Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Segregation distortion in homozygous lines obtained via anther culture and maize doubled haploid methods in comparison to single seed descent in wheat (Triticum aestivum L. )

Background The quality of wheat grain depends on several characteristics, among which the composition of high molecular weight glutenin subunits, encoded by Glu-1 loci, are the most important. Application of biotechnological tools to accelerate the attainment of homozygous lines may influence the proportion of segregated genotypes. The objective was to determine, whether the selection pressure generated by the methods based on in vitro cultures, may cause a loss of genotypes with desirable Glu-1 alleles. Results Homozygous lines were derived from six winter wheat crosses by pollination with maize (DH-MP), anther culture (DH-AC) and single seed descent (SSD) technique. Androgenetically-derived plants that originated from the same callus were examined before chromosome doubling using allele-specific and microsatellite markers. It was found that segregation distortion in SSD and DH-MP populations occurred only in one case, whereas in anther-derived lines they were observed in five out of six analyzed combinations. Conclusions Segregation distortion in DH-AC populations was caused by the development of more than one plant of the same genotype from one callus. This distortion was minimized if only one plant per callus was included in the population. Selection of haploid wheat plants before chromosome doubling based on allele-specific markers allows us to choose genotypes that possess desirable Glu-1 alleles and to reduce the number of plants in the next steps of DH production. The SSD technique appeared to be the most advantageous in terms of Mendelian segregation, thus the occurrence of residual heterozygosity can be minimized by continuous selfing beyond the F6 generation.

Cloning of high molecular weight gluten subunit promoter and study on its function in wheat

The aim of this work was to study the cloning and characterization of HMW-GS 1Dx2 promoter from Triticum aestivum. A 1050 bp partial promoter fragment including a putative TATA box and 5' encoding sequence of the gene was cloned by amplifying the upstream sequences using the nest-PCR with appropriate primers. The analysis of the promoter sequence against the PLACE (Plant cis-acting Regulatory DNA Elements) database showed the presence of certain putative endosperm-specific regulatory cis-elements in the sequence along with the TATA and CAAT boxes. The histochemical method detected the transient expressions of GUS in the seeds of wheat. The results showed that HMW-GS 1Dx2 promoter had the endosperm-specific transcription activity in the wheat seeds.