Silicon carbide fiber-mediated DNA delivery into cells of wheat (Triticum acstivum L.) mature embryos

We have demonstrated that foreign DNA can be delivered into cells of mature embryos of wheat (Triticum aestivum L.) using silicon carbide fibers (SCF). The highest transient expression of thegusA (GUS) gene was detected when dry embryos were vortexed for 10–30 min in a SCF-DNA solution containing 90–120 g/l of sucrose. Up to 100 (on average 20–40) blue expression units per embryo were observed. Scutellum side and epiblast of the intact wheat embryos are preferentially transformed. When embryos with the coleoptilar tip removed were treated and allowed to germinate, GUS staining was observed in emerging leaf tissues. The potential of this new approach for stable transformation of wheat is under investigation. It has been found that callus tissues induced from the SCF treated embryos contain GUS-expressing sectors one month after treatment.     

Defensin promoters as potential tools for engineering disease resistance in cereal grains

Engineering of plant protection in cereals requires well characterized tissue-specific and wounding/pathogen-inducible promoters for targeted expression of pathogen responsive and resistance genes. We describe the isolation of seven wheat and rice defensin genes expressed in early developing grain and during grain germination, two developmental stages that are particularly vulnerable to pathogens and insects. Comparison of three-dimensional (3D) models of these rice and wheat PRPI defensins indicated variations in spatial architectures that could reflect their functional diversities. Wheat and rice were stably transformed with promoter–GUS fusion constructs and the spatial and temporal activities of four promoters were studied using whole-mount and histological assays. PRPI promoters were active before and at anthesis in both transgenic wheat and rice with activity mainly in the ovary. In rice, GUS activity was also observed in vascular tissue of the lemma, palea and anthers. After fertilization, GUS was strongly expressed in the outer cell layers of the pericarp and in the main vascular bundle of the grain. During, and a short time after, seed germination, wheat promoters were active in transgenic rice embryos, roots and/or coleoptiles. All wheat and rice promoters were strongly induced by wounding in leaf, stem and grain of transgenic rice plants. These results suggest that PRPI promoters will be useful for specific targeting and accumulation of proteins conferring resistance to pathogens in vulnerable tissues of developing and germinating grain.     

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the -glucuronidase (GUS) gene, under the control of the doubled enhancer element (the –208 to –46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to –389 bp from ATG) promoter of wheat, -amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 10⁶ protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.     

Stochasticity in the adaptive dynamics of evolution: the bare bones

First a population model with one single type of individuals is considered. Individuals reproduce asexually by splitting into two, with a population-size-dependent probability. Population extinction, growth and persistence are studied. Subsequently the results are extended to such a population with two competing morphs and are applied to a simple model, where morphs arise through mutation. The movement in the trait space of a monomorphic population and its possible branching into polymorphism are discussed. This is a first report. It purports to display the basic conceptual structure of a simple exact probabilistic formulation of adaptive dynamics.     

An accurate model for genetic hitchhiking

We suggest a simple deterministic approximation for the growth of the favored-allele frequency during a selective sweep. Using this approximation we introduce an accurate model for genetic hitchhiking. Only when Ns<10 (N is the population size and s denotes the selection coefficient) are discrepancies between our approximation and direct numerical simulations of a Moran model notable. Our model describes the gene genealogies of a contiguous segment of neutral loci close to the selected one, and it does not assume that the selective sweep happens instantaneously. This enables us to compute SNP distributions on the neutral segment without bias.     

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice

An HD‐Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD‐Zip IV family. The 3′ untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full‐length gene containing a 3‐kb‐long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter‐GUS fusion constructs in transgenic wheat, barley and rice plants. Whole‐mount and histochemical GUS staining patterns revealed grain‐specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm‐dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.     

Novel tumorigenic rearrangement,Delta rfp/ret,in a papillary thyroid carcinoma from externally irradiated patient

Molecular analysis of cDNA derived from a papillary thyroid carcinoma (PTC) (follicular variant of papillary thyroid carcinoma on histology) which developed in an externally irradiated patient 4 years after exposure identified a portion of the 5' region, exons 1-3, of the rfp gene juxtaposed upstream of the fragment encoding the tyrosine kinase (TK) domain of the ret gene. The fusion gene, termed Delta rfp/ret, was the result of a balanced chromosomal translocation t(6;10) (p21.3;q11.2) confirmed by interphase FISH painting, with breakpoints occurring in introns 3 and 11 of the rfp and ret genes, respectively. Both Delta rfp/ret and reciprocal ret/rfp chimeric introns had small deletions around breakpoints consistent with presumed misrepair of a radiation-induced double-strand DNA break underlying the rearrangement. No extensive sequence homology was found between the fragments flanking the breakpoints. The fusion protein retained the propensity to form oligomers likely to be mediated by a coiled-coil of the RFP polypeptide as assessed by a yeast two-hybrid system. NIH 3T3 fibroblasts stably transfected with a mammalian expression vector encoding full-length Delta RFP/RET readily gave rise to the tumors in athymic mice suggestive of high transforming potential of the fusion protein. Thus, the Delta rfp/ret rearrangement may be involved in a causative manner in cancerogenesis and provides additional evidence of the role of activated ret oncogene in the development of a subset of papillary thyroid carcinoma.