HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors.

Improved efficiency of doubled haploid generation in hexaploid triticale by in vitro chromosome doubling.

BACKGROUND: Doubled haploid production is a key technology in triticale research and breeding. A critical component of this method depends on chromosome doubling, which is traditionally achieved by in vivo treatment of seedlings with colchicine. RESULTS: In this study we investigated the applicability of an in vitro approach for chromosome doubling based on microspore culture. Our results show a pronounced increase in the proportion of doubled haploid triticale plants compared to the spontaneous doubling rate, but also compared to the doubling obtained by the standard in vivo approach. In addition, the frequency of plants surviving from culture medium to maturity is also much higher for the in vitro approach. Colchicine concentrations of 1 mM for 24 h or 0.3 mM applied for 48 or 72 h during the first hours of microspore culture performed best. CONCLUSIONS: Our results suggest that for triticale, in vitro chromosome doubling is a promising alternative to the in vivo approach.

Natural selection in common bean microsatellite alleles and identification of QTLs for grain yield

Natural selection acts to select better adapted individuals or alleles in segregating population and help plant breeding. The objective of this work was to verify the effect of natural selection on microsatellite alleles as indicators of better adaptation and identification of quantitative trait loci (QTLs) for grain yield. This study evaluated 107 progenies from the F8 and 107 from the F24 generation derived from crossing Carioca MG and ESAL 686 lines, carried out by the bulk method, and evaluated in three different seasons: winter 2001; rainy 2001 and dry 2002. It was utilized 22 polymorphic markers and the natural selection acted in all of them. The frequency of the alleles of the parent Carioca MG, the most adapted, was increased in all of the 22 loci in F8 and 19 loci in F24. Selection affected each locus with different intensities in different generations. All of the selected alleles can be important for breeding program. QTLs were identified in generation F8 and F24 at varied magnitudes. The best marker PVttc002 explained 11.76 percent of variation in grain yield. However, an elevated interaction between QTLs and the environments was observed, showing the great difficulty in assisted selection.

Direct embryogenesis and green plant regeneration from isolated microspores of hexaploid triticale (x Triticosecale Wittmack) cv. Bogo.

The use of doubled haploids improves the efficiency of cultivar development in many crops and can be helpful in genetic and molecular studies. The major problem with this approach is the low efficiency of green plant regeneration. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of hexaploid triticale (x Triticosecale Wittmack) cv. Bogo. The absence of growth regulators in the induction medium was the most effective condition for the formation of embryo-like structures. The highest induction rates were observed at microspore densities of 1.5x10(5) microspores and 2x10(5) microspores per milliliter. Such cultures produced an average of 54.9 green plants per single donor spike. The frequency of albino plants ranged from 9.3% to 22.9%. Among the green progeny tested, 30.8% were spontaneously doubled haploids.

Analysis of avenin proteins and the expression of their mRNAs in developing oat seeds.

We have isolated and characterized cDNA clones encoding avenins, the prolamine storage proteins of oat seeds. Sequence analysis shows that avenins are a related group of polypeptides and that their mRNAs differ from each other by point mutations and small insertions and deletions. Avenin proteins have structural homology to the alpha/beta-gliadins and gamma-gliadins of wheat, the B-hordeins of barley, and the gamma-secalins of rye. Hybridization analysis of DNA from various diploid, tetraploid, and hexaploid oat species shows that the oat genome contains more globulin storage protein genes than avenin genes and that some restriction fragments containing these genes are conserved between species with common genomes. We estimate that there are 25 avenin genes and 50 globulin genes per haploid genome in Avena sativa and similar ratios of globulin to avenin genes in other Avena species. Avenin and globulin polypeptides begin to accumulate between 4 days and 6 days after anthesis. Messenger RNAs encoding avenin and globulin proteins become abundant 4 days after anthesis and reach peak concentrations at 8 days after anthesis. Avenin mRNAs are present in somewhat greater molar amounts than globulin mRNAs beginning at 4 days after anthesis. Because there is considerably more globulin than avenin in the mature oat seed, the expression of globulin and avenin genes may be regulated both transcriptionally and post-transcriptionally.