Ab stractBar ley trans for ma tion me di ated by Agrobacterium tume faciens is rou tinely per formed in a num ber of lab o ra to ries. However, elim i na tion of selectable marker genes and for ma tion of plants ho mo zy gous for the transgene via con ven tional seg rega tion is la bo ri ous and time-con sum ing. Here we sug gest a con cept that in cludes the pro duc tion of pri mary trans gen ic plants via in fec tion of im ma ture em bryos with A. tumefaciens fol lowed by androgenetic gen er a tion of a seg re gat ing pop u lation of en tirely ho mo zy gous plants. Selectable marker-free, true breeding plants car ry ing a sin gle-opy transgene integrant may thus be ef fi ciently and rap idly ob tained. How ever, amenability to Agrobacterium-me di ated trans for ma tion as well as androgenetic po ten tial is ge no type-de pend ent. Ef ficient ge netic trans for ma tion by in fec tion of im ma ture em bryos is so far con fined to the spring type cultivar 'Golden Prom ise' which, how ever, turned out to be re cal ci trant in pol len embryogenesis. To fa cil i tate androgenetic gen er a tion of ho mo zy gous segregants from pri mary transformants, we have es tab lished a method for embryogenic pol len cul ture in cv. Golden Prom ise that in cludes con ven tional cold-treat ment and sub se quent preculture of im ma ture pol len un der star va tion con di tions prior to trans fer to com plete nu tri ent me dium. Fur ther we show that con di tion ing of the pol len cul ture me dium by co-cul ture of im ma ture wheat pis tils as well as ad di tion of pis til-pre con ditioned me dium con sid er ably sup port androgenetic de vel opment. Em ploy ment of the es tab lished method us ing im ma ture pol len of pri mary trans gen ic plants dem on strates that selectable marker-free, true-breed ing trans gen ic prog eny can be rap idly ob tained pur su ing the con cept pro posed. The pro tocol pre sented will be use ful in func tional genomics as well as in mo lec u lar breed ing ap proaches. In tro duc tionSta ble ge netic trans for ma tion is a stan dard method em ployed for the func tional char ac teri sa tion of reg u la tory and pro tein-cod ing nu cleic acid sequences. More over, it of fers a wealth of op por tu nities to con trib ute to crop breed ing. Bar ley is one of the eco nom i cally most im por tant and most widely dis trib uted crop spe cies world wide. Ini tially, di vers trans for ma tion meth ods based upon di rect DNA-trans fer were de vel oped for this spe cies (Wan and Lemaux 1994, Jaehne et al. 1994, Funatsuki et al. 1995, Koprek et al. 1996, Zhang et al. 1999, Holm et al. 2000, how ever, the reproducibility and ef fi ciency of these pro to cols turned out to be rather poor. More over, due to frequent deg ra da tion of the DNA-con structs prior to 591 ACTA PHYSIOLOGIAE PLANTARUM

Background The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. Results In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. Conclusions The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

Following the production of transgenic plants, the selectable marker gene(s) used in the process are redundant, and their retention may be undesirable. They can be removed by exploiting segregation among the progeny of co-transformants carrying both the selectable marker gene and the effector transgene. Here we show that the doubled haploid technology widely used in conventional barley breeding programmes represents a useful means of fixing a transgene, while simultaneously removing the unwanted selectable marker gene. Primary barley co-transformants involving hpt::gfp (the selectable marker) and gus (a model transgene of interest) were produced via Agrobacterium-mediated gene transfer to immature embryos using two respective T-DNAs. These plants were then subjected to embryogenic pollen culture to separate independently integrated transgenes in doubled haploid progeny. A comparison between 14 combinations, involving two Agrobacterium strains carrying various plasmids, revealed that the highest rate of independent co-transformation was achieved when a single Agrobacterium clone carried two binary vectors. Using this principle along with Agrobacterium strain LBA4404, selectable marker-free, gus homozygous lines were eventually obtained from 1.5 per 100 immature embryos inoculated. Compared to the segregation of uncoupled T-DNAs in conventionally produced progeny, the incorporation of haploid technology improves the time and resource efficiency of producing true-breeding, selectable marker-free transgenic barley.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9988-9) contains supplementary material, which is available to authorized users.

BackgroundDoubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited.ResultsWe have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers.ConclusionsEnzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.

The modifcation of crop genomes employing functional components of the microbial CRISPR/Cas immune system is a rapidly developing area of applied research. Site-directed plant genome modifcation by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products and regeneration of plants from modifed cells. The utilization of this technology in local breeding programs is mainly limited by the typically strong genotype dependence of gene transfer andin vitroregeneration procedures, which holds particularly true in cereals. In the present study, an evaluation ofin vitroregeneration efciency of immature embryos of ten Siberian barley cultivars revealed that only one of these is on a par with the experimental standard cultivar Golden Promise. This cultivar, namely cv. Aley, was consequently chosen for further experiments on site-directed mutagenesis in leaf mesophyll protoplasts. Two genes controlling hulledvsnaked (Nud) and two-rowedvssix-rowed barley (Vrs1) were used as targets to be modifed via polyethyleneglycol-mediated cellular uptake of guide-RNA/Cas9-encoding plasmid DNA. Deep-sequencing of amplicons obtained from protoplast genomic DNA revealed that 6 to 22 percent of the target sites were mutated. The detected modifcations comprised deletions in all three target sites and of various sizes, whereas insertions were observed in only one of the target genes (Vrs1) and were confned to the size of 1 nucleotide. This study demonstrates the possibility of site-directed genome modifcation in Siberian barley. Further steps in technology advancement will require the development of protocols with reduced genotype dependence in terms of both the gene transfer to totipotent cells and the subsequent plant regeneration originating from such cells.

The Eukaryotic Translation Initiation Factor 4E (EIF4E) is a well-known susceptibility factor for potyvirus infections in many plant species. The barley yellow mosaic virus disease, caused by the bymoviruses Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), can lead to yield losses of up to 50% in winter barley. In autumn, the roots of young barley plants are infected by the soil-borne plasmodiophoraceous parasite Polymyxa graminis L. that serves as viral vector. Upon viral establishment and systemic spreading into the upper parts of the plants, yellow mosaics occur as first symptoms on leaves. In the further course of plant development, the disease entails leaf necrosis and increased susceptibility to frost damage. Thanks to the rym4 and rym5 allelic variants of the HvEIF4E gene, more than two thirds of current European winter barley cultivars are resistant to BaYMV and BaMMV. However, several strains of BaYMV and BaMMV have already overcome rym4- and rym5-mediated resistance. Accordingly, new resistance-conferring alleles are needed for barley breeding. Therefore, we performed targeted mutagenesis of the EIF4E gene by Cas9 endonuclease in BaMMV/BaYMV-susceptible winter barley cv. “Igri”. Small insertions were generated, resulting in a shift of the translational reading frame, thereby causing the loss-of-function of EIF4E. The mutations occurred in the homozygous state already in the primary mutants. Their progeny proved invariably homozygous and fully resistant to mechanical inoculation with BaMMV. EIF4E knockout plants showed normal growth habit and produced grains, yet exhibited a yield penalty.

The temperate cereal barley is grown as a source of food, feed, and malt. The development of a broad range of genetic resources and associated technologies in this species has helped to establish barley as the prime model for the other Triticeae cereals. The specific advantage of the transformation method presented here is that transgene homozygosity is attained in the same generation as the transgenic event occurred through the coupling of haploid technology with Agrobacterium-mediated transformation. Pollen is haploid and, following transformation, can be induced to regenerate into haploid plantlets, which can subsequently subjected to colchicine treatment to obtain diploid, genetically fixed plants. The routine application of the method based on the winter-type barley cultivar 'Igri' over a period of over 10 years has achieved an average yield of about two transgenic plants per donor spike. The whole procedure from pollen isolation to non-segregating transgenic, mature grain takes less than 12 months.

In this chapter, a detailed protocol for the induction of targeted mutations in barley using customized transcription activator-like effector nucleases (TALENs) is provided. As explant for the introduction of TALEN expression constructs, embryogenic pollen cultures are used which consist primarily of haploid cells able to be converted via pollen embryogenesis and genome duplication into fertile doubled haploid plants. Thanks to the haploid nature of these target cells, a mutation induced by TALE nucleases in just one gfp allele can be readily detected in vitro and thus genetically fixed primary mutant plants obtained in one generation.