V. PRELIMINARY TEST OF THE USE OF TREHALOSE SYNTHASE GENE AS A SELECTABLE MARKER
5.1 Introduction
Selectable marker, is a gene which are intentionally introduced into cells as developmental tools in recombinant DNA technology e.g. to transfer genes for
desired traits between organisms. Selectable marker is a gene that is introduced into a cell that confers a trait for artificial selection en.Wikipedia.org. The
selectable marker is extremely important tool during transformation protocol acting as “genetic tag” to identify easily the successfully modified cells with
incorporated desired trait genes that have been introduced. Selection markers that are widely used are antibiotic resistance genes by
which their proteins degrade antibiotics such as kanamicin, hygromycin, and herbicide phosphynotrycin. These antibioticsherbicide resistance genes might
not fit well with public concern about the possibility of gene flow and horizontal gene transfer may occur. Hence, non-toxic selectable markers that are more
natural are currently developing. Furthermore, more concern is needed when crop plants are the target of gene transfer particularly when the yield is expected
for foodstuff, although it is unlikely that there will be a gene transfer from GM food to bacteria within intestine Darbani et al. 2007. Methods to obtain marker free
transgenic has been developed, however, its implementation is still difficult. For this reason, selectable marker engineering to suite such purpose is necessary.
Trehalose has been used in many aspects of human daily life: food ingredient, cosmetics, pharmaceutics, and medical treatments as well as used as
biological stabilizer in laboratory works. TfTreS expressing lines Arabidopsis have been obtained Chapter 4. The lines are resistant to high level of trehalose 100
mM, hence this study is conducted to explore the possibility of the use TfTreS as a non-antibiotic selectable marker on trehalose containing medium. Using TfTreS
gene as a selectable marker is beneficial, since this product is edible and environmentally friendly. Additionally, there is an indication that the presence of
TfTreS increases plant capability withstand drought Chapter 4 and might also
other stresses, as Garg et al. 2002 and Jang et al. 2003 found that transgenic rice bearing E. coli trehalose synthases OtsA-OtsB tolerate drought, salt and
cold. The similar case but using TreY and TreZ gene from Grifola frondosa that
were introduced into Tobacco and sugar cane also make the transgenic plants tolerated to drought, salt and cold Zhang et al 2005; Zhang et al 2006.
To be able to use TfTreS as a selectable marker, the transgenic plants have to have a very strong activity to combat the effect of high level trehalose on
the medium. This experiment tested the capability of TfTreS expressing lines to grow on medium supplemented with trehalose and trehalase inhibitor
Validamycin A. Validamycin A is an antibiotic that inhibits trehalase activity, therefore, trehalose added to the medium is remained intact, and endogenous
trehalose level within the tissue can be increased Godijn et al. 1997, Muller et al. 2001. When the plants resistant to trehalose or trehalose and validamycin A in
such combination that the Wild type plants are unable to survive, it means that TfTreS
can be used as a selectable marker on trehalose or trehalose and validamycin A.
5.2 Material and methods 5.2.1 Place and time of research
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