Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack) |
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Authors: | A Nadolska-Orczyk A Przetakiewicz K Kopera A Binka W Orczyk |
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Institution: | (1) Plant Transformation and Cell Engineering Lab, Plant Breeding and Acclimatization Institute, Radzikow, Blonie, 05-870, Poland |
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Abstract: | Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two
of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes
the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin
pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation
with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants
obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was
examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T0 transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed
Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian
and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species
are discussed. |
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Keywords: | Triticale Agrobacterium tumefaciens Cereal transformation Transgene expression PCR analysis |
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