Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2
This project includes two parts. The first part was transformation of Escherichia coli and Agrobacterium tumefaciens with a vector containing the GA2ox gene. The second part was molecular analyses of the apple (Malus domestica) rootstock A2 transformed with the rolC gene. The rolC and GA2ox genes ar...
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| Formato: | Otro |
| Lenguaje: | sueco Inglés |
| Publicado: |
2007
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| Materias: | |
| Acceso en línea: | https://stud.epsilon.slu.se/12497/ |
| _version_ | 1855572075077435392 |
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| author | Emanuelsson, Carin |
| author_browse | Emanuelsson, Carin |
| author_facet | Emanuelsson, Carin |
| author_sort | Emanuelsson, Carin |
| collection | Epsilon Archive for Student Projects |
| description | This project includes two parts. The first part was transformation of Escherichia coli and Agrobacterium tumefaciens with a vector containing the GA2ox gene. The second part was molecular analyses of the apple (Malus domestica) rootstock A2 transformed with the rolC gene. The rolC and GA2ox genes are two potential genes for dwarfing plants.
E. coli was transformed with a binary vector, which was supposed to contain the GA2ox and nptII genes. Transformation events were confirmed by restriction enzyme digestion and by PCR analysis, which led to the discovery that the GA2ox gene was not present in the original vector but the gus gene. A. tumefaciens was later transformed with the vector, isolated from the above transformed E. coli. Transformation of A. tumefaciens could not be confirmed by PCR analysis, even though the colonies grew well on the selectable medium containing kanamycin.
In the second part of the project Southern blot was conducted to further confirm integration of the rolC and nptII genes in the transgenic clones of the apple rootstock A2 and to determine the copy number of the transgenes. The Southern results confirmed that six clones contained the nptII and rolC genes. Two of the transformed clones have a single copy of the T-DNA integrated. |
| format | Otro |
| id | RepoSLU12497 |
| institution | Swedish University of Agricultural Sciences |
| language | Swedish Inglés |
| publishDate | 2007 |
| publishDateSort | 2007 |
| record_format | eprints |
| spelling | RepoSLU124972017-10-27T07:28:52Z https://stud.epsilon.slu.se/12497/ Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 Emanuelsson, Carin Plant Breeding and Biotechnology (until 121231) Plant physiology and biochemistry This project includes two parts. The first part was transformation of Escherichia coli and Agrobacterium tumefaciens with a vector containing the GA2ox gene. The second part was molecular analyses of the apple (Malus domestica) rootstock A2 transformed with the rolC gene. The rolC and GA2ox genes are two potential genes for dwarfing plants. E. coli was transformed with a binary vector, which was supposed to contain the GA2ox and nptII genes. Transformation events were confirmed by restriction enzyme digestion and by PCR analysis, which led to the discovery that the GA2ox gene was not present in the original vector but the gus gene. A. tumefaciens was later transformed with the vector, isolated from the above transformed E. coli. Transformation of A. tumefaciens could not be confirmed by PCR analysis, even though the colonies grew well on the selectable medium containing kanamycin. In the second part of the project Southern blot was conducted to further confirm integration of the rolC and nptII genes in the transgenic clones of the apple rootstock A2 and to determine the copy number of the transgenes. The Southern results confirmed that six clones contained the nptII and rolC genes. Two of the transformed clones have a single copy of the T-DNA integrated. Det här projektet är uppdelat i två delar. Den första delen innefattar transformering av Escherichia coli och Agrobacterium tumefaciens med GA2ox genen. Den andra delen av projektet består av utförandet av molekyläranalyser av äpplegrundstammen A2 transformerad med rolC genen. rolC och GA2ox är två potentiella gener för att reducera plantstorleken. E. coli blev transformerad med en plasmidvektor, vilken var tänkt att innehålla GA2ox och nptII generna. Genomförd PCR analys bekräftade transformering av E. coli men samtidigt upptäcktes det att vektorn innehöll gus genen istället för GA2ox genen. Vektorn som isolerats från E. coli transformerades senare in i A. tumefaciens. Transformering av vektorn i A. tumefaciens kunde inte bekräftas efter utförd PCR-analys, trots att bakteriekolonier växte normalt på medium med kanamycin. I den andra delen av projektet analyserades A2 grundstamkloner som transformerats med rolC och nptII generna, genom utförandet av Southern blot hybridisering för att bestämma antalet kopior av transgenerna som integrerats i respektive klongenom. Det bekräftas att sex kloner är transformerade med nptII och rolC generna. Två av klonerna innehåller endast en kopia av de båda generna, som kan vara av intresse för tillämpningen i framtiden. 2007-09-24 Other NonPeerReviewed application/pdf sv https://stud.epsilon.slu.se/12497/1/emanuelsson_c_171027.pdf Emanuelsson, Carin, 2007. Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2. UNSPECIFIED, Alnarp. Alnarp: (NL, NJ) > Dept. Of Plant Breeding <https://stud.epsilon.slu.se/view/divisions/LMNR=3A642.html> urn:nbn:se:slu:epsilon-s-8328 eng |
| spellingShingle | Plant Breeding and Biotechnology (until 121231) Plant physiology and biochemistry Emanuelsson, Carin Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title | Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title_full | Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title_fullStr | Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title_full_unstemmed | Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title_short | Transformation of E. coli and Agrobacterium and molecular analysis of transformed apple (Malus domestica) rootstock A2 |
| title_sort | transformation of e. coli and agrobacterium and molecular analysis of transformed apple (malus domestica) rootstock a2 |
| topic | Plant Breeding and Biotechnology (until 121231) Plant physiology and biochemistry |
| url | https://stud.epsilon.slu.se/12497/ https://stud.epsilon.slu.se/12497/ |