Environmental Engineering Reference
In-Depth Information
rice. In the initial reports, rice transformation efficiencies were less than 1%, and through the efforts
of many groups over several years, efficiency was improved to the more than 40% commonly
achieved today (Tyagi and Mohanty 2000).
The highly efficient transformation methods developed for Brachypodium lay a strong foundation
for Brachypodium as a model system to study fundamental aspects of grass biology. In addition,
efficient transformation means that Brachypodium is an excellent test bed for transgenic approaches
in the grasses. By using Brachypodium, researchers can much more rapidly test constructs for
expression, efficacy, etc., before moving into biomass or other grass crops.
23.3.4 g
EnEration
of
B
rachypodium
m
utant
p
opulationS
Genome-saturating mutant populations provide a means to explore the relationship between a
phenotype and a gene of interest, and both forward and reverse genetic strategies require large
populations of mutagenized plants. Diverse approaches for the efficient generation of mutant
populations include treatment with mutagenic chemicals, exposure to high-energy radiation, and
random DNA insertions. Researchers have had success applying these protocols to Brachypodium,
and it appears that there are no limitations in applying common mutagens used with other plants.
The development of populations of mutants, similar to those available for
Arabidopsis
and rice, will
provide valuable tools for the Brachypodium research community.
23.3.4.1 ethyl methanesulfonate mutagenesis
Ethyl methanesulfonate
(EMS) is an efficient chemical mutagen that introduces single base changes
and has been used widely to mutagenize plants. In
Arabidopsis
, an extensive review of mutations in
192 genes verified the random nature of EMS mutations and estimated the frequency at one mutation
per 170 kb of genomic DNA (Greene et al. 2003). An advantage of the single base changes derived
from EMS mutagenesis is that these simple changes can result in partial loss-of-function alleles that
may be particularly useful when studying essential genes. We have mutagenized Brachypodium
with EMS by adapting a method used to create a population of barley mutants (Caldwell et al. 2004).
The frequency of albino plants is often used to measure the success of EMS mutagenesis, and over a
mutant population of 2000 M
2
plants, we have observed 2% albinos. This rate is comparable to that
typically observed in successful EMS treatments of
Arabidopsis
seeds (Kim et al. 2006). Our initial
screens have focused on identifying mutations that alter cell wall and biomass phenotypes, and we
have identified more than 25 mutants of interest to date, indicating that the EMS mutagenesis was a
success. Visit http://brachypodium.pw.usda.gov/ for our current EMS mutagenesis protocol.
23.3.4.2 Fast neutron radiation
Fast neutron radiation
(FNR) introduces short deletions into the genomic DNA and is therefore a
complementary mutagen to EMS, which induces single base changes. The larger disruptions generated
by FNR permit rapid cloning of the mutant loci using a genome tiling array. This is an advantage over
EMS mutagenesis, in which the genes must be identified using map-based cloning. However, because
the region deleted by FNR typically encompasses several genes, this method is not suitable for the
identification of essential genes or genes located adjacent to essential genes. In early experiments, 1-2%
of mutagenized plants were albinos, indicating that Brachypodium can be efficiently mutagenized by
FNR (D. Laudencia-Chingcuanco and M. Byrne, personal communication).
23.3.4.3 Insertional mutagenesis
Insertional mutagenesis is a natural complement to chemical and radiation mutagenesis because,
although the mutational load is low (only one or a few mutations per line), the ability to sequence
DNA flanking the insertion site enables rapid identification of the affected genes, which is required
for reverse genetic approaches. The insertion of transferred DNAs (T-DNAs) via
Agrobacterium
-
mediated transformation and the movement of transposons are the two most common approaches
