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GrainGenes Reference Report: JPY-159-1131

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Reference	JPY-159-1131
Title	Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA :: barley genomic DNA junctions
Journal	Journal of Plant Physiology
Year	2002
Volume	159
Pages	1131-1138
Author	Fang YD Akula C Altpeter F
Abstract	Summary: Agrobacterium-mediated barley transformation promises many advantages compared to alternative gene transfer methods, but has so far been established in only a few laboratories. We describe a protocol that facilitates rapid establishment and optimisation of Agrobacterium-mediated transformation for barley by instant monitoring of the transformation success. The synthetic green fluorescent protein (sgfpS65T) reporter gene was introduced in combination with the hpt selectable marker gene into immature embryos of barley (Hordeum vulgare L.) by cocultivation with Agrobacterium tumefaciens strain AGLO harboring binary vector pYF133. Using green fluorescent protein (GFP) as a nondestructive visual marker allowed us to identify single-cell recipients of T-DNA at an early stage, track their fate and evaluate factors that affect T-DNA delivery. GFP screening was combined with a low level hygromycin selection. Consequently, transgenic plantlets ready to transfer to soil were obtained within 50 days of explant culture. Southern blot- and progeny segregation analyses revealed a single copy T-DNA insert in more than half of the transgenic barley plants. T-DNA/barley genomic DNA junctions were amplified and sequenced. The right T-DNA ends were highly conserved and clustered around the first 4 nucleotides of the right 25 bp border repeat, while the left T-DNA ends were more variable, located either in the left 25 bp border repeat or within 13 bp from the left repeat. T-DNAs were transferred from Agrobacterium to barley with exclusion of vector sequence suggesting a similar molecular T-DNA transfer mechanism as in dicotyledonous plants
Keyword	[ Hide all but 1 of 29 ] agrobacterium agrobacterium-mediated transformation barley chromosome culture dna efficient transformation fertile transgenic barley gene gene transfer gfp green fluorescent protein hordeum hordeum vulgare hygromycin marker mechanism molecular-weight dna particle bombardment protein screenable marker segregation selection sequence soil t-dna transformation transgenic vector

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