Through a single genetic transformation in onion (Allium cepa), a crop recalcitrant to genetic transformation, we suppressed the lachrymatory factor synthase gene using RNA interference silencing in six plants. This reduced lachrymatory synthase activity by up to 1,544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatory factor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-L-cysteine sulfoxide breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of this raised thiosulfinate level was a marked increase in the downstream production of a nonenzymatically produced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported in trace amounts or had not been detected in onion. The consequences of this dramatic simultaneous down-and up-regulation of secondary sulfur products on the health and flavor attributes of the onion are discussed.
Transgenic onion plants (Allium cepa) tolerant to herbicides containing active ingredients glyphosate and phosphinothricin were recovered from immature embryos of open pollinated and hybrid parent onion lines at a maximum transformation frequency of 0.9%. Transformants of different onion cultivars, grown on different selective agents and confirmed by Southern analysis, thrived with no apparent ill effects when sprayed with the respective herbicides at double the recommended field dosage for weed eradication. This study demonstrates that the transformation process described previously can be used with different selective agents and is cultivar independent.
Onion exhibits wide genetic and environmental variation in bioactive organosulfur compounds that impart pungency and health benefits. A PCR-based molecular marker map that included candidate genes for sulfur assimilation was used to identify genomic regions affecting pungency in the cross 'W202A' x 'Texas Grano 438'. Linkage mapping revealed that genes encoding plastidic ferredoxin-sulfite reductase (SiR) and plastidic ATP sulfurylase (ATPS) are closely linked (1-2 cM) on chromosome 3. Inbred F(3) families derived from the F(2 )population used to construct the genetic map were grown in replicated trials in two environments and bulb pungency was evaluated as pyruvic acid or lachrymatory factor. Broad-sense heritability of pungency was estimated to be 0.78-0.80. QTL analysis revealed significant associations of both pungency and bulb soluble solids content with marker intervals on chromosomes 3 and 5, which have previously been reported to condition pleiotropic effects on bulb carbohydrate composition. Highly significant associations (LOD 3.7-8.7) were observed between ATPS and SiR Loci and bulb pungency but not with bulb solids content. This association was confirmed in two larger, independently derived F(2) families from the same cross. Single-locus models suggested that the partially dominant locus associated with these candidate genes controls 30-50% of genetic variation in pungency in these pedigrees. These markers may provide a practical means to select for lower pungency without correlated selection for lowered solids.
Transgenic leek (Allium porrum) and garlic (Allium sativum) plants have been recovered by the selective culturing of immature leek and garlic embryos via Agrobacterium-mediated transformation using a method similar to that described by Eady et al. (Plant Cell Rep 19:376-381, 2000) for onion transformation. This method involved the use of a binary vector containing the m-gfp-ER reporter gene and nptII selectable marker, and followed the protocol developed previously for the transformation of onions with only minor modifications pertaining to the post-transformation selection procedure which was simplified to have just a single selection regime. Transgenic cultures were selected for their ability to express the m-gfp-ER reporter gene and grown in the presence of geneticin (20 mg/l). The presence of transgenes in the genome of the plants was confirmed using TAIL-PCR and Southern analysis. This is the first report of leek and "true seed" garlic transformation. It now makes possible the integration of useful agronomic and quality traits into these crops.
A rapid and effective lateral flow assay (LFA) for detection of avian influenza virus (AIV) was developed. For antigen capture, the assay used monoclonal antibody specific for a conserved nuclear protein (NP) epitope, immobilized on a cellulose acetate matrix, in conjunction with a second NP monoclonal antibody chemically linked to either coloured latex beads or colloidal gold particles contained in a sample pad for detection. Virus sample added to the sample pad flowed into the trapping antibody to form a visible band as well as a second, control band further along the acetate strip. The control band consisted of recombinant protein A/G, also immobilized on the matrix. A second LFA for detection of chicken antibody to AIV was developed where NP antigen was immobilized on the matrix with recombinant protein A/G immobilized as a control band. Latex beads or colloidal gold particles to which monoclonal anti-chicken antibody was attached, were used as the indicator system.
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