Plant Biotechnology and Genetics: Principles, Techniques and Applications

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Transformed cells can, however, be selected in other ways. “Positive selection” refers to
the ability of a cell to survive by utilizing nutrient sources that are unavailable to nontrans-
formed cells. As an example, a sugar such as mannose cannot be metabolized by most cells,
unless mannose can be converted to the useful form of fructose, using a transgene that
encodes phosphomannose isomerase. Cells containing this gene can grow on a medium
containing mannitol as the sole carbon source, while the nontransformed cells will
starve. A toxin is not required for selection. Another selection scheme utilizes genes that
allow cells to be identified and physically isolated from nontransformed cells.
Introduction of “reporter genes” allows scientists to identify transformed cells through a
unique characteristic, such as a new color or emission of fluorescence or phosphorescence.
Introduction of the gene encoding the green fluorescent protein imparts a fluorescent green
color to plant cells, when viewed under high-energy blue or UV light. Cells or clusters of
cells containing the green fluorescent protein can be visually detected and physically iso-
lated (see previous chapter).
Once transformed cells have been recovered and purified from nontransformed cells,
whole genetically engineered plants can be recovered through the tissue culture regener-
ation process (Chapter 5).


10.3 Agrobacterium


Agrobacteriumis a soilborne bacterium that has been rightfully called the “natural plant
genetic engineer.” Over its evolutionary journey, this bacterium has developed the
unique ability to transfer part of its DNA into plant cells. The DNA that is transferred is
called theT-DNA(fortransferredDNA) and this DNA is carried on an extrachromosomal
plasmid called theTi(tumor-inducing) plasmid. Through intervention of scientists, the Ti
plasmid no longer causes tumor formation in infected plant cells, but the T-DNA region is
still transferred. As opposed to DNA transfer methods that utilize direct uptake of DNA into
plant cells, the use ofAgrobacteriummay appear to be more complex because two different
biological systems (bacteria and the target plant cells) are involved. This might have been
true in the early years of plant transformation, but today,Agrobacteriumprovides the
method of choice for most plant transformation efforts. With methods utilizing introduction
of DNA without a biological vector (direct DNA uptake), it appears to be necessary to
deliver the DNA to the nucleus of the target cell, but withAgrobacterium, the T-DNA
itself possesses the necessary signals for delivery there.
Most direct DNA introduction systems require expensive instrumentation, but
Agrobacteriumis simply prepared by growth on an appropriate medium and inoculated
on the plant tissue. Additional claims of simpler foreign DNA insertions and more consist-
ent transgene function in plants transformed withAgrobacteriummay or may not be valid,
and this appears to depend more on how the DNA is delivered with direct DNA introduc-
tion systems than on any inherent problem with the method. Considering primarily overall
transformation efficiency, advances since the mid-1980s in our understanding of the
Agrobacterium-mediated DNA transfer process have led to tremendous increases in effi-
ciency and use of this transformation vector.


10.3.1. History ofAgrobacterium


Agrobacterium tumefaciensis naturally occurring bacterium that causes a disease in plants
calledcrown gall. Crown gall disease remains a problem with many horticultural plants,


10.3. AGROBACTERIUM 249
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