Principles of Plant Genetics and Breeding

244 CHAPTER 14
Steps in a DNA microarray experiment
To conduct a DNA microarray experiment, six general
steps are followed:
1 Probe: the experimenter first selects the genetic
material of known identity (e.g., cDNAs or small
oligos) to be used as probes.
2 Fabrication of array: a fabrication format is selected.
3 Sample preparation: the sample (cDNA, RNA) to be
used to interrogate the spotted and immobilized
probes is prepared and fluorescently labeled.
4 Assay: the assay is then conducted.
5 Readout: the results are read by, for example, electronic
devices.
6 Informatics: the data may be submitted to a variety
of data management systems to obtain useful and
desired information according to the objectives of the
researcher.
Applications of DNA microarrays in plant breeding
DNA microarrays may be used in two ways: (i) for
sequence identification (normal genes and detection of
mutations); and (ii) to examine gene expression (level
or abundance of gene expression). One of the attributes
of genes of great interest to microarray research is their
expression. The expression pattern of a gene provides
indirect information about its function.
The general steps followed in conducting a gene
expression study are summarized as follows. Consider
a study in which gene expression between two samples,
A and B, are being compared.
1 Prepare fluorescently labeled cDNA of the total pool
of mRNA from each cell population by reverse transcription
in the presence of fluorescently labeled
cDNA precursors. Use different fluors to allow distinction
between their effects.
2 Mix two fluorescently labeled cDNAs.
3 Hybridize with a DNA microarray in which a distinct
spot of DNA represents each gene. The cDNA
sequences representing each individual transcript will
hybridize with only the corresponding gene sequence
in the array, regardless of the fluorescent labels.
The result is a pattern in which the relative abundance
of the transcripts from each gene corresponds to the
ratio of the two fluors used. It should be mentioned
that gene expression data have certain limitations. For
example, mRNA levels do not always reflect protein
levels, and also, the expression of a protein may not
always have a physiological consequence.
Genetic use restriction systems
Plant breeders may protect their inventions (cultivars)
by seeking patents or plant variety protection. However,
the legal provisions are effective in protecting proprietary
material from abuse only if they are enforced or the
farmers are trusted to abide by the legal restrictions.
Researchers have been working on protection systems
that are self-regulatory, needing no policing for enforcement.
The first of such systems was unveiled in 1998.
Developed jointly by the US Department of Agriculture
(USDA) and the Delta and Pine Land Company, the
technology protection system (TPS) was awarded a
patent in 1998. The nature of the patent allows each
party to act independently from the other. The original
genetic molecular switch was inserted into tobacco, and
then cotton. Delta and Pine Land is the world leader in
cotton seed production. Soon after the announcement,
the technology was greeted by negative attacks from
activist and other sources. The Rural Advancement
Foundation International (RAFI) (now the ETC Group)
described it in near derogatory terms as “terminator
technology”, a term that appears to have stuck. To
avoid this unscientific term, a new term was proposed
and introduced in 1999, the genetic use restriction
system (GURT). The term is broadly used to describe
the use of exogenous substances as inducers to control
the expression of a plant’s genetic traits (e.g., trait
for sterility, color, ripening, and cold tolerance). The
restriction of a specific trait in a plant is called the T-
GURT (also derided by activist as “traitor technology”);
the V-GURT refers to the use of genetic engineering
of plants to produce sterile seeds (i.e., the terminator
technology).
How the technology protection system works
TPS may be deployed in three basic steps:
1 The terminator genes are spliced into the genome of
the target crop.
2 The seed company initiates the terminator process
prior to selling the seed to farmers, by treating the
seed with a substance (an inducer).
3 Farmers plant and harvest the seed in the usual way;
however, the seed is sterile and will not germinate
upon replanting.
Seed sterilization by TPS may be accomplished by one
of three scientific approaches. Generally, all approaches
use known gene mechanisms to control the expression