and target it to the nucleus? Also, where are the cells that need to be targeted; ones that can
either give rise to whole plants directly or give rise to the pollen or egg (germ line) for suc-
cessful transmission of the introduced DNA to progeny?
10.2.2 DNA Delivery
To consider DNA delivery into plant cells for the production of transgenic plants, the plant
cell wall, cell membrane, and the nuclear membrane represent formidable barriers. The cell
wall surface can be visualized as a stainless-steel scouring pad, with the steel fibers repre-
senting cellulose fibers. The cell wall (especially the young cell wall) has some level of
flexibility to allow cell elongation and movement but is a fairly rigid structure, held together
with cement of pectin and other crosslinking materials. Although there are “holes” in the
cell wall, the plasmodesmata connect the protoplasm of adjacent cells and do not provide
open access for DNA introduction. In order to deliver DNA across the cell wall, the cell
wall must first be physically breached. This is also the case for passing DNA across the
cytoplasmic membrane. Holes or breaks in the cell wall cannot be so severe that the
target cell is irreparably damaged, but damage at some level must be done, to get a relatively
large molecule of DNA into the cell. To complicate matters, plant cells are almost always
hypertonic, which means that there is internal pressure pushing the cytoplasm against the
cell wall, keeping plant tissues rigid. The pressure can be temporarily reduced or eliminated
by lowering the osmotic pressure within the plant cells, causing the plant tissue to “wilt.”
By either drying the tissue or placing it on a medium containing sugars, the osmotic poten-
tial of the tissues can be temporarily lowered and DNA introduction efficiencies are then
improved by reducing leakage of cytoplasm from holes or breaks in the cell wall (Vain
et al. 1993).
Introduction of DNA into the cell is only part of the story as the nucleus is the desired
destination in most cases. The chloroplast and mitochondria also contain genetic infor-
mation and can take up and incorporate DNA, separately from the nucleus. However, we
will focus on nuclear transformation here since it is the predominant mode to produce trans-
genic plants. So, how does the introduced DNA get to the nucleus, and what happens when
it finally arrives? With the physical methods of DNA delivery, it appears that the DNA is
actually delivered to an area either adjacent to the nucleus or into the nucleus itself. Naked
DNA (introduced DNA is almost always uncoated and unprotected as opposed to native
chromosomal DNA, which is specifically folded, organized, and coated with proteins)
probably does not survive long outside the nucleus. For biological methods of DNA intro-
duction, the DNA is naturally coated with proteins, which protect the DNA from degra-
dation and escort it to the nucleus. Even if the introduced DNA reaches the nucleus, it is
not precisely known what happens to this foreign DNA or how exactly it is incorporated
into the plant genome. It appears that the natural machinery of the cell, which repairs, mod-
ifies, and replicates DNA, is involved with sewing the foreign DNA into the genomic fabric
of the target cell. Regions of native DNA are constantly being stripped of protective pro-
teins, unfolded, accessed, and reassembled. DNA can be tightly coiled and precisely
ordered, but access to chromosomal DNA is needed for it to function. If foreign DNA is
in the right place at the right time, it may slip into the reassembly process and become incor-
porated into the native DNA. Although presented here as a moderately haphazard process,
foreign DNA must be precisely configured and introduced, show a necessary functionality,
and behave as endogenous plant DNA in order to be retained.
10.2. BASIC COMPONENTS FOR SUCCESSFUL GENE TRANSFER TO PLANT CELLS 247