The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants

 Although it is possible to transform wheat, broad application of the technology is limited because of the low overall efficiency and the lack of reliability of the technique. In addition there is little published data on transgene integration patterns and inheritance in wheat. We have generated a population of transgenic wheat and tritordeum lines under different auxin regimes and show that, under the conditions described, the presence of picloram results in higher transformation efficiencies than the presence of 2,4-D. Molecular analysis shows low-copy numbers and simple integration patterns to be prevalent in the transgenic lines. Mendelian inheritance of transgenes in T ₁ progeny was observed for the majority of lines. Received: 2 December 1997 / Accepted: 12 March 1998     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.     

Genetic or pharmacological blockade of noradrenaline synthesis enhances the neurochemical, behavioral, and neurotoxic effects of methamphetamine

N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine (DSP-4) lesions of the locus coeruleus, the major brain noradrenergic nucleus, exacerbate the damage to nigrostriatal dopamine (DA) terminals caused by the psychostimulant methamphetamine (METH). However, because noradrenergic terminals contain other neuromodulators and the noradrenaline (NA) transporter, which may act as a neuroprotective buffer, it was unclear whether this enhancement of METH neurotoxicity was caused by the loss of noradrenergic innervation or the loss of NA itself. We addressed the specific role of NA by comparing the effects of METH in mice with noradrenergic lesions (DSP-4) and those with intact noradrenergic terminals but specifically lacking NA (genetic or acute pharmacological blockade of the NA biosynthetic enzyme dopamine β-hydroxylase; DBH). We found that genetic deletion of DBH (DBH−/− mice) and acute treatment of wild-type mice with a DBH inhibitor (fusaric acid) recapitulated the effects of DSP-4 lesions on METH responses. All three methods of NA depletion enhanced striatal DA release, extracellular oxidative stress (as measured by in vivo microdialysis of DA and 2,3-dihydroxybenzoic acid), and behavioral stereotypies following repeated METH administration. These effects accompanied a worsening of the striatal DA neuron terminal damage and ultrastructural changes to medium spiny neurons. We conclude that NA itself is neuroprotective and plays a fundamental role in the sensitivity of striatal DA terminals to the neurochemical, behavioral, and neurotoxic effects of METH.     

Involvement of dopamine receptors and beta-arrestin in metamphetamine-induced inclusions formation in PC12 cells

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of β-adrenergic receptors is promoted by β-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether β-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) β-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of β-arrestin; (iii) dopamine antagonists reduced both MA-induced β-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the β-arrestin dominant negative mutant, βarrV53D and was increased by the persistently ubiquitinated β-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of β-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.     

A population of wheat and tritordeum transformants showing a high degree of marker gene stability and heritability

The stability and heritability of three marker genes was investigated in a population of twelve independent transgenic cereal lines (six wheat and six tritordeum). Integration patterns, inheritance of structural transgenes and inheritance of expression were analysed in the T₀ and T₁ generations for all 12 lines. Transmission and expression were analysed in the T₂ generation for 9 lines and in the T₃ generation for the six wheat lines. Inheritance of integration patterns was highly stable, and transmission of the transgenes and inheritance of their expression followed Mendelian ratios in the majority of lines. A gradual reduction in uidA expression was observed over three generations, which was not accompanied by a similar reduction in bar expression. Some unexpected phenomena associated with transgene inheritance were also observed and are discussed. Received: 9 February 1999 / Accepted: 11 February 1999     

Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues

 The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to wheat (Triticum aestivum L.) scutellum and inflorescence tissue. The main factors studied were the DNA/gold precipitation process, bombardment parameters and tissue culture variables. Efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS expression in bombarded tissues. Of the parameters analysed, amount of plasmid DNA, spermidine concentration, presence of Ca⁺⁺ ions, calcium chloride concentration, amount of gold particles, gold particle size, acceleration pressure, chamber vacuum pressure, bombardment distance, osmotic conditioning of tissues and type of auxin had a clear influence on transient gene expression. A bombardment procedure suitable for elite wheat varieties was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues. Received: 6 May 1998 / Revision received: 10 August 1998 / Accepted: 2 February 1999     

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T₂ plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.     

Distribution of the vanilloid (capsaicin) receptor type 1 in the human stomach

Vanilloid receptor type 1 (TRPV1) is expressed in a capsaicin-sensitive and peptide-containing sub-population of primary sensory nerves that in the rat stomach seems involved in regulation of chlorhydropeptic secretion and gastroprotection. Our aim was to identify which cell types express TRPV1 in the human stomach in order to gain a better insight in the role of this receptor in the regulation of HCl secretion. Immunohistochemistry, by using three different commercially available anti-capsaicin antibodies, in situ hybridisation and Western blot analysis were performed on fragments surgically obtained from the gastric body on the large curvature. TRPV1 labelling was found in the parietal cells at the level of intra-cytoplasmatic granules matching mitochondrial features and distribution. Immunolabelled neurons and nerve fibres were also seen, the latter numerous in the submucosa and mucosa and often ending close to the parietal cells. TRPV1 presence was confirmed by Western blot analysis and in situ hybridisation. TRPV1 presence in nerve structures and parietal cells suggests the possibility of a combined effect of both neuronal and epithelial TRPV1 on chlorhydropeptic secretion. The presumed TRPV1 mitochondrial location inside parietal cells is in favour of the existence of a local pathway of auto-regulation of HCl secretion. Therefore, TRPV1 might modulate chlorhydropeptic secretion in the human stomach through more complex pathways than previously thought.     

Genetic manipulation of gamma-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.)

A robust Agrobacterium-mediated transformation procedure was developed for Rigel, a commercial cultivar of evening primrose, and used to deliver a cDNA encoding a Delta(6)-desaturase from borage under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Analysis of the transformed plants demonstrated an altered profile of polyunsaturated fatty acids, with an increase in gamma-linolenic acid and octadecatetraenoic acid in leaf tissues when compared with control lines.