Plant Biotechnology and Genetics: Principles, Techniques and Applications

(Brent) #1

(El Ouakfaoui and Miki 2005). This gene has been the most extensively studied among the
selectable marker genes, and the largest body of information on its use in plants has been
accumulated.
HygromycinB is the second most commonly used antibiotic for selection of transformed
plant cells (Waldron et al. 1985) after kanamycin; however, it is very toxic to plant cells
relative to kanamycin and more difficult to apply without “overkill.” It is an aminocyclitol
antibiotic that also inhibits protein synthesis with a broad spectrum of activity. The bacterial
gene,aphIV(also referred to ashph,hpt), codes for hygromycin phosphotransferase (HPT,
EC 2.7.1.119), which acts by ATP-dependent phosphorylation of hygromycin B. It has
been used extensively over a wide range of species.
Other antibiotics that have been used include streptomycin, bleomycin, streptothricin, and
chloramphenicol. Generally, resistance is conferred by genes coding for enzymes that act by
detoxification of the antibiotic through a modification to the molecular structures.


9.4.1.2. Selection on Herbicides. Phosphinothricin(PPT) or glufosinate ammonium
is the active component of several commercial herbicides. As an analog ofL-glutamic acid,
it is a competitive inhibitor of glutamine synthase (GS) that is essential for the assimilation
of ammonia into plants. By inhibition of glutamine synthase ammonia accumulates to toxic
levels. The enzyme phosphinothricinN-acetyltransferase (PAT) will detoxify PPT by acetyl
CoA-mediated acetylation and thus confer resistance. Two genes coding for the enzyme
have been cloned: thebargene (for bialophos resistance, where bialaphos consists of
twoL-alanine residues and PPT) fromStreptomyces hygroscopicusand thepatgene (for
phosphinothricin acetyltransferase) from Streptomyces viridochromogenes. Both have
been used extensively as selectable marker genes, particularly among cereal species
where kanamycin selection may be less efficient. Typically, kanamycin does not kill mono-
cots very effectively, whereas bialophos or PPT does.
Plants containing thebarorpatgenes have been among the first to receive regulatory
approval for unconfined field production and have been assessed as safe by a number of
international regulatory agencies. A number of other herbicides, including glyphosate, imi-
dazolinones, and bromoxynil, can also be used in combination with their corresponding
resistance genes for selection of transgenic plants.


9.4.1.3. Selection Using Nontoxic Metabolic Substrates.Most conditional posi-
tive selection systems use toxic substrates for selection of the transformed tissues; however,
the use of nontoxic metabolic intermediates has emerged as an alternative. This type of
system differs from the use of antibiotics, herbicides, or drugs in that the substrates are
not inhibitors but rather carbon sources that are restricted from use by the plant cell
unless provided with an enzyme that allows entry of the carbon source into primary meta-
bolism. Examples of such selective agents include mannose andD-xylose. Bacterial genes
coding for phosphomannose isomerase (PMI, EC 5.3.1.8) fromE. coli(manA) or xylose
isomerase from (EC 5.3.1.5)Streptomyces rubiginosusandThermoanaerobacterium ther-
mosulfurogenes(xylA) provide the enzymes that allow entry into glycolysis. The apparent
advantage is that it works with a wide range of plant species and appears to yield higher
transformation frequencies because the selection is not as harsh as with toxic substrates
(Reed et al. 2001). This approach differs fundamentally from the others discussed so far
in that the novel trait encoded by the selectable marker gene alters a basic aspect of
plant metabolism.


9.4. SELECTABLE MARKER GENES 229
Free download pdf