tomato, Flavr Savr, was modified by Calgene (a biotechnology company) using Flavr Savr tomatoes are designed so they can ripen on the vine longer while. Flavr Savr Tomato Disguised in the cover of a favorite summer time topic — why don’t store-bought tomatoes taste good? — The New York Times has printed a. The FLAVR SAVR™tomato was developed through the use of antisense RNA to regulate the expression of the enzyme polygalacturonase (PG) in ripening.
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The Flavr Savr tomato was introduced as the first genetically engineered whole food in The commercial event, resulting from transformation with an antisense tokato cassette of the endogenous polygalacturonase gene, was sequenced and found to contain two contiguous, linked, transfer DNA insertions. Of the 64 plants screened, the ratio was 43 flvr 21 NptII positive to negative plants, which is consistent with a 3: PG enzyme activity assays, measured as an A absorbance, were tomaot in replicates of six according to Redenbaugh et al.
The average enzyme activity of the three transgenic tomatoes, was 0. Sequences used were as follows:. Probe D—Last bp of the PG sequence. Plants from commercial hybrid seed CR Bands were observed in the transgenic plants of and bp corresponding to the PG region of the locus, in addition to a 2-kb band of the native PG gene present in all lanes.
The transgene locus and flanking genomic sequence were obtained by inverse PCR amplification, cloning, and sequence analysis of resulting fragments. These results were further confirmed by amplifying the T-DNAs with flanking genomic and unique junction region primers Fig. Fkavr bp band lane 1 contains the truncated left T-DNA. The bp band lane 3 contains the full-length right T-DNA. We were unable to amplify across the insert 14,bp, lane 5likely due to the inverted repeat sequence.
In the null sibling, these primers amplify a bp band lane 6 verifying insert location. The bp band lane 7 is the product across the unique T-DNA junction sequence. Taken together, these results show the commercial Flavr Savr tomato contained aavr T-DNAs linked in an inverted orientation. Characterization of siblings of the Flavr Savr tomato. Transgenic, and and non-transgenic, and siblings were analyzed.
The T-DNA insertion corresponds to the 3.
A putative CYP82 family cytochrome P mono-oxygenase gene in the adjacent genomic sequence is indicated gray box. Sizes of the marker bands lane L are indicated at right. Left T-DNA, lanes 1 and 2 primers 1 and fpavr ; svar T-DNA, lanes 3 and 4 primers 2 and 3 ; full insert, lanes 5 and 6 primers 1 and 2 ; unique junction region, lanes 7 and 8 primers 5 and 6.
PG enzyme activity assays on ripe tomatoes using a polygalacturonic acid substrate Redenbaugh et al.
A fold reduction in PG activity is consistent with previously described pCGN transgenic tomato events Redenbaugh et al.
The majority of the siRNAs from the coding regions belong to the nt tmoato class, although lower abundant nt siRNAs were also observed. Faint accumulation of NptII mRNA in ripe transgenic, tomatoes and not in null, sibling tomatoes.
We found transgenic siblings of the commercial Flavr Savr tomato event contain two linked T-DNAs; however, unlike the eight right-border linkage events described by Sanders and Hiattthese plants contain T-DNAs linked at the left borders. While this configuration results in inverted PG repeats, the promoter orientation would not be expected to generate a dsRNA precursor.
Previous reports show using a single copy of an antisense transgene can trigger RNAi Fagard and Vaucheret, ; Stam et al. The nt class, associated with transcriptional repression Brodersen and Voinnet, ; Fagard and Vaucheret, ; Hamilton et al.
In contrast, if suppression were due to transcriptional or post-transcriptional gene silencing, accumulation of the message would be reduced, suggesting transgene-associated suppression was not due to repression of endogenous PG mRNA transcription but to RNAi Brodersen and Voinnet, Potential promoter or enhancer elements associated with this gene may be associated with transcription across the T-DNA locus and could be responsible for contributing to the initial production of a dsRNA tmato the coding regions, thus resulting in the production of the siRNAs.
It has been shown that there are several overlapping, and potentially hierarchical, mechanisms and pathways involved in plant gene silencing Deleris et al. Without direct evidence of transcription across the transgene, we hypothesize an RdRP-dependent mechanism may be a contributor to enhancing siRNA production and gene silencing Wassenegger and Krczal, compared with a dsRNA formed directly via readthrough transcription.
It has been demonstrated repeatedly that expression of an antisense cDNA alone does not result in gene silencing as efficiently as expressing a dsRNA Stam et al. Thus, the silencing mechanism of the Flavr Savr events first reported by Sanders and Hiatt might not be the only mechanism responsible for silencing in the commercial Flavr Savr tomato, event CR PG gene suppression was achieved using an antisense savg cassette of tomqto endogenous PG gene resulting in an RNAi-mediated pathway and yielding the phenotype of the Flavr Savr tomato.
This research supports the need for precise characterization of gene insertion structures in relationship to understanding the mechanisms of gene suppression.
Flavr Savr Tomato
User Name Password Sign In. Krieger 1Edwards AllenLarry A. Gilbertson and James K. William Hiatt and Rick A. Previous Section Next Section. Southern blots and inverse PCRs. PG enzyme activity assays. RNA purification and Northern blots. Sequences used were as follows: In this window In a new window. PG gene suppression is the result of RNAi-mediated pathways.
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