Plant transformation vector: Difference between revisions

Revision as of 06:33, 20 October 2022

Revision as of 13:44, 5 September 2023

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==Steps in plant transformation==

Note: There are many variations to these steps. A custom DNA plasmid sequence can be created and replicated in more than one way, this item describes the general steps.

Propagate binary vector in ''E. coli''

Plant transformation using plasmids starts with the propagation of the binary vector in ''E. coli.'' When the bacterial culture reaches the appropriate density, the binary vector is isolated and purified. Then a foreign gene can be introduced. The engineered binary vector, including the foreign gene, is re-introduced in ''E. coli'' to amplify.

Isolate binary vector from ''E.coli'' and engineer (introduce a foreign gene)

The engineered binary factor is isolated from E. coli again and can be introduced into ''Agrobacteria'' containing a modified (relatively small) Ti plasmid. This engineered ''Agrobacteria'' can be used to infect plant cells. The T-DNA containing the foreign gene gets inserted into a plant cell genome. In each infected cell, the T-DNA gets integrated at a different site in the genome.

Re-introduce engineered binary vector into ''E. coli'' to amplify

To develop a plant that carries the transformation DNA integrated in the same way in all its cells, a transformed cell is selected, from which an entire plans is regenerated.

Isolate engineered binary vector and introduce into ''Agrobacteria'' containing a modified (relatively small) Ti plasmid

Infect plant tissue with engineered ''Agrobacteria'' (T-DNA containing the foreign gene gets inserted into a plant cell genome)

In each cell T-DNA gets integrated at a different site in the genome

Note: There are many variations to these steps. A custom DNA plasmid sequence can be created and replicated in more than one way.

=== Consequences of the insertion ===

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Transformation DNA fed to rodents ends up in their [[phagocyte]]s and rarely other cells. Specifically this is bacterial and [[M13 bacteriophage|M13]] DNA. (This preferential accumulation in phagocytes is thought to be real and not a detection artifact since these DNA extents are thought to provoke [[phagocytosis]].) However no [[gene expression]] is known to have resulted, and this is not thought to be possible.<ref name="Goldstein-et-al-2005">{{cite journal | last1=Goldstein | first1=Daniel A. | last2=Tinland | first2=Bruno | last3=Gilbertson | first3=Lawrence A. | last4=Staub | first4=J.M. | last5=Bannon | first5=G.A. | last6=Goodman | first6=R.E. | last7=McCoy | first7=R.L. | last8=Silvanovich | first8=A. | title=Human safety and genetically modified plants: a review of antibiotic resistance markers and future transformation selection technologies | journal=[[Journal of Applied Microbiology]] | publisher=[[Society for Applied Microbiology]] ([[Wiley Publishing|Wiley]]) | volume=99 | issue=1 | year=2005 | issn=1364-5072 | doi=10.1111/j.1365-2672.2005.02595.x | pages=7–23| pmid=15960661 | doi-access=free }}</ref><ref name="Lemaux-2008">{{cite journal | last=Lemaux | first=Peggy G. | title=Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I) | journal=[[Annual Review of Plant Biology]] | publisher=[[Annual Reviews (publisher)|Annual Reviews]] | volume=59 | issue=1 | year=2008 | issn=1543-5008 | doi=10.1146/annurev.arplant.58.032806.103840 | pages=771–812 | pmid=18284373}}</ref>

=== Problem ===

We want to transform the whole organism, not just one cell. This is done by transforming plant cells in culture, selecting transformed cells and regenerating an entire plant from the transformed cell (e.g. tobacco)

==Plasmid selection==