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GrainGenes Reference Report: PRT-21-429

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Reference	PRT-21-429
Title	Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium
Journal	Plant Cell Reports
Year	2003
Volume	21
Pages	429-436
Author	Khanna HK Daggard GE
Abstract	Summary: Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes - vir B, -C and -G. In both cases, transient #-glucuronidase (GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes. Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9%. The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium. Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses. T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants. Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios
Keyword	[ Hide all but 1 of 37 ] agrobacterium agrobacterium tumefaciens bar beta-glucuronidase binary vector calli callus cultures cocultivation gene improvement increase indica rice inheritance insertion media molecular analyses plant regeneration polyamine progeny ratios regeneration segregation single-copy spermidine spring spring wheat stable expression strain superbinary vector t-dna transformation transformation frequency transgene transient triticum triticum aestivum vector

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