Inositol trisphosphate 5/6 kinases (ITPK) constitute a small group of enzymes participating in the sequential phosphorylation of inositol phosphate to inositol hexakisphosphate (IP6), which is a major storage form of phosphate in cereal grains. The development of lines with reduced IP6 content could enhance phosphate and mineral bioavailability. Moreover, plant ITPKs participate in abiotic stress signaling. To elucidate the role of HvITPK1 in IP6 synthesis and stress signaling, a barley itpk1 mutant was created using programmable nuclease Cas9. Homozygous single bp insertion and deletion mutant lines were obtained. The mutants contained altered levels of phosphate in the mature grains, ranging from 65% to 174% of the wild type (WT) content. Homozygous mutant lines were tested for their response to salinity during germination. Interestingly, insertion mutant lines revealed a higher tolerance to salinity stress than deletion mutants. Mature embryos of an insertion mutant itpk1-2 and deletion mutant itpk1-33 were cultivated in vitro on MS medium supplemented with NaCl at 50, 100, and 200 mM. While both mutants grew less well than WT on no or low salt concentrations, the itpk1-2 mutant was affected less than the WT and itpk33 when grown on the highest NaCl concentration. The expression of all ITPKs was induced in roots in response to salt stress. In shoots, the differential effect of high salt on IPTK expression in the two iptk1 mutants was consistent with their different sensitivities to salt stress. The results extend the evidence for the involvement of ITPK genes in phosphate storage and abiotic stress signaling.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Summary: Although many genetic manipulations of crops providing biofortified or safer food have been prepared, the acceptance of biotechnology crops still remains limited. We report on a transgenic barley expressing the multi-functional protein osmotin that improves plant defense under stress conditions. An Agrobacterium–mediated technique was used to transform immature embryos of the spring barley cultivar Golden Promise. Transgenic barley plants of the T0 and T1 generations were evaluated by molecular methods.Transgenic barley tolerance to stress was determined by chlorophyll, total protein, malondialdehyde and ascorbate peroxidase content. Transgenic plants maintained the same level of chlorophyll and protein, which significantly declined in wild-type barley under the same stressful conditions. Salt stress evoked higher ascorbate peroxidase level and correspondingly less malondialdehyde. Methanol extracts of i) Fusarium oxysporum infected or ii) salt-stressed plants, were characterized by their acute toxicity effect on human dermal fibroblasts (HDF). Osmotin expressing barley extracts exhibited a lower cytotoxicity effect of statistical significance than that of wild-type plants under both types of stress tested on human dermal fibroblasts. Extract of Fusarium oxysporum infected transgenic barley was not able to damage DNA in Comet assay, which is in opposite to control plants. Moreover, this particular barley did not affect the local biodiversity interactions, which was tested through monitoring barley natural virus pathogen – host interactions – the BYDV and WDV viruses transmitted to the plants by aphids and leafhoppers. Our findings provide a new perspective which could help to evaluate the safety of products from genetically modified crops.

Phytic acid is the main storage of phosphate in grains of staple crops. As phytic acid is hardly digestible for non-ruminants microbial phytases are used to supplement animal feed to enhance phosphate digestibility. A fungal phytase gene was introduced into barley with the aim of enhancing phosphate digestibility. Transgenic homozygous barley over-expressing fungal phytase phyA showed a 3.3fold increase in mature grain phytase activity. Field trials at two locations in the Czech Republic were conducted in a five-year experiment to test transgene stability and activity under field conditions. Increased phytase activity gradually decreased over the generations showing the most significant drop in the initial years of field trials. Molecular analysis revealed methylation in the coding sequence of the phyA transgene, suggesting transcription gene silencing. On the other hand, herbicide resistance used for selection of transgenic plants was functional over all generations. The feasibility of crossing the transgene into the feeding cultivar Azit was demonstrated with subsequent stabilization of hybrid progeny through androgenesis. Our results indicate that the Azit genetic background tended to reduce phytase activity in mature grains of hybrids. Grain-specific over-expression of fungal phytase driven by an amylase promoter improved phosphate levels during germination. Unfortunately, a malting experiment revealed that phytase over-expression did not significantly improve malting parameters. In fact, the higher nitrogen content in unmalted grain negatively affected the quality of the malt produced from them.