Plant transformation vector: Difference between revisions

Revision as of 10:20, 7 September 2023

Revision as of 16:55, 2 October 2023

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==Steps in plant transformation==

A custom DNA plasmid sequence can be created and replicated in more than one way, thisbut itemall describesmethods generally share the generalfollowing stepsprocesses.

Plant transformation using plasmids startsbegins 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'' for amplification.

The engineered binary factor is isolated from ''E. coli'' and is 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.

The entire plant will regenerate from a single transformed cell, which results in an organism with transformation DNA integrated identically across all cells.

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 plant is regenerated.

=== Consequences of the insertion ===

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Insertional mutagenesis (but not lethal for the plant cell – as the organism is diploid)

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 artefact 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>

==Plasmid selection==

A selector gene can be used to distinguish the successfully genetically bacterial modified cells from unmodified ones. A selector gene is integrated into the plasmid together with the desired target gene and provides the cells with resistance to an antibiotic, such as [[kanamycin]], [[ampicillin]], [[spectinomycin]] or [[tetracycline]]. The desired cells (along with any other organisms growing within the culture) can be treated with an antibiotic allowing only the modified cells to survive. The antibiotic gene is not usually transferred to the plant cell but instead remains within the bacterial cell.

==Plasmids replication==

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T-DNA contains two types of genes: the oncogenic genes encoding for enzymes involved in the synthesis of auxins and cytokinins and responsible for tumor formation; and the genes encoding for the synthesis of opines. These compounds, produced by condensation between amino acids and sugars, are synthesized and excreted by the crown gall cells and consumed by A. tumefaciens as carbon and nitrogen sources.

Outside the T-DNA, are located the genes for the opine catabolism, the genes involved in the process of T-DNA transfer from the bacterium to the plant cell, and the genes involved in bacterium-bacterium plasmid conjugative transfer. (Hooykaas and Schilperoort, 1992; Zupan and Zambrysky, 1995). The T-DNA fragment is flanked by 25-bp direct repeats, which act as a cis -element signal for the transfer apparatus. The process of T-DNA transfer is mediated by the cooperative action of proteins encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The Ti plasmid also contains the genes for opine catabolism produced by the crown gall cells and regions for conjugative transfer and for its own integrity and stability. The 30 kb virulence (vir) region is a regulon organized in six operons that are essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE) (Hooykaas and Schilperoort, 1992; Zupan and Zambryski, 1995, Jeon et al., 1998). Different chromosomal-determined genetic elements have shown their functional role in the attachment of ''A. tumefaciens'' to the plant cell and bacterial colonization: the loci chvA and chvB, involved in the synthesis and excretion of the b -1,2 glucan (Cangelosi et al., 1989) the {{not a typo|chvE}} required for the sugar enhancement of vir genes induction and bacterial chemotaxis (Ankenbauer et al., 1990, Cangelosi et al., 1990, 1991) the cell locus, responsible for the synthesis of cellulose fibrils (Matthysse 1983); the {{not a typo|pscA (exoC)}} locus playing its role in the synthesis of both cyclic glucan and acid succinoglycan (Cangelosi et at. 1987, 1991) and the att locus, which is involved in the cell surface proteins (Matthysse, 1987).

==References==