Genetic Basis of Grain Size and Weight in Rice, Wheat, and Barley.

Grain size is a key component of grain yield in cereals. It is a complex quantitative trait controlled by multiple genes. Grain size is determined via several factors in different plant development stages, beginning with early tillering, spikelet formation, and assimilates accumulation during the pre-anthesis phase, up to grain filling and maturation. Understanding the genetic and molecular mechanisms that control grain size is a prerequisite for improving grain yield potential. The last decade has brought significant progress in genomic studies of grain size control. Several genes underlying grain size and weight were identified and characterized in rice, which is a model plant for cereal crops. A molecular function analysis revealed most genes are involved in different cell signaling pathways, including phytohormone signaling, transcriptional regulation, ubiquitin-proteasome pathway, and other physiological processes. Compared to rice, the genetic background of grain size in other important cereal crops, such as wheat and barley, remains largely unexplored. However, the high level of conservation of genomic structure and sequences between closely related cereal crops should facilitate the identification of functional orthologs in other species. This review provides a comprehensive overview of the genetic and molecular bases of grain size and weight in wheat, barley, and rice, focusing on the latest discoveries in the field. We also present possibly the most updated list of experimentally validated genes that have a strong effect on grain size and discuss their molecular function.

Genome-Wide Identification of Barley Long Noncoding RNAs and Analysis of Their Regulatory Interactions during Shoot and Grain Development.

Long noncoding RNAs (lncRNAs) are a class of RNA molecules with gene regulatory functions in plant development and the stress response. Although the number of lncRNAs identified in plants is rapidly increasing, very little is known about their role in barley development. In this study, we performed global identification of barley lncRNAs based on 53 RNAseq libraries derived from nine different barley tissues and organs. In total, 17,250 lncRNAs derived from 10,883 loci were identified, including 8954 novel lncRNAs. Differential expression of lncRNAs was observed in the developing shoot apices and grains, the two organs that have a direct influence on the final yield. The regulatory interaction of differentially expressed lncRNAs with the potential target genes was evaluated. We identified 176 cis-acting lncRNAs in shoot apices and 424 in grains, while the number of trans-acting lncRNAs in these organs was 1736 and 540, respectively. The potential target protein-coding genes were identified, and their biological function was annotated using MapMan ontology. This is the first insight into the roles of lncRNAs in barley development on the genome-wide scale, and our results provide a solid background for future functional studies.

TaCKX2.2 Genes Coordinate Expression of Other TaCKX Family Members, Regulate Phytohormone Content and Yield-Related Traits of Wheat.

TaCKX gene family members (GFMs) play essential roles in the regulation of cytokinin during wheat development and significantly influence yield-related traits. However, detailed function of most of them is not known. To characterize the role of TaCKX2.2 genes we silenced all homoeologous copies of both TaCKX2.2.1 and TaCKX2.2.2 by RNAi technology and observed the effect of silencing in 7 DAP spikes of T1 and T2 generations. The levels of gene silencing of these developmentally regulated genes were different in both generations, which variously determined particular phenotypes. High silencing of TaCKX2.2.2 in T2 was accompanied by slight down-regulation of TaCKX2.2.1 and strong up-regulation of TaCKX5 and TaCKX11, and expression of TaCKX1, TaCKX2.1, and TaCKX9 was comparable to the non-silenced control. Co-ordinated expression of TaCKX2.2.2 with other TaCKX GFMs influenced phytohormonal homeostasis. Contents of isoprenoid, active cytokinins, their conjugates, and auxin in seven DAP spikes of silenced T2 plants increased from 1.27 to 2.51 times. However, benzyladenine (BA) and abscisic acid (ABA) contents were significantly reduced and GA3 was not detected. We documented a significant role of TaCKX2.2.2 in the regulation of thousand grain weight (TGW), grain number, and chlorophyll content, and demonstrated the formation of a homeostatic feedback loop between the transcription of tested genes and phytohormones. We also discuss the mechanism of regulation of yield-related traits.

Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology.

Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields.

Specificity of expression of TaCKX family genes in developing plants of wheat and their co-operation within and among organs.

Multigene families of CKX genes encode cytokinin oxidase/dehydrogenase proteins (CKX), which regulate cytokinin content in organs of developing plants. It has already been documented that some of them play important roles in plant productivity. The presented research is the first step of comprehensive characterization of the bread wheat TaCKX gene family with the goal to select genes determining yield-related traits. The specificity of expression patterns of fifteen formerly annotated members of the TaCKX family was tested in different organs during wheat development. Based on this, the genes were assigned to four groups: TaCKX10, TaCKX5 and TaCKX4, highly specific to leaves; TaCKX3, TaCKX6 and TaCKX11, expressed in various levels through all the organs tested; TaCKX1, TaCKX2.3, TaCKX2.2, TaCKX2.1, TaCKX2.4 and TaCKX2.5 specific to developing spikes and inflorescences; TaCKX9, TaCKX8 and TaCKX7, highly specific to roots. Amplification products of tested genes were mapped to the chromosomes of the A, B or D genome using T. aestivum Ensembl Plants. Based on analysis of TaCKX transcripts as well as encoded amino acids in T. aestivum and Hordeum vulgare the number of CKX genes in wheat was limited to 11 and new numbering of selected TaCKX genes was proposed. Moreover, we found that there were developmental differences in expression of TaCKX in the first and the second spike and expression of some of the genes was daily time dependent. A very high and significant correlation was found between expression levels of TaCKX7 and TaCKX9, genes specific to seedling roots, TaCKX1, TaCKX2.1 and TaCKX2.2, specific to developing spikes, and the group of TaCKX3, 4, 5, 6, 10 and 11, highly expressed in leaves and other organs. The genes also co-operated among organs and were included in two groups representing younger or maturating stages of developing plants. The first group was represented by seedling roots, leaves from 4-week old plants, inflorescences and 0 DAP spikes; the second by developing spikes, 0 DAP, 7 DAP and 14 DAP. The key genes which might determine yield-related traits are indicated and their possible roles in breeding strategies are discussed.

A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (Hordeum vulgare L.).

BACKGROUND: Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley. RESULTS: We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T0 plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T1 lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T1 plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T1 plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T0 plants. CONCLUSIONS: We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

Major genes determining yield-related traits in wheat and barley.

KEY MESSAGE: Current development of advanced biotechnology tools allows us to characterize the role of key genes in plant productivity. The implementation of this knowledge in breeding strategies might accelerate the progress in obtaining high-yielding cultivars. The achievements of the Green Revolution were based on a specific plant ideotype, determined by a single gene involved in gibberellin signaling or metabolism. Compared with the 1950s, an enormous increase in our knowledge about the biological basis of plant productivity has opened new avenues for novel breeding strategies. The large and complex genomes of diploid barley and hexaploid wheat represent a great challenge, but they also offer a large reservoir of genes that can be targeted for breeding. We summarize examples of productivity-related genes/mutants in wheat and barley, identified or characterized by means of modern biology. The genes are classified functionally into several groups, including the following: (1) transcription factors, regulating spike development, which mainly affect grain number; (2) genes involved in metabolism or signaling of growth regulators-cytokinins, gibberellins, and brassinosteroids-which control plant architecture and in consequence stem hardiness and grain yield; (3) genes determining cell division and proliferation mainly impacting grain size; (4) floral regulators influencing inflorescence architecture and in consequence seed number; and (5) genes involved in carbohydrate metabolism having an impact on plant architecture and grain yield. The implementation of selected genes in breeding programs is discussed, considering specific genotypes, agronomic and climate conditions, and taking into account that many of the genes are members of multigene families.

Agrobacterium-Mediated Transformation of Leaf Base Segments.

Agrobacterium-mediated transformation has become a routine method of genetic engineering of cereals, gradually replacing the biolistic protocols. Simple integration patterns of transgenic loci, decent transformation efficiency, and technical simplicity are the main advantages offered by this method. Here we present a detailed protocol for the production of transgenic oat plants by Agrobacterium-mediated transformation of leaf base segments. The use of leaf explants as target tissues for transformation and in vitro regeneration of transgenic plants may be a good alternative for genotypes which are not susceptible to regeneration from immature or mature embryos. We also describe the biochemical and molecular analysis procedures of the transgenic plants including a GUS histochemical assay, and Southern blot, both of which are optimized for application in oat.

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.

Oat (Avena sativa L.).

Agrobacterium-mediated transformation is a suitable method to transform different cultivars using different systems of A. tumefaciens strains and binary vectors as well as selection cassettes. We describe here a detailed protocol for two cultivars, one naked and one husked, using the AGL1 strain and the pGreen vector containing the nptII selection cassette ( http://www.pgreen.ac.uk/ ), suitable for oat as well as other cereals. The pGreen vector system was recently developed for pBract ( http://www.bract.org/ ) and its transformation ability for cereals was proved. Assuming our experience and the latest knowledge on Agrobacterium-mediated transformation of cereals, we suggest using in the protocol one of the newly developed pBract or pCAMBIA ( http://www.cambia.org/daisy/cambia/ ) vector systems which carry different selection cassettes. The commonly used selection genes nptII, bar, and hpt were proved to be applicable for oat transformation and might be used as needed.

Expression patterns of HvCKX genes indicate their role in growth and reproductive development of barley.

Cytokinin oxidase/dehydrogenase proteins (CKX) are encoded by a multigene family of CKX genes with a varying number of members depending on species. For some of the genes, spectacular effects on grain production in selected cereals have been observed. Despite the fact that partial or full length sequences of most HvCKX genes in barley (Hordeum vulgare) have already been published, in most cases their specific biological functions have not been reported. Detailed expression patterns for five HvCKX genes in different organs/tissues of developing barley plants coupled with analysis of RNAi silent for two genes are presented to test the hypothesis that these expression profiles might indicate their function. Elevated expression for four of them - HvCKX1, HvCKX9, HvCKX4, and HvCKX11 - was found in developing kernels of wild-type plants compared to other tissues. HvCKX5 was mainly expressed in leaf tissue. Lower expression was noted for HvCKX1 in seedling roots and for HvCKX9 in leaves. The documented effect of RNAi silencing of HvCKX1 and a trend for HvCKX9 was higher plant productivity, and the trait was inherited through four generations. Higher plant yield was determined by higher numbers of seeds and spikes. Increased productivity was significantly greater in HvCKX1 silenced plants showing higher relative expression of HvCKX1 in developing kernels of wild-type plants compared to the expression of HvCKX9. Both HvCKX1 silenced T1 seedlings of cv. Golden Promise and the newly transformed breeding line STH7308 showed greater root mass, but this trait was not inherited in the next generation. Similarly HvCKX9 silenced T1 seedlings exhibited greater plant height without inheritance in the next generation. It is suggested that these effects were not inherited because of compensation by other genes co-ordinately regulating reproductive development. One line with untypically changed, inherited phenotype, which was selected from several dozen silenced lines showing stable and common phenotypes is presented.

Sina and Sinb genes in triticale do not determine grain hardness contrary to their orthologs Pina and Pinb in wheat.

BACKGROUND: Secaloindoline a (Sina) and secaloindoline b (Sinb) genes of hexaploid triticale (x Triticosecale Wittmack) are orthologs of puroindoline a (Pina) and puroindoline b (Pinb) in hexaploid wheat (Triticum aestivum L.). It has already been proven that RNA interference (RNAi)-based silencing of Pina and Pinb genes significantly decreased the puroindoline a and puroindoline b proteins in wheat and essentially increased grain hardness (J Exp Bot 62:4025-4036, 2011). The function of Sina and Sinb in triticale was tested by means of RNAi silencing and compared to wheat. RESULTS: Novel Sina and Sinb alleles in wild-type plants of cv. Wanad were identified and their expression profiles characterized. Alignment with wheat Pina-D1a and Pinb-D1a alleles showed 95% and 93.3% homology with Sina and Sinb coding sequences. Twenty transgenic lines transformed with two hpRNA silencing cassettes directed to silence Sina or Sinb were obtained by the Agrobacterium-mediated method. A significant decrease of expression of both Sin genes in segregating progeny of tested T1 lines was observed independent of the silencing cassette used. The silencing was transmitted to the T4 kernel generation. The relative transcript level was reduced by up to 99% in T3 progeny with the mean for the sublines being around 90%. Silencing of the Sin genes resulted in a substantial decrease of secaloindoline a and secaloindoline b content. The identity of SIN peptides was confirmed by mass spectrometry. The hardness index, measured by the SKCS (Single Kernel Characterization System) method, ranged from 22 to 56 in silent lines and from 37 to 49 in the control, and the mean values were insignificantly lower in the silent ones, proving increased softness. Additionally, the mean total seed protein content of silenced lines was about 6% lower compared with control lines. Correlation coefficients between hardness and transcript level were weakly positive. CONCLUSIONS: We documented that RNAi-based silencing of Sin genes resulted in significant decrease of their transcripts and the level of both secaloindoline proteins, however did not affect grain hardness. The unexpected, functional differences of Sin genes from triticale compared with their orthologs, Pin of wheat, are discussed.

HvCKX2 gene silencing by biolistic or Agrobacterium-mediated transformation in barley leads to different phenotypes.

BACKGROUND: CKX genes encode cytokinin dehydrogenase enzymes (CKX), which metabolize cytokinins in plants and influence developmental processes. The genes are expressed in different tissues and organs during development; however, their exact role in barley is poorly understood. It has already been proven that RNA interference (RNAi)-based silencing of HvCKX1 decreased the CKX level, especially in those organs which showed the highest expression, i.e. developing kernels and roots, leading to higher plant productivity and higher mass of the roots [1]. The same type of RNAi construct was applied to silence HvCKX2 and analyze the function of the gene. Two cultivars of barley were transformed with the same silencing and selection cassettes by two different methods: biolistic and via Agrobacterium. RESULTS: The mean Agrobacterium-mediated transformation efficiency of Golden Promise was 3.47% (±2.82). The transcript level of HvCKX2 in segregating progeny of T(1) lines was decreased to 34%. The reduction of the transcript in Agrobacterium-derived plants resulted in decreased CKX activity in the developing and developed leaves as well as in 7 DAP (days after pollination) spikes. The final phenotypic effect was increased productivity of T(0) plants and T(1) lines. Higher productivity was the result of the higher number of seeds and higher grain yield. It was also correlated with the higher 1000 grain weight, increased (by 7.5%) height of the plants and higher (from 0.5 to 2) numbers of spikes. The transformation efficiency of Golden Promise after biolistic transformation was more than twice as low compared to Agrobacterium. The transcript level in segregating progeny of T(1) lines was decreased to 24%. Otherwise, the enzyme activity found in the leaves of the lines after biolistic transformation, especially in cv. Golden Promise, was very high, exceeding the relative level of the control lines. These unbalanced ratios of the transcript level and the activity of the CKX enzyme negatively affected kernel germination or anther development and as a consequence setting the seeds. The final phenotypic effect was the decreased productivity of T(0) plants and T(1) lines obtained via the biolistic silencing of HvCKX2. CONCLUSION: The phenotypic result, which was higher productivity of silenced lines obtained via Agrobacterium, confirms the hypothesis that spatial and temporal differences in expression contributed to functional differentiation. The applicability of Agrobacterium-mediated transformation for gene silencing of developmentally regulated genes, like HvCKX2, was proven. Otherwise low productivity and disturbances in plant development of biolistic-silenced lines documented the unsuitability of the method. The possible reasons are discussed.

The RNA-mediated silencing of one of the Pin genes in allohexaploid wheat simultaneously decreases the expression of the other, and increases grain hardness.

The RNAi-mediated silencing of Pina and Pinb, the two genes responsible for the grain texture of allohexaploid wheat, was induced and analysed in two wheat cultivars, Kontesa and Torka. A characterization of the two genes in non-transgenic plants revealed that Pinb carries a point mutation, designated Pinb-D1c in both cultivars. This mutation does not influence transcript abundance or protein content. Two silencing cassettes of the hpRNA type were constructed and used for stable transformation via Agrobacterium. In total, 43 transgenic lines representing the two cultivars were obtained, transformed with the silencing cassettes for Pina or for Pinb or co-transformed with both cassettes. The relative transcript levels of the two genes in the same progeny plant were found to be similar, independent of the silencing cassette used. The reduction in the Pina and Pinb transcript levels in the segregating T(1) progeny of Kontesa and Torka transformed with one of the silencing cassettes exceeded 80%. Co-transformation with the silencing cassettes for both genes resulted in a reduction of over 91% of Pina and Pinb transcripts in some segregating T(1) progeny of Kontesa. The silencing was transmitted to the T(4) kernel generation of the T(3) lines. A significant reduction or lack of both puroindoline proteins in the silenced lines correlated with an essential increase in grain hardness. The discussion covers some new insights into the function of the Pin genes, including the simultaneous silencing of both, independent of the siRNA signal.

Silencing of the HvCKX1 gene decreases the cytokinin oxidase/dehydrogenase level in barley and leads to higher plant productivity.

Stable RNA interference-based technology was used to silence the expression of the HvCKX1 gene in barley and the TaCKX1 gene in wheat and triticale. The silencing cassettes containing the fragments of these genes in the sense and antisense orientations were cloned into the pMCG161 binary vector and used for Agrobacterium-based transformation. Out of the five cultivars representing the three studied species, transgenic plants were obtained from one barley cultivar Golden Promise, one wheat cultivar Kontesa, and one triticale cultivar Wanad. Almost 80% of 52 regenerated lines of Golden Promise exhibited significantly decreased cytokinin oxidase/dehydrogenase (CKX) enzyme activity in bulked samples of their T(1) roots. There was a positive correlation between the enzyme activity and the plant productivity, expressed as the yield, the number of seeds per plant, and the 1000 grain weight. Additionally, these traits were associated with a greater root mass. Lower CKX activity led to a higher plant yield and root weight. This higher plant productivity and altered plant architecture were maintained in a population of segregating T(1) plants. The levels of HvCKX1 transcript accumulation were measured in various tissues of Golden Promise and Scarlett non-transgenic barley plants in order to choose the most appropriate plant organs to study the expression and/or silencing of the gene in those transgenic lines. The highest levels of the HvCKX1 transcript were detected in spikes 0 days after pollination (0 DAP), 7 DAP, and 14 DAP, and in the seedling roots. The analysis of HvCKX1 gene expression and CKX enzyme activity and the evaluation of the phenotype were performed in the progeny of seven selected transgenic T(1) lines. The relative expression of HvCKX1 measured in the spikes 0 DAP and 14 DAP, respectively, ranged from 0.52+/-0.04 to 1.15+/-0.26 and from 0.47+/-0.07 to 0.89+/-0.15. The lowest relative values were obtained for the enzyme activity in the spikes at 0 DAP, which ranged from 0.15+/-0.02 to 1.05+/-0.14 per single progeny plant. Based on these three values, the coefficient of HvCKX1 silencing in the spikes was estimated. Possible mechanisms leading to higher plant productivity via the silencing of HvCKX1 and a decrease in CKX enzyme activity are discussed.

Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants.

This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T(0) plants and 27.5% of the T(1) showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T(0) plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T(0) and T(1) showed simple integration pattern with the low copy number of the introduced transgenes.