Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with two different plasmids containing the E. coli tn5 neomycin phosphotransferase II gene (npt II). Selection was accomplished using the antibiotic paromomycin sulfate following microprojectile bombardment. From two independent experiments, 88 paromomycin-resistant tissue cultures were shown to be transgenic based on Southern blot analysis and detection of the neomycin phosphotransferase (NPT II) protein using ELISA. Copy numbers of the npt II gene ranged from one to eight copies per haploid oat genome integrated into high molecular weight DNA of the paromomycin-resistant cultures. Plants were regenerated from 32 of the 88 transgenic tissue cultures. Plants from 17 of the 32 regenerable cultures exhibited fertility. Stable transformation was shown by segregation patterns of the NPT II protein in R1 seedlings produced from 16 fertile culture lines that were tested. The overall results demonstrate that the combination of the npt II gene and paromomycin provides efficient selection of transgenic oat tissue cultures. Oat plants transformed with the npt II gene present reduced ecological risk compared to the previously used herbicide-resistance selection system.
Mature embryos of oat (Avena sativa L.) have been used to establish regenerable tissue cultures with potential use for transformation. The objective of this study was to investigate tissue cultures established from mature embryos of oat as an alternative source of totipotent target cells for microprojectile bombardment-mediated transformation. Mature embryos of a specific genotype, GAF/Park-1, were incubated on a tissue culture induction medium for 1, 4, 8, or 9 wk before either being directly bombarded after 1 and 4 wk, or bombarded as tissue cultures initiated after 8 and 9 wk incubation. The 8-and 9wk-old tissue cultures yielded the greatest numbers of transgenic tissue cultures (3.2 transgenic tissue cultures per microprojectile bombardment treatment). Three additional transformation experiments were conducted with mature embryo-derived 8-to 9-wk-old tissue cultures to determine the regeneration capacity and production of fertile transgenic plants. Overall, fertile plants were regenerated from 35 of 85 independently derived transgenic tissue cultures. Identification of mature embryo-derived tissue cultures as a source of transformable totipotent cells should reduce the expense and labor involved in oat transformation. Moreover, the uniformity and convenience of this explant likely will stimulate further investigations in oat transformation efficiency.
Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.
Barley yellow dwarf viruses (BYDVs) are the most serious and widespread viruses of oats, barley, and wheat worldwide. Natural resistance is inadequate. Toward overcoming this limitation, we engineered virus-derived transgenic resistance in oat. Oat plants were transformed with the 5' half of the BYDV strain PAV genome, which includes the RNA-dependent RNA polymerase gene. In experiments on T2- and T3-generation plants descended from the same transformation event, all BYDV-inoculated plants containing the transgene showed disease symptoms initially, but recovered, flowered, and produced seed. In contrast, all but one of the BYDV-PAV-inoculated nontransgenic segregants died before reaching 25 cm in height. Although all of the recovered transgenic plants looked similar, the amount of virus and viral RNA ranged from substantial to undetectable levels. Thus, the transgene may act either by restricting virus accumulation or by a novel transgenic tolerance phenomenon. This work demonstrates a strategy for genetically stable transgenic resistance to BYDVs that should apply to all hosts of the virus.
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