Factors influencing successful Agrobacterium-mediated genetic transformation of wheat.
The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration. We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables. Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media. We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties. The transformation frequency ranged from 0.3% to 3.3%. Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios.
Wu
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CPI Division, AL5 2JQ, Rothamsted, Harpenden, UK.
Sparks
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Amoah
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Jones
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D000098:Acetophenones / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D012038:Regeneration; D012231:Rhizobium / Q000235:genetics* / Q000254:growth & development; D012639:Seeds / Q000235:genetics / Q000502:physiology; D014170:Transformation, Genetic; D014908:Triticum / Q000235:genetics*
D000098:Acetophenones; D004269:DNA, Bacterial; C036483:T-DNA; C051667:acetosyringone; D005966:Glucuronidase