Brachypodium - Bioenergy Crop Plants - page 603

relatively simple insertions (e.g., cloning flanking DNA or promoter tagging) and may lead to silencing
of transgenes in later generations. Attempts to minimize the complexity of biolistic loci by using linear
DNA instead of circular plasmid DNA have produced mixed results (Fu et al. 2000; Loc et al. 2002).
23.3.3.2 agrobacterium-mediated transformation
Compared with biolistic transformation, Agrobacterium -mediated transformation has been shown
to yield much simpler and lower copy number insertion patterns in rice and Arabidopsis (for a direct
comparison of methods see Dai et al. 2001 and Travella et al. 2005). Furthermore, transgenic plants
contain an average of approximately 1.5 insertions per line, averting the challenges to downstream
analyses encountered with the complex loci of the biolistic lines (Feldmann 1991; Jeon et al. 2000).
Instead, the difficulties of establishing an efficient Agrobacterium -mediated transformation system
reside in the host limitations of Agrobacterium . Fortunately, Brachypodium has proven amenable
to Agrobacterium -mediated transformation. In the first report of Agrobacterium -mediated
transformation, 16 polyploid accessions and 3 diploid accessions were evaluated for transformability
(Vogel et al. 2006b). The highest transformation efficiency (14% of the callus pieces cocultivated
with Agrobacterium -produced transgenic plants) was achieved with the polyploid line Bd17-2. A
diploid accession, PI 254867, was transformed at a much lower efficiency (2.5%).
Subsequent studies have made considerable progress at improving the efficiency of the
Agrobacterium -mediated transformation of Brachypodium. In 2007, three studies reported very high
transformation efficiencies for three different Brachypodium lines. The first two papers used the inbred
lines Bd21-3 (Vogel and Hill 2008) and Bd21 (Vain et al. 2008), which were separately derived from
USDA accession PI 254867 as previously described in Section 23.3.1. The methods described in these
two papers share a number of important similarities, including media types, Agrobacterium strains,
and use of immature embryos as initial explants. Dissecting out immature embryos is the most labor-
intensive step in these processes, and both methods take advantage of multiple subculture steps to
amplify the callus before transformation so that each dissected embryo gives rise to many transgenic
plants. The differences between the methods lie in the following: the use of desiccating conditions
to improve transformation of Bd21-3, the formation of a yellow embryogenic callus in Bd21-3 that
allows selection of the appropriate callus type without the aid of a microscope, the use of very small
embryos and copper sulfate to improve the quality of the Bd21 callus, and visual selection of green
fluorescent protein (GFP) and the subculturing callus under a microscope to improve the efficiency of
Bd21 selection. In these studies, transformation efficiency was calculated as the percentage of calluses
cocultivated with Agrobacterium that produced fertile transgenic plants, and the average efficiencies
achieved were 37% for Bd21-3 and 17% for Bd21. The third Brachypodium transformation paper
reports an extremely high average transformation eficiency of 55% for accession BDR018 (Pˇ curar
et al. 2007). The authors achieve this high efficiency by placing immature embryos on callus-inducing
media for 17 days and then cocultivating those embryos with Agrobacterium . Efficiency is calculated
from the percentage of dissected embryos that form fertile transgenic plants. The limitation of this
method is that the embryogenic callus is not subcultured, and therefore no more than one independent
transgenic line can arise from each dissected embryo. This increases the labor involved in generating
transgenic plants when compared with the methods for Bd21-3 and Bd21 transformation.
The publication of three high-efficiency Agrobacterium -mediated transformation methods signals
that Brachypodium transformation technology has matured, yet the lessons learned from these three
papers inspire continued studies to optimize Brachypodium transformation. For example, adoption
of the use of very small embryos (0.3-0.7 mm) and a callus initiation medium that includes 0.6 µg/
mL copper sulfate has further increased the average transformation efficiency of Bd21-3 to more
than 50% (J. Bragg unpublished; protocol available at http://brachypodium.pw.usda.gov/). Further
improvements in Brachypodium transformation will undoubtedly emerge both from investigating
the genetic diversity within the growing collections of Brachypodium accessions and from other
technical advances. It is noteworthy that the rapid pace of Brachypodium transformation technology
development compares very favorably with the development of high-efficiency transformation in