Environmental Engineering Reference
In-Depth Information
“shattering” (Opanowicz et al. 2008). Diploid seed sizes range from an average of 2.5 mg/seed for the
Tek-10 inbred line to 5.9 mg/seed for Kah-7 (Vogel et al. 2009).
Brachypodium also exhibits intraspecific diversity in chromosome number. Most diploid
Brachypodium lines have a base chromosome number of 5 (1
n
= 5) (Draper et al. 2001; Vogel et al.
2006b; Filiz et al. 2009). However, accessions with chromosome numbers of 1
n
= 10 and 1
n
= 15
have also been described (Draper et al. 2001; Hasterok et al. 2004). Studies utilizing FISH and GISH
techniques indicate that the 1
n
= 10 and 1
n
= 15 cytotypes are not merely autopolyploids derived
from the 1
n
= 5 cytotype (Hasterok et al. 2004). Chromosomes of the 1
n
= 10 accession ABR114
are smaller than those of the 1
n
= 5 accession ABR1, and the FISH-visualized pattern of rDNA
loci in ABR114 is inconsistent with ABR114 being an autotetraploid arising from ABR1 (Hasterok
et al. 2004). Thus, ABR114 seems to be a diploid with a base chromosome number of 10. Similar
cytogenetic analyses have led to the idea that the 1
n
= 15 accession ABR113 is an allotetraploid
that arose from the hybridization of ABR1- and ABR114-like parents, with base chromosome
numbers of 5 and 10, respectively (Hasterok et al., 2004, 2006). Thus, the different Brachypodium
cytotypes should probably be considered different species rather than a simple polyploid series.
When examined carefully, the polyploids characterized to date can be easily distinguished from the
1
n
= 5 diploid accessions. For example, among the Turkish lines analyzed, one group of polyploids
was distinguished by large seeds and thick, hairy stems and a second group of polyploids was
characterized by a deep, longitudinal crease in the seed (Vogel et al. 2009). In both groups, anthers
exerted more frequently than in the diploid lines. It should be noted that the 1
n
= 5 diploid is the
cytotype being used for genome sequencing and resource development and that the 1
n
= 10 form is
known from only one collection to date (Garvin et al. 2008).
23.3.2 g
rowth
r
EquirEmEntS
The simple requirements for growing Brachypodium make it easy to culture under laboratory
conditions. Brachypodium can be grown in growth chambers or greenhouses used for
Arabidopsis
,
wheat, barley, switchgrass, or other plants. Our standard conditions for growth chambers are 20-h
light:4-h dark photoperiod, 24°C during the day, and 18°C at night with cool-white fluorescent
lighting at a level of 150 µEm
-2
s
-1
. Our standard greenhouse conditions are no shading, 24°C
in the day and 18°C at night, and supplemental lighting to extend day length to 16 h. Although
Brachypodium grows well in a number of different soil types, we have observed that it is highly
susceptible to
Pythium
root rot. Plants that are watered excessively or left in standing water often
develop disease symptoms under our conditions. We also have observed disease symptoms that
correlated with the use of one brand of commercial potting mix and recommend that Brachypodium
growers test a few soil formulations before selecting one to grow large numbers of plants (Vogel
and Bragg 2009).
Vernalization has been shown to induce flowering in all diploid accessions studied to date, but,
as noted above, the time required to induce flowering varies greatly between accessions (Vogel
et al. 2006b, 2009; Vogel and Hill 2008). Providing the appropriate conditions to induce flowering
is critical to preventing excessive vegetative growth. For a combined stratification and vernalization
treatment, we typically sow the seeds and then place them at 4°C for the desired number of weeks
(inbred lines Bd21 and Bd21-3 require 2-3 weeks of vernalization to reliably induce flowering
unless grown under very long day lengths). After approximately 3 weeks in the cold, seeds
begin to germinate. Thus, for vernalization times greater than 4 weeks, we place the pots under
fluorescent lighting. Vernalizing seeds/seedlings induces the plants to flower quickly while still
small. Alternatively, one can vernalize larger plants if more seed from individual plants is desired.
Inbred lines Bd21 and Bd21-3 are particularly responsive to growth under very long day conditions
(20 h light:4 h dark) and go from seed to seed in as little as 8 weeks to yield nearly six generations
per year. Under these conditions, the plants flower and set seed when they are approximately 15 cm
tall—a size that is compatible with high-density planting.