Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi
An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty...
| Main Authors: | , , , , , |
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| Format: | Journal Article |
| Language: | Inglés |
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Springer
2016
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| Online Access: | https://hdl.handle.net/10568/76626 |
| _version_ | 1855541154819342336 |
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| author | Orbegozo, Jeanette P. Solorzano, Dennis Cuéllar, Wilmer Jose Bartolini, Maria Lupe Ghislain, M. Kreuze, Jan F. |
| author_browse | Bartolini, Maria Lupe Cuéllar, Wilmer Jose Ghislain, M. Kreuze, Jan F. Orbegozo, Jeanette P. Solorzano, Dennis |
| author_facet | Orbegozo, Jeanette P. Solorzano, Dennis Cuéllar, Wilmer Jose Bartolini, Maria Lupe Ghislain, M. Kreuze, Jan F. |
| author_sort | Orbegozo, Jeanette P. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB. |
| format | Journal Article |
| id | CGSpace76626 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2016 |
| publishDateRange | 2016 |
| publishDateSort | 2016 |
| publisher | Springer |
| publisherStr | Springer |
| record_format | dspace |
| spelling | CGSpace766262025-03-13T09:45:39Z Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi Orbegozo, Jeanette P. Solorzano, Dennis Cuéllar, Wilmer Jose Bartolini, Maria Lupe Ghislain, M. Kreuze, Jan F. potato leaf roll luteovirus genetic transformation viruses hybridization transgenic plants virus transformación genética hibridación plantas transgénicas An inverted repeat construct corresponding to a segment of the potato leaf roll virus coat protein gene was created under control of a constitutive promoter and transferred into a transformation vector with a heat inducible Cre-loxP system to excise the nptII antibiotic resistance marker gene. Fifty-eight transgenic events were evaluated for resistance to PLRV by greenhouse inoculations, which lead to the identification of 7 highly resistant events, of which 4 were extremely resistant. This resistance was also highly effective against accumulation in subsequent tuber generations from inoculated plants, which has not been reported before. Northern blot analysis showed correlation of PLRV specific siRNA accumulation with the level of PLRV resistance. Heat mediated excision of the nptII antibiotic resistance gene in PLRV resistant events was highly efficient in one event with full excision in 71 % of treated explants. On the other hand 8 out of 10 analyzed events showed truncated T-DNA insertions lacking one of the two loxP sites as determined by PCR and confirmed by sequencing flanking regions in 2 events, suggesting cryptic LB sites in the non-coding region between the nptII gene and the flanking loxP site. Accordingly, it is proposed to modify the Cre-loxP vector by reducing the 1 kb size of the region between nptII, loxP, and the LB. 2016-12 2016-08-29T18:27:26Z 2016-08-29T18:27:26Z Journal Article https://hdl.handle.net/10568/76626 en Open Access Springer Orbegozo, Jeanette; Solorzano, Dennis; Cuellar, Wilmer J.; Bartolini, Ida; Roman, Maria Lupe; Ghislain, Marc; Kreuze, Jan. 2016. Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi . Transgenic Research 25(6): 813-828. |
| spellingShingle | potato leaf roll luteovirus genetic transformation viruses hybridization transgenic plants virus transformación genética hibridación plantas transgénicas Orbegozo, Jeanette P. Solorzano, Dennis Cuéllar, Wilmer Jose Bartolini, Maria Lupe Ghislain, M. Kreuze, Jan F. Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title | Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title_full | Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title_fullStr | Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title_full_unstemmed | Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title_short | Marker-free PLRV resistant potato mediated by Cre-loxP excision and RNAi |
| title_sort | marker free plrv resistant potato mediated by cre loxp excision and rnai |
| topic | potato leaf roll luteovirus genetic transformation viruses hybridization transgenic plants virus transformación genética hibridación plantas transgénicas |
| url | https://hdl.handle.net/10568/76626 |
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