10.2.3 Target Tissue Status
For successful production of transgenic plants, plant cells, which have the ability to grow
(differentiate) into whole plants, should be targeted. The ability of a single cell to grow
into a whole plant is calledtotipotency, and the cell that is naturally totipotent is the fer-
tilized egg. Although it is probably true that all plant cells have thepotentialto grow into
whole plants, that potential has not yet been reached for most cells. At this point in trans-
genic plant history, scientists can regenerate plants only from specific cell types in most
plants. With a few plants, many different cell types are more easily manipulated to
grow into whole plants through the tissue culture process (see Chapter 5). Successful pro-
duction of genetically engineered plants is dependent on the coordination of DNA delivery
with generation of a whole plant from the single cell, which is targeted for DNA
introduction.
An ideal target would therefore be the fertilized egg or even the pollen that gives rise to
the fertilized egg. Unfortunately, these ideal targets do not appear to be responsive for
almost all plants with the exception of the model plant,Arabidopsis thaliana. The next
most suitable target for DNA delivery might be the shoot meristem, which gives rise to
the aboveground parts of the plant. Although the meristem has been successfully targeted
for DNA introduction, it is a complex multicellular structure, and the most appropriate
target cells are located in the center of the structure, buried under quite a few cell layers.
Surface cells are obviously more accessible for DNA delivery. In the clear majority of
cases, the target tissue used for production of transgenic plants consists of rapidly
growing specialized plant cells, which have been induced to form whole plants. These
cells should be physically accessible, actively dividing (DNA replication accelerates
DNA integration into the genome), and able to give rise to whole plants. These cells
should also be resilient enough to tolerate the breach of the cell wall and membrane by
the DNA, which is truly an intrusive event in the life of a plant cell.
10.2.4 Selection and Regeneration
Because of the nature of DNA introduction, only a small percentage of plant cells can
usually be successfully targeted. The clear majority of cells therefore just get in the way.
How do scientists pick out the rare cell that contains the foreign DNA? For almost all trans-
formation efforts, selection is the key. Along with the gene of interest, another gene, encod-
ing resistance to an antibiotic or herbicide, is introduced (see Chapter 9 for a more thorough
description). The mixture of transformed and nontransformed cells is then exposed to the
antibiotic or herbicide, and only those cells containing the resistance gene will survive
and grow.Selectionrefers to the ability of the transformed cells to proliferate in the presence
of otherwise toxic selective agents. Resistance genes will encode for proteins that either
detoxify a toxin or produce an alternate form of a target enzyme that is insensitive to the
toxin. The most commonly used antibiotic resistance genes are neomycin phosphotransfer-
ase and hygromycin phosphotransferase, which provide resistance to the antibiotics kana-
mycin and hygromycin, respectively. The most commonly used herbicide resistance gene is
thebargene (sometimes referred to as thepatgene), which encodes for phosphinothricin
acetyl transferase. This enzyme inactivates the herbicides glufosinate and bialaphos, which
were originally discovered for their antibiotic properties. Selection for growth in the pre-
sence of toxic agents is the most common form of selection and is often called “negative
selection” (as noted in the last chapter, the terminology used to describe selection of
marker genes is variable among researchers).
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