11.1 Introduction
After a particle bombardment orAgrobacterium-mediated transformation experiment to
generate transgenic plants is conducted (see Chapter 10), several tests must be conducted
to determine whether the experiment yielded transgenic plants and to determine a host of
other features about the plants. In the perfect world, an experiment will yield hundreds
oftransgenic eventsfrom which to choose, and at least one event with a single insertion
and high level of gene expression. The unique placement of the transgenic construct
within the plant genome constitutes a transgenic event, along with the progeny of that
plant. Therefore, each transformation event is said to beindependentof the other events.
Of equal importance is the fact that the phenotype of the transgenic plant will be
changed only in the way expected. Independent transformants (or transgenic events)
must be identified among the nontransformed plants and the validity of each transformation
event established (e.g., Are my plants really transgenic?). This is accomplished through a
series of physical, phenotypic, and genetic analyses (Birch 1997; Potrykus 1991). New
tools have been developed to validate transformation events. Throughout this chapter, we
will explore the various tests biotechnologists use to analyze transgenic events.
11.2 Directionally Named Analyses: As the Compass Turns
A number of requirements must be met before declaring that a plant is transformed, and the
best way to do this is through molecular analyses. The analyses that molecular biologists
have devised to characterize segments of DNA, RNA, and protein in organisms have
unique names that are often confusing to a newcomer in the field because they are in no
way descriptive of the molecules described: a secret nomenclature of molecular biologists.
In 1975 Edwin Southern at the University of Oxford described a way to transfer DNA to a
membrane and then probe it with homologous DNA of interest (aprobe); henceDNA gel
blotanalysis is also known asSouthern blotanalysis (Southern 1975). Southern blot analy-
sis is performed to demonstrate that the transgene is physically integrated into the genome,
usually nuclear, which also determines the number ofinsertionsorcopy numberof trans-
genes. It also shows whether the entire gene was integrated into the plant genome. A trun-
cated or rearranged copy will not be functional. After determining that a transgene is stably
integrated, the next step is often to see if its specific mRNA is produced. There are several
ways to do that.RNA gel blot analysis, also known asnorthern blot, is the conventional
method. This test is not named after a person, but is a cute derivative of its seemingly oppo-
site direction on a compass. Not that RNA is opposite of DNA, but in a moment of scientific
frivolity, northern blots must have seemed like a nifty name to refer to RNA blots. Another
assay for mRNA transcript detection and quantification isreal-time reverse transcriptase
(RT)-PCR. Real-time RT-PCRdoes not need as large a sample and is more rapid.
Immunoblotanalysis, also known asprotein blotorwestern blotanalysis (or did you
think it would be “eastern”?) identifies transgenic proteins with antibodies that indicates
bands of discernible sizes that can be semiquantified by their intensities. An alternative
way to see if the specific transgenically produced protein is produced is by enzyme-
linked immunosorbent assay (ELISA), which uses a specific antibody to probe a protein
mixture in a microtiter plate.
Without providing Southern blot analysis showing integration of the transgene, pheno-
typic data are only partially qualifying evidence and not proof that a transformation event
276 TRANSGENIC PLANT ANALYSIS