Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

BACKGROUND: While the genetic transformation of the major cereal crops has become relatively routine, to date only a few reports were published on transgenic triticale, and robust data on T-DNA integration and segregation have not been available in this species. RESULTS: Here, we present a comprehensive analysis of stable transgenic winter triticale cv. Bogo carrying the selectable marker gene HYGROMYCIN PHOSPHOTRANSFERASE (HPT) and a synthetic green fluorescent protein gene (gfp). Progeny of four independent transgenic plants were comprehensively investigated with regard to the number of integrated T-DNA copies, the number of plant genomic integration loci, the integrity and functionality of individual T-DNA copies, as well as the segregation of transgenes in T(1) and T(2) generations, which also enabled us to identify homozygous transgenic lines. The truncation of some integrated T-DNAs at their left end along with the occurrence of independent segregation of multiple T-DNAs unintendedly resulted in a single-copy segregant that is selectable marker-free and homozygous for the gfp gene. The heritable expression of gfp driven by the maize UBI-1 promoter was demonstrated by confocal laser scanning microscopy. CONCLUSIONS: The used transformation method is a valuable tool for the genetic engineering of triticale. Here we show that comprehensive molecular analyses are required for the correct interpretation of phenotypic data collected from the transgenic plants

VII Jornadas de Expania

Sección: Noticias. Noticias externasLos días 27 y 28 de mayo se celebraron en Santiago de Compostela las VII Jornadas de Expania, la Asociación de Usuarios de Ex Libris en España.N

Agrobacterium-Mediated Gene Transfer to Cereal Crop Plants: Current Protocols for Barley, Wheat, Triticale, and Maize

The development of powerful “omics” technologies has enabled researchers to identify many genes of interest for which comprehensive functional analyses are highly desirable. However, the production of lines which ectopically express recombinant genes, or those in which endogenous genes are knocked down via stable transformation, remains a major bottleneck for the association between genetics and gene function in monocotyledonous crops. Methods of effective DNA transfer into regenerable cells of immature embryos from cereals by means of Agrobacterium tumefaciens have been modified in a stepwise manner. The effect of particular improvement measures has often not been significantly evident, whereas their combined implementation has resulted in meaningful advances. Here, we provide updated protocols for the Agrobacterium-mediated generation of stably transgenic barley, wheat, triticale and maize. Based upon these methods, several hundred independent transgenic lines have been delivered, with efficiencies of inoculated embryos leading to stably transgenic plants reaching 86% in barley, 10% in wheat, 4% in triticale, and 24% in maize

Two Lateral organ boundary domain transcription factors HvCRL1 and HvCRL1L1 regulate shoot-borne root formation in Barley (Hordeum vulgare L.)

Plant-specific LOB-domain (LBD) transcription factors are crucial in post-embryonic root initiation. In cereals, the fibrous root system comprises primary and seminal roots that develop during embryogenesis and lateral- and crown roots that develop post-embryonically from root or stem, respectively. In rice, the CROWNROOTLESS1 (CRL1) LBD transcription factor is the core regulator of crown root initiation and a direct target of the auxin response factor (ARF)-mediated auxin signaling pathway. Orthologs of CRL1 have been identified and characterized in several species, where their role in crown root initiation has been validated. In barley, we identified two genes phylogenetically closely related to the rice CRL1 genes that we named HvCRL1 and HvCRL1L1. Using a crown root inducible system (CRIS), we identified that both HvCRL1 and HvCRL1L1 are expressed in response to auxin during the early steps of crown root differentiation in stem base, with HvCRL1 transcripts being accumulated quickly during the first hour of treatment. Transient activation assays in rice proto- plast showed that HvCRL1 could bind the LBD-box, a consensus DNA sequence recognized by LBD transcription factors, whereas HvCRL1L1 did not. Both genes can partially complement the crl1 rice mutant. Loss-of-function mutation in each gene drastically impairs crown root formation in barley. These data show that HvCRL1 and HvCRL1L1 are both involved in the regulation of crown root formation in barley but that these two transcription factors likely act through distinct and complementary pathways in this developmental process

Electromagnetic Tracking in High Dose Rate Brachytherapy - A Composite Analysis Model

Electromagnetic tracking (EMT) in high dose rate Brachytherapy has to face a number of signal processing challenges which we summarize in this study. We propose a coherent signal processing chain which encompasses a particle filter tracking of the state space trajectory of the sensors inside catheters implanted surgically into the breast of female patients. Singular spectrum analysis is employed to remove high amplitude artifact signals from the recordings as well as to decompose simultaneously recorded signals from additional fiducial sensors used to monitor breathing motions. Ensemble empirical mode decomposition is applied to both, the fiducial and solenoid sensor signals to decompose them into their intrinsic modes. Information-theoretic similarity measures serve to identify those intrinsic modes which carry information about the breathing mode contamination of the observed solenoid sensor signals. Finally, a multi-dimensional scaling achieves a common principal coordinate system where both, the various EMT signals and related data deduced from an initial X-ray CT imaging can be compared quantitatively to identify any deviations from the treatment plan established with the CT data. We also consider the distributions of such deviations and demonstrate their heavy-tailed character. A Hartigan dip test is employed to establish a uni- or bi-modal character of these distributions which we approximate by alpha-stable distributions

EMTLAB: A Toolbox for the Analysis of Electromagnetic Tracking Data in Brachytherapy

Background: High dose rate brachytherapy (HDR-BT) of female breast cancer patients relies on electromagnetic tracking (EMT) for localizing the prescribed dwell positions of the radiation source. A collection of machine learning techniques like Particle Filtering (PF), Singular Spectrum Analysis (SSA), Ensemble and Multivariate Empirical Mode Decomposition (EEMD/MEMD) represent powerful signal processing techniques and are employed in this study to achieve this goal. Information-theoretic similarity measures allow comparing extracted signal components for artifact identification and elimination. New toolbox: We present a new toolbox, called EMTLAB, which is designed as an extensible toolbox for electromagnetic tracking data analysis. It contains all machine learning techniques mentioned above and is written in MATLAB®. Results: EMTLAB offers the practitioner a convenient way to easily and efficiently perform particle filtering, signal decomposition and manual or automatic artifact removal with an SSA, an EEMD or MEMD in combination with three similarity measures: Pearson correlation, Jensen-Shannon divergence or Kull back-Leibler divergence. As demonstrated with illustrative examples, EMTLAB offers a complete and almost fully automatic signal processing chain for an analysis of EMT data sets collected during a HDR-BT. In addition, EMTLAB represents a user-friendly graphical user interface (GUI), which also provides convenient means to visualize the results in illustrative graphs. A number of screen shots helps in understanding the functioning of the signal processing chain and the use of the GUI. Conclusion: EMTLAB is a reliable, efficient and automated solution for processing and analyzing EMT sensor data from a HDR-BT, while employing different physical models of system dynamics. This sensor tracking by particle filtering allows to adapt the analysis to different dynamical models and the SSA and EMD algorithms provide an easy means to remove from the data artifacts stemming from breathing modes or measurement device malfunctioning

Modification of Barley Plant Productivity Through Regulation of Cytokinin Content by Reverse-Genetics Approaches

Barley is one of the most important cereals, which is used for breweries, animal and human feeds. Genetic manipulation of plant hormone cytokinins may influence several physiological processes, besides others stress tolerance, root formation and crop yield. In planta, endogenous cytokinin status is finely regulated by the enzyme cytokinin dehydrogenase (EC 1.5.99.12; CKX), that irreversible degrades the side chain of adenine-derived isoprenoid cytokinins. Increasing grain yield by mean of manipulation of endogenous cytokinin content was assayed by the silencing of the HvCKX1 gene. Moreover, to elucidate the putative role of HvCKX1 gene on grain production, knocked-out Hvckx1 mutant plants were generated using the RNA-guided Cas9 system. Homozygote transgenic plants with silenced HvCKX1 gene and azygous knock-out Hvckx1 mutants have been selected and analyzed. Both reduced expression of HvCKX1 gene and CKX activity were measured in different stages of barley grain development. Phenotyping of the transgenic lines revealed reduced root growth, however, plants produced more tillers and grains than azygous wild-type controls and the total yield was increased up to 15 per cent. Although plant productivity was increased, total grain biomass was decreased to 80% of WT grains. RNA-seq analysis of knock-down transgenic lines revealed that several important macronutrient transporters were downregulated in the stage of massive starch accumulation. It suggests that local accumulation of cytokinins negatively affected nutrients flow resulting in reduced grain biomass. Obtained results confirmed the key role of HvCKX1 for regulation of cytokinin content in barley