Plant Biotechnology and Genetics: Principles, Techniques and Applications

(Brent) #1

  1. Why is there a need for negative selectable markers in experimental plant science?
    Can you design experiments that would employ them?


9.1 Introduction


The genetic transformation of plant cells is known to occur in nature, but the technologies
for the reproducible generation of transgenic plants in the laboratory are only about 25 years
old. Many questions about the fundamental nature of transgenic plants are therefore still
being raised. In part, this reflects the more recent emergence of the technologies but
more importantly it reflects our limited understanding of the plant genome and the
genetic mechanisms that govern how it works. Indeed, it was shown only as recently
as 2005 how remarkably stable the transcriptional patterns and programming mechanisms
are in plants and how impervious they are to the insertion of marker genes (El Ouakfaoui
and Miki 2005). Such understanding is critical if transgenic plants are to be used as a
vehicle to study the functions of unknown genes isolated from genomics studies.
Furthermore, the biosafety of transgenic food or feed is evaluated by their equivalence to
other plants that are already being used and known to be safe; therefore, it is important
to determine whether nontargeted, unanticipated genetic changes could be induced
during the transformation process. Marker genes allow scientists to study such fundamental
processes and also provide a pivotal ingredient needed to generate transgenic plants.
Several kinds of marker genes have been developed and are needed for the diverse roles
they play in biotechnology as well as in experimental plant science. These include the
recovery of transgenic plants, the experimental manipulation of plant tissues, the assess-
ment of plant gene regulatory mechanisms, the intracellular trafficking of proteins, and
the assessment of biosafety of transgenic plants. The marker genes can be grouped into
different categories depending on whether they (1) are selectable, (2) promote or suppress
tissue growth and differentiation, and (3) are conditional on external substrates. As the com-
plexity and needs of research increase, there might also arise a requirement to selectively
remove marker genes from transgenic plants to create marker-free plants. This chapter
also covers important promoters that are used in transgenic plants as they often represent
the difference between a successful plant biotechnology project and one that remains
in obscurity.


9.2 Definition of Marker Genes


Various marker genes have played crucial roles in facilitating the production of transgenic
plants, the subsequent identification of the transgenic plants, and the fine-tuning of
procedures needed to increase the transformation frequencies.Marker genesfall into two
categories: selectable marker genes and nonselectable(also referred to as scorable
markerorreporter) genes (see Table 9.1 for examples).


9.2.1 Selectable Marker Genes: An Introduction


The first selectable marker genes enabled the production of transgenic plants by using
chemicals in the plant growth media that allowed transgenic tissues to grow, but not non-
transgenic tissues. Because only a few cells are transformed in a population of target cells in


218 MARKER GENES AND PROMOTERS
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