Environmental Engineering Reference
In-Depth Information
The inbreeding nature of Brachypodium simplifies the maintenance of independent lines
under laboratory conditions. The anthers of diploid accessions rarely exert, suggesting a low rate
of outcrossing. This was confirmed by measuring pollen flow from transgenic to nontransgenic
plants under growth chamber and greenhouse conditions. In a population of more than 2000
progeny, no outcrossing was observed (Vogel et al. 2009). The inbreeding nature of Brachypodium
in the wild was confirmed by analyzing SSR profiles of 62 wild individuals. These individuals
were overwhelmingly homozygous, despite the presence of multiple SSR alleles in the population,
indicating that Brachypodium primarily self-pollinates in the wild (Vogel et al. 2009).
23.3.3 t
ranSformation
The utility of a modern model plant system depends greatly on the development of efficient methods
to introduce foreign DNA into its genome. The dicot model
Arabidopsis
benefits from an extremely
facile transformation method in which flowers are simply dipped into a solution of
Agrobacterium
tumefaciens
for several seconds (Clough and Bent 1998). As a result, the
Arabidopsis
research
community has access to invaluable tools such as stable knockout lines for most genes in the
genome (Pan et al. 2003). In contrast, grass transformation is a more challenging and labor-intensive
endeavor. Routine transformation of grasses requires extensive tissue culture manipulations, and
transformation of almost all grasses is very inefficient. Supporting its utility as a model system,
Brachypodium has proven to be very responsive to in vitro culture, and current transformation
efficiencies are on par with rice, the present gold standard for grass transformation. A major step
toward achieving Brachypodium transformation was the development of a method for the induction
of embryogenic callus (Figure 23.1g) from Brachypodium seeds and the regeneration of fertile plants
(Figure 23.1, h and i) from this callus. In 1995, Bablak et al. established the optimal callus-inducing
medium to contain LS salts, 3% sucrose, and 2.5 mg L
-1
2,4-Dichlorophenoxyacetic acid (Bablak
et al., 1995). Mature seeds from three diploid accessions (B200, B373, and B377) were incubated on
callus-inducing media. All were found to produce embryogenic callus, along with several other types
of callus, and regeneration was observed on several common media, indicating that Brachypodium
had no unusual requirements for regeneration.
Particle bombardment and
A. tumefaciens
-mediated transformation are the two methods most
commonly used for plant transformation, and both have been used to successfully transform
Brachypodium. Each technique offers unique advantages and disadvantages.
23.3.3.1 Biolistic transformation
The regeneration of plants from bombarded explants represents the primary determinant of
successful biolistic transformation. The first published Brachypodium transformation involved
particle bombardment of a polyploid Brachypodium accession (ABR100). In these experiments,
the average efficiency was five transformants per gram of starting embryogenic callus (Draper
et al. 2001). A subsequent, more detailed, account of biolistic transformation answered the question
of whether a diploid accession could be transformed (Christiansen et al. 2005). In this study, the
authors successfully transformed the diploid accession BDR018 with an average efficiency of 5.3% of
bombarded calluses producing transgenic plants. The authors' failed attempts to transform a second
diploid accession (BDR001) demonstrate that, as for other plants, genotype plays a critical role in
determining transformation efficiency. These initial Brachypodium studies compare favorably with
early reports of biolistic rice transformation that showed an average efficiency of 3.75% (Christou
et al. 1991). However, biolistic transformation commonly results in complex transgenic loci. Typically,
these loci contain multiple copies of the inserted DNA, including truncated pieces of the target DNA
interspersed with genomic DNA (Svitashev and Somers 2002; Kohli et al. 2003). These biolistic
insertions often contain many repeats of inserted DNA and can span several megabases of host
DNA (Svitashev and Somers 2002). The complexity of these insertions represents a major drawback
of biolistic transformation because they can interfere with downstream applications that require
