Man Bo Lee

Effect of gamma radiation on growth and lignin content in Brachypodium distachyon

Brachypodium distachyon has been highlighted as a model monocot plant with small genome and short life cycle. Biofuels are being developed as renewable energy sources to replace fossil fuels. Bioethanol production is negatively correlated with lignin content. Here, Brachypodium was acutely or chronically irradiated at doses of 50, 100, 150, 200, and 250 Gy. The effect of radiation on plant growth and generation of mutant populations was explored. The lethal effect of radiation was higher in acutely irradiated M0 populations. A dose-dependent negative effect in plant height, tiller number, floral spikelet, and total seed number was observed, with a positive effect in days to heading. The phenotype of 1,773 M1 plants was evaluated, with 417 plants being selected to construct the M2 population. The 31 M2 plants that showed the least staining with phloroglucinol were selected. These mutants could be useful materials for studies such as identification of nucleotide substitutions in genes involved in lignin biosynthesis pathway, monitoring of mutant physiological traits, and evaluation of fitness for bioethanol production. As biological resources, the M2 populations generated in this work will contribute to studies of functional genomics of Brachypodium and to the breeding of grass crops.

Employment of hordein subunit polymorphisms in establishing selection criteria for high quality malting barley (Hordeum vulgare L.)

Cereal seed storage proteins are encoded by complex multigene families and their subunit profiles are highly related to end-use qualities. Each fraction of albumin and hordein was extracted and its subunit profile was evaluated in related to malt and grain quality parameters. The purpose of this study was to provide selection criteria for high quality malting barley using grain and malt quality parameters and biochemical-genetic information. Grain and malt quality of 13 local adaptability test (LAT) lines were evaluated for malting process. A total of 16 germplasm accessions of high or low seed storage protein content were also evaluated for biochemicalgenetic analysis. The correlation coefficients among quality parameters were analyzed. Several important quality parameters in brewing process showed significant positive or negative correlations. Seed storage protein subunits of albumin and hordein of all tested lines and accessions were evaluated using 12% 1D SDS-PAGE. Scored data of protein subunit’s presence or absence was applied to Agglomerative Hierarchical Clustering (AHC) for statistical analysis and showed specific grouping patterns among tested lines. Clustered lines with subunit information were highly related with agricultural performance and grain and malt qualities. Based on the profiles of seed storage protein subunits, association of hordein subunit of 38, 43, and 65 kDa with high malt scored lines was found. The obtained results would provide improved selection criteria for high quality malting barley in the malting barley breeding program.

Identification of candidate genes for the seed coat colour change in a Brachypodium distachyon mutant induced by gamma radiation using whole-genome re-sequencing

Brachypodium distachyon has been proposed as a model plant for agriculturally important cereal crops such as wheat and barley. Seed coat colour change from brown-red to yellow was observed in a mutant line (142-3) of B. distachyon, which was induced by chronic gamma radiation. In addition, dwarf phenotypes were observed in each of the lines 142-3, 421-2, and 1376-1. To identify causal mutations for the seed coat colour change, the three mutant lines and the wild type were subjected to whole-genome re-sequencing. After removing natural variations, 906, 1057, and 978 DNA polymorphisms were detected in 142-3, 421-2, and 1376-1, respectively. A total of 13 high-risk DNA polymorphisms were identified in mutant 142-3. Based on a comparison with DNA polymorphisms in 421-2 and 1376-1, candidate causal mutations for the seed coat colour change in 142-3 were selected. In the two independent Arabidopsis thaliana lines carrying T-DNA insertions in the AtCHI, seed colour change was observed. We propose a frameshift mutation in BdCHI1 as a causal mutation responsible for seed colour change in 142-3. The DNA polymorphism information for these mutant lines can be utilized for functional genomics in B. distachyon and cereal crops.

Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes

Brachypodium distachyon (Brachypodium) is a novel model plant for structural and functional genomic studies of temperate grasses. Brachypodium as a model plant has many favorable features, such as small size, small genome, short life cycle, self-fertility, and simple growth requirements. The genome sequence of the standard line Bd21 has been released and genomic resources have been developed.It is imperative to develop a method for efficient Agrobacterium-mediated Brachypodium transformation. Yellowish and compact embryogenic calli derived from immature embryos of the Bd21 were transformed with the Agrobacterium strain AGL1. Seven- and nine-week-old calli were used for transformation with Agrobacterium carrying either pCAMBIA 1301 and pCAUGH. Transformation efficiencies were assessed through histochemical GUS assay. The efficiency of transformation with pCAMBIA 1301 (based on the number of callus lines producing GUS-detected plantlets and the number of calli used for transformation) reached 20.1% (7-week-old calli) and 1.7% (9-week-old calli), and with pCAUGH (based on the number of GUS-detected plantlets and the number of regenerants) 90 and 87% for 7- and 9-week-old calli, respectively. High selection pressure was obtained by using pCAUGH, which is preferred for saving labor and time consumption during the callus selection.