, DNA-coated gold particles are
used and then transferred into target tissues or cells by using high pressure. DNA is
delivered along with heavy metal particle into the target cell, and thus these
particles must be nonreactive and nontoxic to the cell components cell. Naked
DNA is mixed with these microparticles and then released within the cell with the
help of gene gun. In Gene Gun method, DNA-coated tungsten particles were
injected by gunpowder acceleration system, which transfers them through the
plasma membrane into the nucleus, integrated, and ultimately resulted in stable
gene expression. This method was used for transgene expression in plant cells
initially and was further applied to mammalian cells and living tissues as well
(Yang et al.
1990
). However, the major drawback of this method was short-term
and low-level expression of genes and shallow penetration ability of particles into
the plant tissue. The loading of DNA onto the particles, the timing of delivery, and
the velocity of acceleration were the important factors concerning the efficiency of
this method. Moreover, the final distribution of DNA-coated particles is mainly
influenced by the fine-tuning of the acceleration imparted by the particle bombard-
ment method (Uchida et al.
2002
). The efficiency of gene expression was dependent
on the number of DNA-coated particles delivered to the nucleus and on the degree
to which these particles are coated with the desired plasmid (Mehier-Humbert and
Guy
2005
).
